CLIN.
CHEM.
28/12,
2351-2358
(1982)
The Biological Role of EnzymeAttachmentand Immobilization
Mario Werner, Carleton Garrett, Arthur Chiu, and Lev Klempner
The state of enzymes in the living cell is Considered: It is
conditions
not clear whether some, if any, enzymes are naturally
present in the “soluble” state. The evidence for enzyme
binding to cellular constituents, and its effects on function
and properties of enzyme and isoenzymes, is reviewed.
are largely regulated
Additional Keyphrases:
“soluble” and “fixed” enzymes
biochemistry of enzymes as affected by attachment
histochemistry as evidence of enzyme status
arte facts in evaluation of enzymes
lysosomes
.
.
Enzymes immobilized
in vitro are a subject of intense current interest in the development
of analytical procedures
and
instruments
(1). Anyone investigating
these applications
soon
recognizes
that bonding
enzymes
to a carrier
profoundly
changes their properties
(2), but this insight has hardly affected the way in which enzyme action in vivo is viewed. Although some enzymes are known to be anchored
in a rigidly
structured
microenvironment
and others are known to be
associated
with specific organelles-albeit
their action may
not depend on such an attachment-many
more enzymes are
believed to be normally present in and to function in solution
in the cytosol.
This paper
brings
together
information
suggesting that there is a varied spectrum of enzyme bindings,
ranging from tight and irreversible
to transient
and reversible,
all of which are relevant
to function
regardless
of the bond
characteristics.
Some Enzymes Only Function Tightly Bound to
Membranes
Enzymes crucial for cell function are associated
with all of
the various membrane
fractions
of the eukaryotic
cell, including the nuclear
and plasma membranes,
endoplasmic
reticulum,
and Golgi apparatus
(3). In all of these membrane
systems the basic membrane
unit appears to be a bilayer of
phospholipid
in which polar groups are located on the exterior
of the bilayer and hydrophobic
regions are located in the
central region of the bilayer (4). Enzymes
are considered
to
be loosely bound to the membrane
if they are attached
to the
surface of the membrane
through mainly ionic interactions,
and to be tightly bound if a hydrophobic
portion of the enzyme
molecule penetrates
into and permanently
resides within the
hydrophobic
region of the membrane
(4,5).
The plasma
membrane
of the erythrocyte
represents
a
prototype
membrane
system (6, 7). A cytoskeleton
composed
of microfilaments
and actin is located adjacent
to the inner
surface of the plasma membrane
of this and other cell types
as well (8, 9). An extensive listing of enzymes bound in this
system has been published
by Schrier (7). Erythrocyte
shape
and deformability,
which are important
factors in such clinical
Division of Laboratory
versity
Medical
Center,
Medicine,
901 23rd
20037.
Received Apr. 15, 1982; accepted
The George Washington
Street,
N.W.,
Aug. 31, 1982.
Washington,
UniDC
as sickle cell disease
and hereditary
spherocytosis,
by such membrane-associated
enzymes
(6). For instance, membrane-bound
ATPases
regulate intracellular cation concentration
and water content; these in turn
control cytoplasmic
viscosity,
a cardinal
regulator
of erythrocyte
deformability.
(ATPases are considered
in more detail
below.) Calcium-activated
protein kinases, which adhere to
the inner surface
of the plasma
membrane,
alter membrane
flexibility
and mechanical
stability through phosphorylation
of cytoskeletal
proteins. Erythrocyte aging and the membrane
loss seen in hereditary spherocytosis
may be in part due to the
activation
of another
membrane
enzyme, phospholipase
A
(EC 3.1.1.4), which causes the accumulation
of 1,2-diacylglycerol, thus perturbing
the structure
of the lipid Inlayer and
resulting in echinocyte formation and the release of membrane
vesicles (6). These examples are restricted
to the erythrocyte
plasma membrane,
but similar interactions
between enzymes
and the plasma membrane
of other cells have been implicated
in the membrane
shedding
observed in tumor cells and cells
of the immune system (10) and in mechanisms
of tumor-cell
metastases
(11).
The binding of enzymes to membranes
imposes a topography on the workings of the cell (3, 12). It reduces the orientation of the enzymes’ interactions
from a three-dimensional
space to a quasi two-dimensional
space. Thus membranes
provide a matrix on which multienzyme
systems catalyzing
coupled reactions
can more readily be arranged
spatially:
for
instance,
the electron-transport
chain, or systems involving
multiple intermediates
such as steroidogenesis,
are embedded
in membranes
(12, 13). Furthermore,
membranes
establish
compartments
that define inside and outside regions in cells
and subcellular
organelles.
If one abandons
the notion that
biological
membranes
are passive matrices,
bound enzymes
can be perceived to create gradients
or vectorial relationships
across biological
barriers.
Examples
would include the hydrogenLion gradient
associated
with adenosine
triphosphate
(ATP) generation
(13, 14) or the gradients associated with the
active transport
of inorganic
ions, sugars, and amino acids
(15-17). Analogously,
the “information”
contained
in hormone
signals
is transduced
from
extra-
to intracellular
space
across a lipid bilayer that is relatively
impermeable
to hydrophilic
“messenger”
molecules.
This is accomplished
by
activation
of an enzyme bound to the inner surface of the
plasma membrane
(18-20) when a hormone attaches to a receptor at its outer surface (21-25).
In essence,
then, there are three key features
of membrane-bound
enzymes to be considered:
(a) orientation
by
restriction
on a two-dimensional
plane,
(b) transfer
of
chemical energy from outside to inside and vice versa, and (c)
similar transduction
of information.
Asymmetric
orientation
of enzyme
in membranes.
Macromolecules
bound to membranes
have relatively
free lateral
movement
(26, 27) but essentially
no transverse
mobility,
preventing
a “flip” or transverse
reorientation
within the
membrane (26-29). These restrictions maintain crucial spatial
CLINICALCHEMISTRY,Vol. 28, No. 12, 1982 2351
relationships
in cases where proteins interact directly rather
than
through
mobile
low-molecular-mass
intermediates.
Further,
enzyme activity generally
is localized on only one
surface of the membrane
(12,30-34),
so the reaction catalyzed
potentially
can be influenced
by the flow of reactants
through
the membrane.
Examples
of this include the modulation
of
substrate
supply to NADH dehydrogenase
(NADH
cytochrome c reductase, EC 1.6.99.3) or of inorganic phosphate
supply for the ATP synthetases
in the mitochondrial
mem-
brane (25, 36); similarly,
enzymes
for the synthesis
and
packaging
of peptide hormones
and secretory
proteins
form
an integral part of the membrane (37,38). Also, the substrates
for some membrane-bound
enzymes
are themselves
membrane components,
an example being the use of arachidonic
acid, a component
of phosphatidylcholine
and ethanolamine,
by membrane-bound
prostaglandin
synthase
(EC 1.14.99.1)
(39).
Energy
transduction
by gradients
across membranes
(Figure 1). Such transfers
typically involve the interconversion of chemical
and binding energy, through the linking of
enzymatic
activity
to morphological
structures.
Electron
transport
in the mitochondrion
is the classically
quoted example, but similar events occur elsewhere.
The mitochondrion
is enclosed by two membranes,
which
define an intermembrane
space and a matrix space inside the
inner membrane
(40,41). The two membranes
differ in both
composition
and structure.
The smooth outer membrane
has
a low surface-to-volume
ratio (42), a high lipid-to-protein
ratio, and is permeable to most molecules of molecular mass
less than 5000 daltons. By contrast, the highly invaginated
inner membrane has a high surface-to-volume
ratio (42) and
a low lipid-to-protein
ratio, which makes
selective. Therefore,
carrier proteins
permeability
embedded
highly
in the mem-
brane must facilitate the passage of specific metabolites,
and
most hydrophilic
substances
indeed cannot traverse the inner
membrane
(43).
ATP
H’
MATRIX
SPACE
INTE RM EM BR AN OUS
,PACE
t
OUTER
MEMBRANE
Fig. 1. Oxidative phosphorylation on the inner mitochondrial
membrane
The membrane-bound ATP synthetase complex comprises two major parts, a
hydrophilic head piece (F1), which projects from the inner membrane into the
matrix and can bind ADP as well as ATP. and a hydrophobic base piece (F0)
embedded in the inner membrane and known to be a proton channel. The head
piece is attached to the base piece and can block the proton channel. When ADP
and inorganic phosphorus are present, the head piece permits protons to flow
through the channel from the intermembrane space to the matrix, and ATP is
concomitantly synthesized. Conversely, when ATP is hy&olyzed in the presence
of uncouplers, protons are pumped In the opposite direction from the matrix into
the intermembrane space. In this way, active transport by the ATP synthetase
complex acts as a reversible proton pump. (OH. NADH dehydrogenase; Q,
coenzyme 0; a,a3,b,c,c,, cytochromes)
2352
CLINICALCHEMISTRY,Vol. 28, No. 12, 1982
The mitochondrial
electron-transport
chain consists
of
three closely coupled enzyme complexes
(Complex I, III, and
IV) and molecules
that transport
electrons
among these
complexes,
cytochrome
such as NADH, ubiquinone (coenzyme Q), and
c (44-46). NADH is formed in the mitochondrial
matrix by the Krebs cycle and then oxidized
to NAD
by
Complex I, which simultaneously
reduces ubiquinone.
Complex III oxidizes ubiquinone
and reduces cytochrome
c, while
Complex IV oxidizes cytochrome c and reduces 02 to H20.
Figure 1 shows the relationship of these complexes to membrane structures. The free energy liberated in these reactions
can be used to alter the conformation of the inner mitochondrial membrane
(47), to transport
tons) across the inner membrane
(13).
cations (particularly
(46), or to synthesize
proATP
The coupling of free energy release to these three functions
by enzymes associated with membrane structures is only incompletely understood, and the elucidation of these mechanisms is still an important
goal of molecular biology (13).
Three theories, involving to various degrees the topographical
differentiation
caused by membrane-bound
enzymes, have
been proposed:
(a) In its simplest form, the chemical
enzyme Complexes
theory
I, III, and IV generate
postulates
that
an intermediate
with a high-energy
bond, and that this bond energy is transferred during an interaction
with ADP and ATP synthetase
(48). This hypothesis
has few adherents
at present. However,
Green anj his collaborators have proposed a model for direct
coupling
between ATP synthetase
and the enzymes of the
electron-transport
chain without the generation
of chemical
intermediates
(49-51).
(b) In 1961, Peter Mitchell formulated
a chemi -osmotic
theory which proposes
that electron transport induces the
transfer
of hydrogen
ions from the mitochondrial
matrix to
the intermembranous
space. This chemi-osmotic
gradient
provides the energy for ATP synthesis, for the active transport
of calcium and other cations into mitochondria,
and for the
mechanical rearrangement
brane (52-54).
of the inner mitochondrial
mem-
(c) According to the conformation
theory, electron transport induces conformational
changes in membrane
proteins,
which result in a transmembrane
proton gradient and at the
same time in generation
of high-energy
ATP bonds (54-56).
Because conformational
membrane
change can be linked to
ATP synthesis
and because the proton motive force can be
linked to cation transport,
the chemi-osmotic
and conformational hypotheses
both view structural
membrane
changes
and the generation
of a proton gradient as the two sides of the
same coin (57). Thus, we may consider the two theories under
a single designation,
the chemi-osmotic
hypothesis of oxidative
phosphorylation.
Support for the chemi-osmotic
model includes: (a) results
obtained by techniques
developed
to measure the postulated
thermodynamic
potentials
(58, 59), (b) evidence
that ATP
synthesis
can be driven by an artificially
imposed
electrochemical hydrogen
ion potential
(60-64), and (c) the reconstitution
in vitro of proton-pumping
activity from individual
electron-transport
complexes
and the ATP synthetase
complex, as well as their coupling in osmotically
intact vesicles
(65).
Another example of the transduction
of binding energy into
chemical energy is provided in skeletal muscle. Here, calcium
ions are actively
transported
from the cytosol
into the sarcoplasmic reticulum
by a membrane-bound
enzyme that has
ATPase (EC 3.6.1.3) activity. In contrast to the mitochondrial
ATP synthetase
system, where energy from a proton gradient
generates
ATP, the sarcoplasmic
ATPase system generates
a calcium gradient
at the expense of ATP. Even so, the re-
versed
calcium
centration
flow that
results
from the high calcium
inside of the sarcoplasmic
reticulum
con-
leads to the
binding of calcium to a low-affinity
site on the ATPase inside
the sarcoplasmic
reticulum
and to ATP generation,
if only
ADP and inorganic phosphorus
are present (66-70). Calcium
binding to the enzyme is enhanced
by high pH, and vice versa
(71-75), and so the existence of a proton gradient
in this system again influences
ATP synthesis
(76). The possibility
of
a similar relationship
in the case of Na-K
ATPase also has
been suggested
(77).
Information
transduction
across biological
membranes.
The synthesis
of adenosine-3’:5’-monophosphate
by intracellular adenylate
cyclase (EC 4.6.1.1) in response to a signal
by an extracellular
hormone
is a classical example of information
transduction
by a membrane-bound
enzyme. The
product
of these events, cyclic AMP, then functions
as an
intracellular
“second messenger”
for the signals provided
by
a clinically heterogeneous
group of hormones.
“Second messenger” activates
a variety of protein kinases, which in turn
alter the activity of other proteins by phosphorylating
them
(78, 79).
The adenylate
cyclase system comprises
at least three in-
proteins: C-protein,
regulatory
GIF protein,
and hormone receptor (80-85). Lateral mobility of the components of this system as well as membrane
fluidity conceivably are important
to the regulation
of cAMP synthesis,
but
the quantitative
aspects have not been clarified (86).
trinsic
membrane
Molecular Markers and Cellular Receptors
Enzyme Binding Efficient
Make
The formation
of lysosomes
is poorly understood.
Traditionally,
they were thought
to be sac-like structures
loosely
filled with hydrolytic
enzymes. Dominant
theory still holds
that newly synthesized
hydrolytic
enzymes are transferred
to the Golgi apparatus
or the “Golgi-endoplasmic
reticulum-lysosome”
complex,
and detached
as primary
where
they
are packaged
lysososmes
into
vesicles
(87). Since these assumptions
have never been substantiated
experimentally,
Neufeld and coworkers
incorporated
new findings in an alternative
hypothesis
(88, 89), which can be summarized
by
three postulates: (a) the presence of a common recognition
marker
on lysosomal
enzymes,
(b) the presence of specific
receptors
on the cell surface,
and (c) the existence
of a
mechanism
by which lysosomal enzymes equipped
with recognition markers are first secreted to the cell exterior, are next
recaptured
by receptor-mediated
endocytosis
and only then
are packaged
into lysosomes.
This secretion-recapture
hypothesis
thus describes a highly structured
process by which
lysosomal enymes are transported
and packaged,
and postulates that they bind through a marker-receptor
complex, first
on the cell surface and then on the lysosomal membrane.
Evidence
for a common recognition
marker on lysosomal
enzymes.
It is well established
that lysosomal
enzymes
in
human cells exist in “high-” and in “low-uptake”
forms (88),
the rate of cellular uptake differing
by one to two orders of
magnitude.
Treatment
with Na104 (88) or digestion
with
a-D-mannosidase
(EC 3.2.1.24) (90) converts
the “high-” to
the “low-uptake”
form without concomitant
loss of catalytic
activity.
Such conversions
of f-N-acetyl-D-hexosaminidase
(EC 3.2.1.52) and /3-D-galactosidase
(EC 3.2.1.23) confirmed
the presence of a recognition
marker and suggested
it to be a
carbohydrate
residue. Later it was also shown that mannose
6-phosphate
is a competitive
inhibitor
of f3-D-glucuronidase
(EC 3.2.1.31) uptake, and that phosphatase
treatment
of the
enzyme decreased its uptake (91). The findings are similar for
such lysosomal enzymes as a-L-iduronidase
(EC 3.2.1.76) (92),
$-hexosaminidase
(93), and a-N-acetyl-D-glucosaminidase
(EC 3.2.1.50) (94). These results suggest that mannose
6-
phosphate is a key structure of the recognition marker common to lysosomal enzymes. Indeed, mannose 6-phosphate has
been chemically identified on the high-uptake
forms of
f3-galactosidase
(95), 19-hexosaminidase
(93), and 3-glucuronidase (96). Finally, failure of cells to equip the lysosomal
enzymes with recognition
markers leads to the diseases known
as mucolipidosis
II and III, which are discussed
below.
Evidence for specific receptors on the cell surface. If en-
zyme internalization
is receptor-mediated,
the mechanism
must be saturable, and saturability
of cellular uptake has been
shown for several lysosomal
enzymes
(88). Attachment
of
lysosomal
enzymes
to the cell surface has also been evidenced
directly by sensitive fluorometric
assay of cell-bound
enzyme
activity (97). Other studies have shown that suppression
of
protein synthesis
leaves the rate of a-L-iduronidase
uptake
unchanged
at near-saturating
enzyme concentration.
Given
a limited number of receptors,
this suggests continuous
regeneration
and recycling of receptors
(97).
Evidence that enzymes
are secreted, recaptured,
and only
then packaged
into lysosomes.
Fibroblasts
of patients
with
mucolipidosis
II (I-cell
disease)
and mucolipoidosis
II
(pseudo-Hurler
zymes
polydystrophy)
(88, 98, 99). These
companied
lack several
intracellular
lysosomal
deficiencies
by an excess of the same enzymes
en-
are ac-
in the body
fluids or in the media of cell cultures (90,98-100).
Therefore,
these diseases are not due to deficient enzyme production, but
rather due to inappropriate
enzyme
localization:
extracellular,
rather than intralysosomal
(101). Further, fibroblasts
from
these patients, presented with “high-uptake” a-L-iduronidase,
internalize
and retain the enzyme with the same nine-day
half-life as normal controls and thus are not “leaky” (102).
Transfer
of 3-hexosaminidase
activity from normal to enzyme-deficient
fibroblasts
in co-culture can be shown by direct
assay of single cells (103). Similarly,
co-cultured
fibroblasts
with different
defects in lysosomal
enzyme production
mutually correct their deficiencies,
which suggests the possibility
of intercellular
“crossfeeding”
of lysosomal
enzymes
(102).
On the other hand, treatment
of normal human fibroblast
cultures with goat antibodies
sults in decreased intracellular
to human a-L-iduronidase
re-
a-L-iduronidase
activity, even
though the immunoprecipitated
enzyme retains full activity
(88). The most probable
explanation
for this finding is competition between antibodies and excreted enzyme for receptors
on the cell surface (88). Analogous explanations
apply to
earlier studies showing that normal fibroblasts
grown in the
presence of antibodies
against lysosomal enzymes assume the
morphological
features of cells from patients
with mucolipidosis (104). The aggregate of these fmdings makes the secretion-recapture
hypothesis
of lysosomal transport believable,
but there are contradictory
data. For instance,
the block of
enzyme recapture
by excess mannose 6-phosphate
or by antibodies against lysosomal enzyme did not decrease intracellular enzyme activity of cultured fibroblasts
as expected (105,
106). Rather,
this suggested
that most lysosomal
enzymes
normally
cycle through
the cell surface in a receptor-bound
form, while only a small fraction
is released into the extracellular space (105). If one accepts this modification
of the
main hypothesis,
it provides the most cohesive explanation
of the largest number of experimental
data.
Bound Enzymes Confer Metabolic
Functions to the Vascular Bed
and Regulatory
The vascular system is commonly
thought
of as a hydromechanical
pipe system. However, the vast endothelial
surface
can also be considered
equivalent
to an organ with singular
metabolic
and regulatory
functions,
because enzymes are attached to it or are associated
with the surface of cells attached
to it, such as the Kupffer cells. The relevance
of this view is
CLINICALCHEMISTRY,Vol. 28, No. 12, 1982 2353
illustrated
by three examples:
the role of bound lipoprotein
lipase in triglyceride
metabolism,
the effects of enzymatic
activities
in the vascular surface on the metabolism
of hormones, and the crucial interactions
between plasma enzymes
and endothelium
in initiating and demarcating
the hemostatic
process.
Assimilation
of lipoprotein
triglyceride.
Triglyceride assimilation
into tissue is controlled
by a dual process: passive
modulation
that changes plasma triglyceride
and active regulation of triglyceride
hydrolysis
by lipoprotein
lipase (LPL;
EC 3.1.1.34). Experiments
with rat adipose and heart tissue
suggest the LPL is synthesized
in parenchymal
cells, perhaps
structurally
altered
before being released
from them, and
subsequently transported
into the vascular bed, where it attaches to the luminal surface (Figure 2). Here LPL can associate with a specific
protein,
apoC-Il,
which
is present
on the
surface of the lipoproteins that are its natural substrate (107,
108). Two key postulates of this sequence are that the endothelial cell would not synthesize
but only bind LPL and that
the LPL activity in vivo is expressed
only at the endothelial
surface of the vascular
bed (109). LPL activity, barely detectable in growing fibroblast
cultures,
increases about 100fold as the cells mature into adipocytes
(110). The persistent
presence of LPL in adipocytes
after their isolation or culture
(111) suggests that these cells synthesize
the enzyme-but
cultured bovine-aorta endothelium does not synthesize LPL
(112). These observations
suggest that the vascular endothelium may only be a secondary LPL receptor site. Conclusive evidence might require tracking the enzyme with, say, a
labeled antibody.
Extracellular
LPL activity and immunologically
assayed
LPL of fat tissue increase in fed and insulin-treated
animals
(113). It is uncertain
whether
this represents
new protein
synthesis,
activation
AMINO
of proenzyme
already present,
ACIDS
or a reg-
VLDL
)c.ll
CAPILI..ARY. ENDOTHELIUM
FIg. 3. Attachment of lipoprotein lipase by glucosaminoglycans
to capillary endothelium and fixation of lipoprotein particle
ulatory role of the plasma membrane of the fat cell in LPL
release. Most LPL is believed to be inside the fat cell in the
fasted animal but extracellular
in the fed (114, 115). When
adipose tissue of fasted rats is incubated in an appropriate
medium at 25 #{176}C,
with protein synthesis decreased to about
5% of the usual rate by cycloheximide,
LPL activity still
doubles or trebles within 2 h (108). Some say this suggests
pro-enzyme activation. Finally, the possibility that the plasma
membrane of the fat cell is involved in LPL regulation is
supported by the observation that LPL secretion by adipocytes is greater in insulin-treated
rats than in controls (116).
Heparmn-releasable LPL in blood presumably corresponds to
the functional and the working LPL of Figure 2. Glucosaminoglycan chains anchor such LPL to capillary luminal endothelium (117). In rat heart, this surface activity has been
shown to turn over in about 2 h (118, 119). Consequently,
maintenance
of enzyme on the endothelium
depends on a
continued supply from parenchymal cells. Thus the efficiency
of renewal can promptly influence two metabolic events, because LPL not only regulates plasma lipoprotein concentrations but it also catalyzes the assimilation of triglyceride fatty
acids by tissues.
Chylomicrons
and very-low-density
lipoproteins
(VLDL)
are LPL substrates,
and the LPL alone catalyzes their entire
degradation,
producing some high-density lipoprotein species
the enzyme binds circulating lipid particles through specific interaction with the apoC-Il apolipoproteins (119) (Figure 3). Other interactions between LPL and
lipoprotein
lipid, and between anchoring
glucosaminoglycan
chains and the lipoprotein surface, may enhance particle attachment. ApoC-Il greatly enhances LPL activity in vitro, and
various studies suggest that the apoC-Il content of VLDL and
(120-1 23). Probably
Synthesis
PRO LIPOPROTEIN
LIPASE
Modification
ACTIVE LIPOPROTEIN
Secretion
LIPASE
from Cell
Transport
Binding to Endothelium
FUNCTIONAL
LIPOPROTEIN
Attachment
of
or Chylomicron
LIPASE
VLDL
WORKING LIPOPROTEIN
LIPASE
I..
Removal
Hepatic
from Endothelium
Uptake
DEGRADATION PRODUCTS
Fig. 2. The metabolism of lipoprotein lipase
See text for details of this sequence
2354
CLINICALCHEMISTRY,Vol. 28, No. 12, 1982
chylomicrons
matches or even exceeds that required
for optimal triglyceride
hydrolysis
(124-127). Absence of apoC-Il,
on the other hand, causes a severe hypertriglyceridemia,
which
can largely be corrected by infusion of normal plasma (128).
Thus apoC-Il is a necessary but not normally rate-limiting
facilitator of catabolism of triglyceride-rich
particles. Conversely apoC-I (129-131), apoC-Ill (129, 132), and apoE (131)
inhibit LPL in vitro.
Contact between working LPL and fat particles rapidly
produces fatty acids from triglycerides. Subsequent hydrolytic
events are less well understood, but LPL may also degrade
diglyceride
to monoglyceride.
Most of the fatty acids are
transported
into tissue, but some escape into the circulation
(133). The hydrolysis
of lipoprotein
triglyceride
is believed
to occur in a series of reversible attachments
of working LPL
to the endothelium
(134). At the end of this process, some
LPL molecules attached to partly degraded chylomicrons and
VLDL may be swept into the circulation. These lipoprotein
remnants may mark such complexes for hepatic uptake (135),
and LPL thus may be cleared from the circulation within
minutes (136). Indeed, the rapidity with which the liver removes it may effectively confine LPL function to the site of
its original binding.
Degradation
of circulating
hormones.
Plasma
hormone
concentration
results both from their release into and clearance from the circulation. Uptake of circulating insulin (137),
glucagon (138), somatotropin (139), lutropin (140), follitropin,
choriogonadotropin
(141), and prolactin (142) by Kupffer cells
phospholipid
sources and
faster and is
into solution
in the liver was documented
a decade ago. Very recent evidence implicates
enzymic activities of the vascular bed in the
factors become
half-lives.
regulation of parathyrin, thyroxin, and angiotensin catabolism.
New findings suggest that Kupffer cells catabolize parathyrin into fragments, which are also seen in blood (143-1 45).
Thus, it has been shown that the liver is the source of carboxy-terminal
parathyrin
fragments but removes neither
amino-terminal
nor carboxy-terminal
fragments from the
circulation (146). Consequently,
one can postulate the presence of an enzymatic system capable of degrading parathyrin
on the surface of Kupffer cells.
Thyroxin (T4) appears to be metabolized on the surfaces
of cells, either within or near the vascular bed (147,148). The
monodeiodination
ofT4 to triiodothyronine
(T3) appears to
be the major source ofT3 in some animal species (149) as well
as man (150-154).
Recently, two independent
studies identified an enzyme that converts T4 to T3 on the plasma membrane of cells from rat liver (148) and rat kidney (147). In
neither instance were the organs fractionated
into parenchymal and nonparenchymal
cells to identify exactly where
Inactivation
by inhibitors in solution, affecting surfaceactivated proteases, is also encountered
in plasminolysis
(175-177)
and in the complement pathway (178). The more
general role of molecular attachments in enzymic conversions
occurring in plasma, however, is again evidenced by an example involving lipid metabolism. Lecithin:cholesterol
acyltransferase (EC 2.3.1.43) is the major enzyme for esterification
of cholesterol in serum (179). It catalyzes the transfer of an
acyl group from the 2 position of lecithin to the 3-hydroxyl
position of cholesterol (180). This enzyme, synthesized in the
liver, is activated by apolipoprotein
A-I (179, 181, 182). Although nominally considered a soluble enzyme (107), it appears to be present in plasma either on the surface of highdensity lipoproteins, where both its activator and substrates
are located as well, or as a complex with apolipoprotein
A-I
and cholesteryl transfer protein (apolipoprotein
D) (183).
Moreover, studies in which synthetic vesicles and multila-
the T4-deiodinase
activity
Angiotensin-converting
is located.
enzyme (dipeptidyl
by endothelial
cells:
soluble
and
targets
for inhibitors
and may have shorter
mellar liposomes were used as substrates
steric relationships
must exist between
particle surface (182).
suggest that specific
this enzyme and the
carboxypep-
tidase, EC 3.4.15.1) catalyzes the conversion of angiotensin
I to angiotensin
II and and the degradation
of bradykinin, a
vasodilator. The combined effect is vasoconstriction
and increased blood pressure
(155-158).
Two forms of it are synthesized
from damaged endothelium,
platelets, or other
activated co-factors are present, coagulation is
localized to that area. In contrast, once released
or removed from phospholipid binding, activated
cell-membrane
bound enzyme (159). The soluble form may be constant
throughout
the vascular system, but the membrane-bound
enzyme seems to vary among organs. The catalytic site of the
bound enzyme has access to circulating blood. Its localization
on vascular endothelium
is evidenced by light microscopywith use of fluorescent-labeled
antibody to the enzyme-in
rabbit lung, liver, adrenal cortex, pancreas, kidney, and spleen
(160, 161) and, with electron microscopy and peroxidaselabeled antibody to the enzyme, in rat lung and cultured endothelial
cells (162, 163).
Interaction
between plasma enzymes
and surfaces in the
vascular bed. In thrombosis and hemostasis, plasma enzymes
act on each other for the production of an insoluble fibrin clot,
which may be dissolved again at the conclusion of the healing
process. Interaction
of coagulation
factors with the exposed
functional
groups in the damaged endothelium
initiates this
enzyme cascade (164, 165). The subsequent processes consist
of several specific and limited cleavages of single peptide
bonds, which results in activation of zymogens, latent cofactors, or fibrinogen. Unchecked, this process is irreversible and
multiplicative, as demonstrated
by blood clotting in a test tube
(166-1 71). In contrast, hemostasis
in situ always remains
topographically confined. Exposed phospholipids of damaged
endothelium
again are essential for localizing the extent of
hemostasis. The mechanism of demarcation
can be through
positive or negative control of proteolysis, or a combination
of both. An example of positive control is the activation of
thrombin by the prothrombin complex. Factors Va and Xa and
Ca2+ can catalyze thrombin formation, but the complete
prothrombin
complex also requires
negatively
charged
phospholipids, and when all four components are present, the
rate of thrombin activation is accelerated by five orders of
magnitude (172). Factor Va interacts with the phospholipids
in a hydrophobic manner (173), and the constant for the dissociation
of Xa from the prothrombin
complex decreases
by
four orders of magnitude
in the presence
of Va (174). Where
Few Working Enzymes May Be in the Freely
Soluble State
“Soluble”
enzymes.
Subcellular
enzyme localization
has
been built on the bedrock
of differential
centrifugation
of
homogenized
tissues (184). The fractions resulting from a
series of increasingly forceful centrifugations
have been correlated-by
morphological characterization-with
different
organelles, and the purity of these cuts is usually assessed from
the activities of enzymes believed to be markers of different
organelles. The final residue, which cannot be precipitated
by conventional
gravity forces, is believed to contain those
substances, including enzymes, that are free in the cytosol.
This approach has supplied substantial new information, but
it must also be recognized as potentially misleading. Cell
disruption and the suspension of cell matter can cause artefacts. The use of aqueous media may cause loss of water-soluble substances from organelles, adsorption of other substances to organelles, and transfer of substances between organelles. Use of sucrose solutions, isopycnic fractionation, and
non-aqueous media lessen these translocations,
but no artificial medium sufficiently
resembles native cytoplasm to
protect all its constituents
from change.
Such limitations can be debilitating when subcellular attachments of enzymes in the cytosol are to be defined, because
these molecules would be expected to be the most susceptible
to redistribution.
Consequently
the description of cytosolic
enzymes as “soluble” should no longer necessarily imply that
these molecules are in fact freely dissolved within the cell, and
“cytosolic enzymes” should simply be considered an operational fraction defined by centrifugation. Subcellular binding
of these enzymes
is believed to be possible in two ways-(a)
through interaction with cellular structures or (b) through
assemblage into multi-enzyme
complexes-but
characterization of such loosely complexed enzymes probably requires
that additional techniques be applied, to complement centrifugal analysis.
Binding
of enzymes
to formed
cellular
structures.
Enzymes
may bind to cell surfaces or to intracellular
structures. Examples of surface attachment pertaining to the vascular bed
have already been presented in the previous section. The small
intestine provides another example of soluble enzymes adCLINICALCHEMISTRY,Vol. 28, No. 12, 1982 2355
sorbing onto a surface and interacting
with
brane proteins.
The “membrane
digestion”
tulates that pancreatic
enzymes are adsorbed
of intestinal
mucosal cells, thus ensuring the
insoluble
memhypothesis
posto the glycocalyx
proximity of exo-
and endopeptidases
to the transport proteins of the brush
border (185, 186). In a test to evidence this association,
crystalline trypsin (EC 3.4.21.4) and chymotrypsin
(EC 3.4.21.1)
were mixed with homogenized
intestinal
mucosa and fractionated
by differential
centrifugation
(187, 188). The fact
that the enzymes were again recovered in the microsomal
fraction without loss of activity was considered proof of the
postulated attachment. The binding was optimal between pH
5 and 8.5, but was diminished by non-ionic detergents such
as Nonidet P-40 and deoxycholate. However, no ultrastructural study has been performed to confirm the postulated
enzyme location.
The association of hexokinase
(EC 2.7.1.1) with mitochondria (189-191) and the associations
of aldolase (EC
4.1.2.13),
lactate
dehydrogenase
(EC
1.1.1.27),
glyceralde-
hyde-3-phosphate
dehydrogenase
(EC 1.2.1.12), pyruvate
kinase (EC 2.7.1.40), and phosphofructokinase
(EC 2.7.1.90)
with cell structures and particulate elements (192-197) have
furnished the most relevant information on the interactions
of cytosolic enzymes with intracellular
structures.
The cogent
characteristics
of these associations
are the presence of specific
binding sites on formed structures,
the reversible attachment
of enzymes,
and the possibility
of simultaneous
conformational change. The resulting biphasic enzyme distribution has
implications
that may touch not only on the metabolic role of
but also, more far reaching, on metabolic control
isoenzymes
at the molecular
level.
If one considers enzyme interactions to be biologically significant, it is not surprising that isoenzymes differ in both
kinetics
and binding
properties
(197-1 99), given that they
function in different cellular microenvironments.
Creatine
kinase (EC 2.7.3.2) can serve as an example. According
to
classical cell-fractionation
data, the bulk of it is recovered in
the soluble cell fraction (200-205). However, more recent data
suggest that cytosolic activity comprises at least three isoenzymes-MM,
MB, and BB-which
carry out identical reactions but are differently distributed
in tissues and possibly
bind to different intracellular
structures as well (206). In
skeletal muscle the MM isoenzyme
binds to a particulate
fraction within the M-band (207,208), where it appears to act
in concert with myosin ATPase. In the mitochondrial
intermembranous
space, another creatine kinase isoenzyme is
bound to the outer surface of the inner membrane, where it
transfers high-energy phosphate bonds out of the mitochondna, working together with the adenylate kinase (EC 2.7.4.3)
bound to the inner surface of the inner membrane (209).
Thus, the different binding sites of the mitochondrial and MM
isoenzymes of creatine kinase (210, 211), and the resulting
anabolic and catabolic interactions with other enzymes, form
a complex system supplying the energy for muscle contraction.
It is generally accepted that binding can significantly alter
the kinetics of an enzyme’s action. To describe the circumstances in which the same enzyme may coexist in both functionally different free and reversibly-bound
forms within the
cell, the term “ambiguity” has been coined. Should this partition vary with changing circumstances, the resulting changes
could have important regulatory implications. In fact, several
investigators
involved
have suggested
in changes
that alterations
of metabolic
status
in ambiguity
are
and in metabolic
control. For instance, experimental
data indicate a rapid and
reversible
shift to increased
proportions
of particulate
enzymes when glycolysis in mouse or chick brain is increased by
ischemia or insulin (212).
2356
CLINICAL CHEMISTRY, Vol. 28, No. 12, 1982
Assemblies
of soluble enzymes.
Soluble multi-enzyme
sequences
include those for fatty acid synthesis,
glutathione
synthesis,
pentose phosphate
shunt, glycolysis (glycogen to
pyruvate), tryptophan catabolism, and pyrmiidine catabolism,
and those in the ribosome (213). Some “soluble” multi-en-
zyme aggregates, such as pyruvate dehydrogenase (EC 1.2.4.1)
or the enzymes of fatty acid synthesis, appear to be relatively
stable and are quite well documented
(214, 215). Other assemblages, such as that comprising the glycolytic enzymes and
mammalian tRNA synthetase (216, 217), are more controversial. Over a decade ago, Green (218) derived preparations
from disrupted yeast cells and erythrocytes that catalyzed the
complete glycolytic sequence more efficiently than did the
whole homogenates.
He proposed that these preparations
contained enzyme aggregates, which he called glycosomes, and
that the aggregates are membrane bound in vivo. More generally, he postulated
that enzymes catalyzing other integrated
metabolic
sequences
might be similarly complexed
to membranes. Other investigators
have since provided experimental
support
for the concept of a glycosome
(219-222)
and, re-
turning to the general postulate, Ureta has suggested that up
to 10 poly-enzyme complexes may be involved in glucose
metabolism (223).
Methodology
Clearly,
phological
Offers No “Free Rides”
the investigation
of enzyme attachments
to morstructures
requires the combined
use of a variety
of analytical
techniques.
Histochemistry
is a promising
complement to differential centrifugation,
because it allows
enzymes to be made visible by light or electron microscopy at
their cellular and subcellular
sites. However, the artefacts
of
tissue fixation and of enzymic detection reactions can render
interpretation
ambiguous,
even in such restricted
observation
of static topography.
Further, the specificity of histochemistry
is limited, because enzymes are usually characterized
solely
by the choice of substrate.
Thus problems
related to tissue
preparation,
as well as problems
related to the detection
of
enzyme
activity,
must
be dealt with.
Problems
related to tissue preparation.
Histological
procedures require that tissue be frozen or chemically
fixed. Either method
perturbs
macromolecular
architecture
by the
denaturation
of proteins
and nucleic acids. Freezing causes
morphological
changes in membrane
structures
such as mitochondria.
In chemical fixation, proteins are cross-linked
by
such reagents as glutaraldehyde
(224) or formaldehyde
(225),
as are fatty acids by osmium tetroxide
(226). Either type of
fixation replaces a dynamic representation
of morphological
structures
(227) with the snapshot of a rigid skeleton. Further,
fixation may cross-link enzymes with structural proteins or
membranes, thereby obscuring the distinction between “soluble” and “fixed” enzymes (228). In addition, enzymic activity
itself may be lost to a varying degree during fixation, owing
to denaturation
or to spatial rearrangements.
In formaldehyde, only a fifth of acid phosphatase
(EC 3.1.3.2) and of
nonspecific esterase activities is lost within 2 to 4 h at 4 #{176}C,
whereas cytochrome c oxidase (EC 1.9.3.1) and succinic dehydrogenase (EC 1.3.99.1) are completely inactivated (229).
On the other hand, freezing partly destroys other enzymic
activities, such as that of succinic dehydrogenase,
by an
unexplained mechanism (230).
Problems
related
to the
detection
of enzyme
activity.
There can be problems related to substrate and problems
related to reaction conditions. To assay all enzyme present
requires zero-order kinetics, and excess substrate must be
present. This limitation
precludes
kinetic studies. Next, certain substrates
are difficult
to solubilize
other than in dimethylformamide,
but this solvent may affect enzymic activity by altering
the dielectric
constant
(231). Further, to
localize enzymes unequivocally, it is necessary to precipitate
the reaction product and so reduce its diffusion after completion of the detection reaction. Such fixation may require
a different pH from the one optimal for enzymic activity.
Therefore,
assay conditions
may represent
a compromise
(232). Together, these technical problems render the histo-
chemistry
of enzymes
qualitative
rather
than quantitative
(233).
Attempted
solutions
and applications.
For avoiding the
problems of specificity and quantitation
in histochemical
enzyme localization, detection by use of immunoreactions
offers one of the most straightforward
solutions. As an additional advantage, enzyme precursors and enzyme breakdown
products
may be made visible
(234,235)
and isoenzymes
may
be distinguished (236237). To avoid the problems of fixation,
flow cytometry can be combined with the use of fluorescent
substrates. In this procedure, cells flow in single file past a
sensor, and a decision can be made whether to select any
particular one for observation or collection (238). This allows
the study of enzyme kinetics, enzyme synthesis, and subcellular enzyme localization in homogeneous viable cell populations. For instance, the kinetics of fluorescein-methotrexate
binding to tetrahydrofolate
dehydrogenase
(EC 1.5.1.3) and
the metabolism
of the complex could be followed in this
fashion. In addition, isolation of homogeneous cell preparations permits the investigation of enzyme kinetics over time,
as well as the study of enzyme turnover
in vitro (239). The
relevance
of such studies
is documented
by the observation
that the kinetics of fluorescein-diacetate
breakdown
by
nonspecific esterase differ in intact EMT6 cells and in homogenates
availability
of the same
(240).
cells,
reflecting
different
cated chain of events transports hydrolytic enzymes synthesized in the rough endoplasmic reticulum, first to cell-surface
receptors and thence following internalization to the lysosomal
membrane. Specificity and efficiency in this pathway is provided by recognition markers, a receptor-mediated
process
of fixation
and transport
A reversible
of bound
surface
enzymes.
attachment
seems to regulate
the
working of many enzymes. This is illustrated
by the binding
of pancreatic
enzymes to the glycocalyx
of intestinal
mucosa
and by the binding of lipoprotein
lipase to the vascular en-
dothelium. Indeed, the vascular bed is the site of action for
many enzymes that control hormone metabolism and, in the
case of hemostasis
and fibrinolysis,
it defines
the topogra-
phical area affected by enzyme action.
Regulation of enzyme activity in general can be viewed in
three phases: first, synthesis and induction; second, activation
or inhibition; and third, inactivation and degradation. Enzyme
binding in multi-enzyme
complexes or attachment
to morphological structures within the cell could influence metabolic
control in the latter two phases by affecting such kinetic parameters
as substrate
affinities or activity optima, by
achieving
a topographic
orientation
with respect
to substrates,
products, or other enzymes and, finally, by modulating degradative attack. Unfortunately,
there are still no simple
methods to avoid artefacts in the study of such enzyme
binding and localization that either are caused by tissue
preparation or are related to the detection of enzymic activity
by morphological methods. However, promising approaches
to the study
of enzymes
in living cells must
be developed.
substrate
A Summing-up
Enzyme action is determined
by events that occur concomitantly
but involve systems of vastly different scale.
Events within and between molecules, on the one hand, are
relatively well understood by the biochemist and the molecular biologist. Events within tissues, organs, and the whole
organism, on the other hand, are relatively well understood
by the physiologist, the pathologist, and the clinician. It is the
events at the interface between these spheres, occurring within
and possibly between cells-a
domain shared by the cell biologist and the biochemist-that
are understood
least, even
though they are certain to have relevant regulatory
implications.
Some general principles emerge from the aggregate evidence
brought together here. Tightly bound enzymes permit complex, closely linked reactions such as those of oxidative
phosphorylation
or steroidogenesis.
Usually, tightly bound
enzymes
are attached
to membranes.
These structures
introduce (a) ariisotropy of three-dimensional
space (i.e., regions
“inside”
and “outside”
the membrane)
and (b) quasi two-
dimensional spaces within the membrane itself. These fundamental features permit establishment
of gradients, control
of metabolite
flow by restricted
diffusion,
and spatial arrangement
of macromolecules.
Enzymes that are tightly associated with biological membranes
exploit these properties
in the maintenance of nutrient and ion gradients as well as in
the transfer of energy and transduction
of biological signals.
Indeed, the enzymes of metabolic pathways, involving multiple steps that generate many low-molecular-mass
intermediates, require tight coupling to function efficiently.
The functional organization achieved by transient and more
reversible attachments
is illustrated
by the intracellular
compartmentalization
of lysosomal enzymes. Cultured cells
can internalize almost any macromolecule, but such nonspecific “bulk” endocytosis is inefficient. By contrast, a compli-
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