ROLE
THE
OF
LYSOSOMAL
J. T.
From
The
not
the
Tissue
presence
until
de
enzymes
organelle
workers,
DINGLE,
Physiology
of digestive
Duve
and
Department,
cells
(the
exceptions
now
led
cells
Research
has
been
investigated
being
to a confirmation
the
context
of recent
on the
THE
In the
last
decade
this
the salient
points
In most cells this
mature
known
for
Cambridge
a long
intracellular
time,
but
localisation
it was
of
of lysosomes
system
has
turnover
in joint
of their
of lysosomes
of skeletal
LYSOSOMAL
been
the
and
these
lysosomal
consists
and
to contain
of protein
synthesis
1010are
in
acids,
at least
capable
polysaccharides,
in the
thirty
tissues,
ribosomes
of the degradation
and proteins.
subject
we
are,
as yet,
macromolecules.
of several
and
primary
vacuoles;
and usually
to be budded
formed
biological
enzymes
1) the
3) the autophagic
is a dynamic
one
endoplasmic
of every known
The lysosomal
publications
components:
enzymes
rough
thousand
in a three volume
work
(Dingle
than present
in a very simplified
recognised
synthesised
of the
though
The
tissues
was
laboratories
SYSTEM
system.
of four easily
newly
spermatozoon).
function
in these
tissues.
This
and their constituent
enzymes
tissue
reviewed
by some seventy
authors
possible
in a short
space to do more
of the
system
the
and pathology
of skeletal
in this and in many other
lysosomes;
2) the digestive
vacuoles
(or secondary
lysosomes);
and 4) residual
bodies.
The interaction
between
these organelles
occurs
by fusion
of their membranes.
The primary
lysosomes-These
organelles
are generally
considered
Golgi
TISSUES
Laboratory,
erythrocyte
of the full complexities
some of the properties
studies
has been comprehensively
1972).
It is therefore
not
the
to the physiology
Laboratory.
Work
of a role
far from
an understanding
article
will deal briefly
with
form
SKELETAL
ENGLAND
Strangeways
within
co-workers
relevance
of the lysosomal
system
first investigated
at the Strangeways
in the
CAMBRIDGE,
enzymes
his
IN
that it was realised
that a variety
of acid hydrolases
coexisted
within
a distinctive
(for review
see de Duve
1969).
This organelle,
named
the “lysosome”
by the Louvain
is now known
to be part of a vacuolar
system
present
in all but the most
highly
specialised
has
ENZYMES
off from
by the usual
reticulum.
These
polymer,
primarily
including
responsible
the
pathways
enzymes
nucleic
for the
degradation
of skeletal
matrix
macromolecules
will be discussed
below.
In most cells the life span
of the primary
lysosome
is thought
to be quite short,
its fate being fusion
with either
digestive
vacuoles
or with the plasma
membrane
of the cell.
In the first case, this fusion
provides
newly
formed
digestive
of the lysosome
circumferential
limited
vacuoles
with
are released
macromolecules.
autolytic
digestion
of endogenous
store of digestive
enzymes.
Digestive
vacuoles-Digestive
the plasma
membrane
of
the
extracellular
The
cell,
other
a complement
secondary
breakdown
VOL.
environment.
receiving
55
lysosomes.
of the
B,
NO.
1,
enclosed
FEBRUARY
their content
of enzymes,
and in the
to the outside
of the cell and may
Primary
lysosomes
may also provide
materials
and
in some
second
case the contents
initiate
the digestion
of
enzymes
for membrane
specialised
vacuoles,
or secondary
lysosomes,
are formed
cell which
is then “pinched
off”
to enclose
newly
formed
vacuole
moves
deeper
into
cells
may
act
as a
by an infolding
a portion
of
the cytoplasm
of
the
of the
of enzymes
by fusion
with
primary
lysosomes
or sometimes
with
The secondary
lysosome
usually
undergoes
dehydration
and rapid
(endocytosed)
materials
occurs.
It is probable
that the conditions
1973
87
88
of pH
and
ionic
for the action
It is known
lysosomal
concentration
of the
from
the molecular
it may pass
are
lysosomal
the specific
cathepsin
4 and 65.
It is
of the substrate
It is known
that
vacuoles,
and it
cell,
that
T. DINGLE
..
D that
controlled
to give
enzymes,
but
intravacuolar
the
a favourable
intravacuolar
the mechanism
of this
inhibition
of digestion
intravacuolar
pH
must,
at least
environment
process
is not understood.
by an antibody
against
part
of the
time,
be between
tempting
to speculate
that conditions
may change
depending
on the nature
and the state of its digestion,
but there
is little
information
on this point.
the degradation
of macromolecules
can be carried
to completion
within
these
seems
probable
that before
the products
of digestion
can be utilised
by the
the
size must
enclosing
be reduced
membrane.
to the equivalent
There
is good
of a disaccharide
or less, in order
evidence
that in the physiology
of
the extracellular
macromolecules
of skeletal
tissues
the secondary
lysosomes
are responsible
for the final stages
of the digestion
of both proteoglycan
and collagen.
The autophagic
vacuoles-Besides
their role in the digestion
of both extracellular
materials
and
material
endocytosed
by cells, the lysosomal
enzymes
are also responsible
for the turnover
of
intracellular
materials.
This often occurs
by the enclosure
of the unwanted
organelle
or other
material
with
by a membrane
primary
and
lysosomes
enclosing
the accretion
or perhaps
membrane.
There
of skeletal
matrix
and
Residual
incomplete
bodies-These
digestion,
is
they
from
evidence
no
will
of lysosomal
directly
not
components
often of lipids
be associated
with enzyme
deficiency
activity.
These
bodies,
although
thought
to be related
to any aspect
be
for
discussed
This
process
is fundamental
lysosomes
materials
may
occur
reticulum,
of autophagic
by fusion
if this
vacuoles
forms
in the
the
turnover
further.
of the lysosomal
or other relatively
system
are thought
to be associated
with
indigestible
materials,
and may possibly
or metabolic
changes
sometimes
associated
of matrix
turnover.
in the cell which
with pathological
impair
its digestive
changes,
are not
FUSION
to the distribution
and in allowing
to the outside
This
endoplasmic
a role
MEMBRANE
forms
of
extracellular
hydrolases.
the
of lysosomal
enzymes
the secretion
of both
of the cell.
The physical
between
lysosomal
processes
the various
enzymes
and
other
have been discussed
by several
authors
(for review
see Dingle
1969) but as yet no definite
conclusions
have been
made as to the factors
which
allow the fusion
of, say, a primary
lysosome
with a newly formed
digestive
vacuole
but not with a functional
mitrochondrion.
However,
it is clear that
the
process
is central
to any understanding
of the control
of lysosomal
function,
and if the thesis
that
lysosomal
enzyme
translocation
is of importance
accepted,
it becomes
of some practical
interest
that cortisol
acts when
preventing
the release
cartilage
skeletal
pathology
is
it is probable
that it is at this
enzymes
and the degradation
physiology
and
level
of
matrix.
EVIDENCE
Lysosomes
have
in
also.
Thus
of lysosomal
the
usual
have
FOR
been
properties
LYSOSOMAL
isolated
and
from
enzyme
FUNCTION
cartilage,
content.
IN
bone
They
SKELETAL
and
have
TISSUES
synovial
also
tissues
been
and
demonstrated
shown
to
to
be
present
by conventional
cytochemical
techniques
very recently
by immunocytochemical
methods.
numbers
of lysosomes
have been demonstrated
(such as staining
for acid phosphatase)
and
Under
pathological
conditions
increased
at the light and electron
microscope
levels in
animal
Increased
rheumatoid
synovium
been
demonstrated
in
and
human
levels
of
tissues,
enzyme
tissues
and material
from
work from this laboratory,
D, have demonstrated
pannus
and cartilage.
particularly
human
activity
have
also
rabbits
using
accumulation
with experimentally
immunocytochemical
of extracellular
produced
methods
enzyme
THE
(Page-Thomas
both
human
1969).
arthritic
arthritis.
Recent
unpublished
specific
for lysosomal
cathepsin
at the junction
JOURNAL
OF
BONE
between
AND
JOINT
invading
SURGERY
THE
The
above
tissues
of
been
carried
environment
collected
ROLE
studies
out
LYSOSOMAL
demonstrate
and
animal
OF
in
human
the
joints
This
vitro.
ENZYMES
presence
IN
SKELETAL
of lysosomes
in vito;
studies
is because
under
of the tissue
can be controlled
and
without
loss.
It is of course
essential
and
1969, Coombs
antiserum,
D. In such
but they synthesise
and release
enzyme
secretion
is correlated
localisation
of the degradation
by hydrocortisone
and Fell
indigestible
experiments
followed
of the
matrix
and
stimulated
their
From
studies
of actively
endocytosis
enzymes
escape
“regurgitation”
fusion
from
theory.
of primary
manner
and
newly
example
formed
in the
a
interaction
the
secretion
It is a
the
same
digestive
osteoclast,
(1969)
of
ofmatrix
manner
pH and
while
enzymes
hydrolases,
general
seem
to be present
deeper
intercellular
conditions,
as well
advantage
matrix.
connective
the extent
normal
proteoglycan,
55 B,
fusion
may
as well
secretion
lead
to
at
an
accelerated
with
a
neutral
in lower
pH
optimum
concentration,
are
Thus
those
synergism
acting
on
and
in the
directed,
local
the
polymers
of
by a
there
is at
can be established.
favourable
for the
collagenase),
in the
with
could
for
physical
action are optimal.
under
conditions
Unfortunately
enzymes
substrates,
into
as
is accompanied
effective
the
same
of enzymes
of
for enzyme
is depressed
more
lysosomal
lysosomes-the
occurs
by
release
of the
(including
between
different
mechanisms.
that
be spatially
rate.
more
is the “two-
membrane,
by osteoclasts
that the local conditions
of organ
culture
medium
is occurring
two
secondary
of enzymes
as to digestion
of enzymes
by
a modification
in which
the pH of the microenvironment
ionic conditions
close to the cell are
material.
as between
and
THE
VOL.
the
fibroblasts.
this
proposed
plasma
of enzyme
to
conclusion
which
turnover
differing
confer
It is
acid
in
of the
optimal
a distinct
on cells whose
function
is the precise
control
of the structure
and physiology
of the
Before
attempting
to identify
the lysosomal
enzymes
involved
in the catabolism
of
tissue
matrix
and the manner
in which
they are allowed
to act, it is first necessary
to consider
The
the
a manner
the pH
the
and
cells;
occur
has
fused
release
of the
permit
expected
the matrix
to
(1972)
portion
The
osteocytes
of these
postulated
of incompletely
suggested
that
that
and
degradation.
of the macromolecules
of the
of lysosomal
enzymes
released
no granulocytes
or macrophages
allowed
diffusion
of products
vacuole
Weissmann
release
that
breakdown
been
reasons
suggested
matrix
has
a modified
physical
surface
no precise
that the
digestive
vacuole.
Such
and may
be
H ions in such
finding
that
common
in which
present
possible
for
with
main
complete
enzymes
to the exterior.
This
studied
with the extent
and
of lysosomal
enzyme
release
by chondrocytes,
the
cells,
the
open
ends
Dingle
(1969)
lysosomes
of the cell
matrix.
Vaes
phagocytosing
the
1969).
Such methods
include
the use ofvitamin
A,
sugars,
hyperoxia,
parathyroid
hormone
and
the cells remain
healthy
and synthetically
active
by inhibition
Digestion
was probably
completed
within
stage”
hypothesis
of matrix
digestion.
The release
of lysosomal
enzymes
conditions
in the
in the
careful
studies
have demonstrated
such in vitro work.
of cartilage
or bone by a variety
from
all the in vitro
studies
was that the initial
degradation
matrix
was due at least in part to the extracellular
function
from viable
cells of cartilage
and bone (in these experiments
were
present).
It was shown
further
that this initial degradation
out
have
products
of digestion
and released
enzymes
to correlate
experiments
on in vitro organ
increased
amounts
of lysosomal
in all the experimental
systems
of the organic
matrix.
Inhibition
is immediately
enzymes
function
culture
culture
of cartilage
and bone with studies
in i’h’o, and many
that it is possible
to predict
accurately
in vivo events
from
In organ
culture
it is possible
to stimulate
the resorption
ofmethods
(Dingle
complement-sufficient
metabolites
ofvitamin
lysosomal
of lysosomal
organ
89
TISSUES
NO.
TURNOVER
turnover
are
1,
very
FEBRUARY
nature
rates
different.
1973
of the
turnover
OF
THE
of the
two
At least
part
of skeletal
MATRIX
major
of the
macromolecules.
MACROMOLECULES
macromolecules
proteoglycan
of matrix, collagen
has a short
half-life-about
and
90
T.
..
eight
days
five
in the
days
case
in young
of adult
fowls.
rabbit
DINGLE
articular
Observations
on
tissue
human
short-term
organ
culture
suggest
that the turnover
also.
It is probable
that
the more
metabolically
predominantly
chondroitin
sulphate,
whereas
the
slowly.
It has
has a half-life
been estimated
of 120 days.
proteoglycan
with
that the keratan
It may be that
collagen
fibres
of proteolytic
enzymes.
The turnover
of mature
might
collagen
It has been
least
that
continual
that
an
suggested
that
the
synthesis
repair
and
processes
organised
degradation
can take
were
directed
at the reduction
of
during
an acute
phase
of articular
the
and secretion
order
to achieve
macromolecules
would
these
under
briefly
the
discusses
breakdown
group
THE
It seems
complete
We
probable
that
degradation
consider
every case
considered
These
that
ENZYMES
there
these
they are
together.
enzymes
enzymes
act
to
from
indicated
Hyaluronidase
including
the
bones
has
of young
are
MATRIX
groups,
shown
rats.
might,
at
half-life
fibres
implies
are degraded,
of this hypothesis
to
leads
to the consideration
the
inhibitors
control
of specific
of enzyme
enzymes
that
conditions.
considered
to be the
catabolise
The
next
principal
agents
CATABOLISM
which
together
are
capable
of the
organic
matrix
of bone
and
and
although
that
the
the
tissue
it is not
established
are,
here
majority
proteinases
and
and
of
the
cartilage.
they
peptidases,
that
in
will
be
which
of collagen
and the protein
backbone
of proteoglycans,
hydrolysis,
the glycosidases.
are lysozyme
and hyaluronidase.
Lysozyme
has been
leucocyte
been
to
it seems essential
collagen.
The
by the short
collagen
term, chemical
pharmacological
of enzymes
it is clear
major
polymorphonuclear
cartilage.
of fifty
degradation
of cartilage
the mature
the mature
which
synergistically,
origin,
in two
of proteoglycan.
component”
to determine
the
and pathological
macromolecules
of lysosomal
are
OF
is a group
of the major
that
the access
collagen.
together
are capable
of the degradation
and the enzymes
capable
of carbohydrate
The two major
endoglycosidases
isolated
than
“slow
to proteolytic
In the short
long term the
of enzymes
and
in
the activity
of the enzymes
catabolising
the
erosion
might
protect
the collagen
framework
be a desirable
goal.
ends it is necessary
both
physiological
of proteoglycan
about
in monkeys.
of collagen
of the tissue
and allow
subsequent
recovery.
enzymes
might
well be useful,
and in the
in
their
obtained
; if, however,
by restricting
slower
for
of
that
therapy
proteoglycan
section
to be much
repair
process
becomes
unlikely.
An extension
articular
damage,
seen for example
in arthritis,
consideration
synthesis
In
matrix
perhaps
proteoglycan.
structure
and physiology
it should
not extend
readily
and
maintained
of the nucleus
pulposus
of the rabbit
association
of the keratan
sulphate
of the proteoglycan
place
1969)
cartilage
of proteoglycan
is high in this tissue
active
pool
of proteoglycan
contains
keratan
sulphate
may be metabolised
more
a half-life
resistance
Lippiello
rate
its catabolism,
is thought
in part,
be due to the surrounding
In terms of the maintenance
of the
when
damage
to the tissue
occurs
and
bronchial
sulphate
the close
retard
In rats, Prockop
and Kivirikko
(1967)
found
300 days.
Similar
findings
of a long half-life
(Mankin
adult
Such
granules
and
to have
a lysosomal
evidence
as there
is known
to
localisation
is does
not
concentrate
within
in a variety
of tissues
lead
one
to think
that
these enzymes
(nor the exoglycosidases)
play a major
role in the initial
stages of the breakdown
of matrix;
however
they are possibly
of importance
in the final stages
of polymer
breakdown
within
the lysosomal
digestive
vacuole.
It seems generally
accepted
that the initial
cleavage
of both the major
macromolecules
matrix
is a proteolytic
diffusion,
alter
of the matrix
one.
the mechanical
macromolecules
In
the
case
properties
with the
of the
proteoglycan
proteolysis
of the tissue,
and
cell surface,
allowing
THE
this
perhaps
affect
endocytotic
JOURNAL
OF BONE
may
of
facilitate
the interaction
activity
to take
AND
JOINT
SURGERY
THE
place.
This
or endocytosis
ROLE
OF
LYSOSOMAL
results
in the second
of both proteoglycan
the
lysosomes.
In
the
proteolytic
enzymes
the
light
of this
that
may
D-Principally
due
IN
SKELETAL
91
TISSUES
phase
of the “two-stage
digestion”-that
and collagen
and its subsequent
complete
hypothesis
it seems
be involved
THE
Cathepsin
ENZYMES
to
in the
ACID
the
important
initiation
to
is, the
breakdown
study
of matrix
in
uptake
within
more
detail
degradation.
PROTEINASES
work
of A.
J. Barrett
in this
laboratory
and
of J. F.
Woessner
in Miami,
this lysosomal
enzyme
has been completely
purified
from several
species.
It has been shown
to be immunologically
identical
within
the organs
of any one species,
but
not to cross-react
between
species.
The enzyme
exists in several
isoenzyme
forms
with acidic
isoelectric
activation.
points,
has a molecular
weight
of approximately
Its spectrum
of inhibitors,
including
pepstatin
45,000
(Barrett
and
and
does
Dingle
compatible
with a catalytic
mechanism
similar
to that of pepsin.
When
acting
on purified
proteoglycan
or on whole
cartilage
cathepsin
optimum
of 5. A similar
pH optimum
is found
for the autolytic
degradation
animal
cartilage
released
from
by endogenous
cartilage
culture
has been studied
or three
polysaccharide
at
perhaps
three
degradation
desulphation.
stimulated
of the
to
resorb
these
in organ
culture
This
damage
changes
as upon
the
locally.
E-This
relative
enzyme
was
immunologically
but one may
like cathepsin
material.
located
various
from
bone
D,
smaller
than
specificity.
This
the pH optimum
those
were
clearly
cathepsin
B!,
result has
localisation,
important
but it
cathepsin
D.
NO.
was
enzyme
this
for
has
Even
1,
implications
is
not
if this
FEBRUARY
yet
is
1973
that
in matrix
known
not
the
this
is required
A.
This
to allow
a very limited
of the tissue.
proteolytic
Cathepsin
sites
enzymes
that
where
marrow
are
of
Webb
on
digestion
and
(Lapresle
than
cathepsin
has shown
its action
the marrow
is
of the other
conditions
synthesised
and
optimum
laboratory
D, but
to be
it
matrix
components,
cavity.
It may
of
of
attack
is
D is clearly
degradation
being
by Lapresle
pH
this
partial
diffusion
its local activity
and that
upon the microenvironmental
more
by
Burleigh
readily
The
it is released
may
D, which
readily
enzyme
dynamics.
it
of
also,
protein-containing
PROTE1NASES
whether
case
evidence
of vitamin
obtained
with cathepsin
has also been
shown
effect was 5, but much
demonstrated
no
addition
that
Bi (Otto
1971), which
has a molecular
weight
of 24,000,
to be capable
of the degradation
of isolated
proteoglycan.
Cathepsin
laboratory
55 B,
There
had two
molecule
found
evidence
of enzymic
the autolytic
system
and in
Little
is known
at present
about
that it has a role in the formation
have
a part
to play in the intravacuolar
THIOL
in organ
material
of each
Morrison
in both
distinct.
speculate
molecules
maintained
released
cleavage
at extracellular
between
depending
neutrality
than was displayed
by cathepsin
D.
Recent
unpublished
work
in this
laboratory
VOL.
the
weight
and a lower
of action,
work
in
THE
digestion
papain-like
cartilage;
by
D has a pH
of human
and
proteoglycan
tissue
backbone.
to be all
of the
isolated
It has a higher
molecular
it has a similar
mode
living
that the
indicate
means
that only
to the integrity
; it is also
amounts
found
would
though
occurred
seems
of the
from
polypeptide
but the quantitative
relationship
is not yet clear ; it may well vary,
secreted
Cathepsin
this
It was
which
the
molecule
distribution
and
side chains,
of degradation
of the matrix.
very extensive
of causing
occurring,
proteinases
1971).
although
along
proteoglycan
the products
out
necessary
to cause
as well
places
size
conditions
in this laboratory.
chains
per fragment,
or four
cartilage
The
the above
of the polysaccharide
A similar
type
cleavage
capable
enzymes.
under
not require
1972),
are
have
a
activity
and
enzyme
was
Lazarus,
is known
in the
been shown
The products
is consistent
degrade
adult
to
degrades
major
has
same
collagen
its
human
towards
using
purified
have
extracellular
catabolic
with
found
collagen
to
in
of
fibrils.
This
a lysosomal
sites
function
as
in
92
J. T. DINGLE
secondary
lysosomes,
parallel
with
the
doubt
little
will
that
focus
perhaps
following
“two-stage
these
recent
increasing
studies
attention
first
degrading
to
that
show
on cathepsin
on this
degrading
with
joint
activity
inhibitors
neutral
Unfortunately
purified
to allow
any
D to be attempted,
cathepsin
Collagenases-These
are
physiological
demonstration
and TCb
collagen
pieces.
molecule
leucocytes.
Daniels,
Brown,
but the identity
immunoenzymic
studies
presence
in this
(1968)
White
synovial
laboratory,
coupled
and animal
of cathepsin
enzymes
has
and localisation
cells
and
of these
techniques
is awaited
well
been
Spilberg
(1960)
were
were
capable
made
it has
Fullmer
with
worked
out
so
been
shown
1968).
interest.
The
using
a new
assay
for
to sepharose,
as substrate,
have
cartilage.
The use of pepstatin
D, whilst
the
use
of specific
localisation
yet known.
capable
of attacking
native
to have
thiol
in other
localisation
cells
may
techniques
at
of the
has
(Lazarus,
also
is still open
to question.
to the study of collagenase
of these
next
collagen
fibrils,
and by
to give TC
and Fullmer
1969).
and in polymorpho-
reactive
but enzymologically
to the immunocytochemical
in the
and
out
breakdown
Collagenase
(Lazarus
1971)
a lysosomal
Its localisation
application
is described
similar
and Nuki
(1971)
fluids
of patients
far been
sufficiently
characterised
studies
similar
to those being carried
granules
with
lysosomes
has begun
to be applied
and
the
of
Bl.
Jeffery
(1971) demonstrated
immunologically
skin.
The extension
of these experiments
enzyme
Lospalluto
membrane
where it is apparently
ofdermal
origin
in rheumatoid
synovium
(Evanson
In these
is
specificity,
turnover.
The pure enzyme
is probably
not capable
of the complete
; this may be completed
by other
enzymes
(see above).
Bladen
The
There
substrate
pH.
The enzyme
is usually
assayed
by its action
on reconstituted
of the characteristic
cleavage
of the helical
part of the molecule
nuclear
in
and
nor is their intercellular
described
as enzymes
usually
been detected
in human
skin
It has also been demonstrated
and
Weissmann
of cathepsin
none
of these
immuno-inhibition
in matrix
Gribetz
and
and synovial
proteoglycan,
in human
in the
action
collagenase.
be close.
its wide
and Wood,
Pryce-Jones,
of cells obtained
from
Recent
labelled
is present
indicating
its role
of
could
PROTEINASES
pH.
disease.
to be measured
precluded
and
NEUTRAL
peritoneal
cells,
activity
in extracts
which
uses S(SO4)
that enzyme
activity
enabled
on
at
on rabbit
CPGase
inflammatory
CPGase,
shown
of
proteoglycan
observations
demonstrated
enzyme
action
hypothesis
Bl,
(CPGases)-Ziff,
human
leucocytes
enzymes
extracts
extracellular
of proteoglycan
THE
Proteoglycan
partial
digestion”
be granular,
The
; Eisen,
use of
Bauer
inactive
localisation
to cathepsin
enzyme
of the
D has
now
section.
IMMUNOENZYMOLOGY
In this
laboratory
origin.
cathepsin
D,
and
human
antisera
mainly
to this.
in matrix
catabolism.
The
by sheep
action
We
is given
that
indicating
when
a stearic
As-D
human
was
was
cartilage,
also
unaffected
precipitation
blockade
been
work
similar
other
CATHEPSIN
was
of the
not
enzyme
necessary
by six
almost
latter
for
inhibition.
antibody
of
major
the
completely
case
there
was
THE
this
B!
of
specific
section
enzymes
will
involved
inhibited
inhibition
specifically
complete
with
Fall
inhibition
fragments,
was
found
occurred.
breakdown
autolytic
residual
raise
D
Complete
molecules
D and
to
remainder
on the
OF
cathepsin
sufficiently
purified
in the
studies
IMMUNOINHIBITION
pure
against
was
be reported
to
to make
raised
which
cathepsin
to inhibit
though
in the
by pepstatin.
found
have
enzyme
D on haemoglobin
is completely
and
D antiserum
(As-D)
(Dingle
et a!. 1971);
similar
proteoglycan
as substrate.
Inhibition
was obtained
pure
anti-cathepsin
with isolated
first
the
and
expect
THE
of
antisera
The
animal
was
refer
specific
activity
JOURNAL
near
OF
BONE
of
neutral
AND
animal
pH,
JOINT
and
which
SURGERY
THE
These
of
experiments
study
the
enzymic
experiments
good
the
was
to
and
confirmed
vitamin
biochemical
through
the
release
of cathepsin
would
suggest
D
intact
D and
that
released
release
A mediated
criteria
undegraded
the enzyme
activity
caused
by vitamin
CPGase
action
cannot
be ruled
out.
therefore
conditions,
that
is when
complete
materials
model
release
was due
with which
; in
resorption
other
could
experiments
to
to
on organ
In some
be demonstrated
much
less
by
effect
was
It was found
that part of the lack of consistency
was due to poor
penetration
into the cartilage,
until significant
degradation
of matrix
had occurred.
This
by the use of 3H labelled
IgG, which
showed
that the molecule
was too large
antibody
diffuse
autolytic
93
TISSUES
the major
breakdown
of matrix
of skeletal
tissues
is a very imperfect
of the
inhibition
histological
demonstrated.
of
under
IN SKELETAL
of
tissues
both
that
ENZYMES
breakdown
of skeletal
matrix.
the immunoinhibition
of living cartilage
resorption
were made
grown
in the
presence
of vitamin
A and As-D
or normal
serum.
Experiments
culture
LYSOSOMAL
had occurred,
the autolysis
from
D.
OF
indicate
cells
However
enzymes
cathepsin
ROLE
to
matrix
matrix.
Inhibition
A is that
The close
breakdown
experiments
in a variety
matrix
breakdown
has
been
established
and
also
spatial
very
it unlikely
the possibility
correlation
of tissues
another
enzyme,
if it exists,
is probably
by the same mechanism
as cathepsin
D. The
immunocytochemical
made
of cathepsin
B!, but
temporal
and spatial
under
several
of lysosomal
relationship
recently
in
that
of neutral
between
the
stimuli
origin,
and
of cathepsin
this
laboratory
by
methods.
IMMUNOCYTOCHEMISTRY
skeletal
Specific
cells
within
cells
A more
It
As-D
and
when
difficult
was
antibody
found
into
labelled
tissues.
As-D
was used
problem
was
possible,
the
with fluorescent
As was expected,
rabbit
dyes
the
in the usual
the
has enabled
us
enzyme
displayed
manner
localisation
to “stain”
of enzyme
by culturing
tissues
knee joint,
to “trap”
to localise
cathepsin
D
a lysosomal
localisation
frozen
in the
or fixed
extracellular
cells
in
or tissues.
environment.
in the presence
of As-D,
and also by injecting
the released
enzyme.
This then appeared
as a
distinctive
“packet”
of enzyme/antibody
complex,
often
very close
to the cell membrane.
The extracellular
localisation
of the enzyme
was found
to occur
in those
regions
in which
matrix
catabolism
could
be demonstrated
histologically.
Increased
extracellular
accumulation
the
around
well
with
chondrocytes
the biochemical
extracellular
arthritis.
but it has
extracellular
replaced
localisation
Similar
studies
been established
accumulation
by bone
could
be visualised
as the vitamin
A effect developed,
correlating
evidence
of increased
extracellular
proteolytic
activity.
Good
was
also
seen
in the pannus
of rabbits
with experimentally
induced
on the release
of enzyme
from
cells of bone
are at an early
stage,
that in normal
(unstimulated)
bone of young
chicks
areas of intense
of enzyme
occurred
where
cartilage
was in the process
of being
mineral.
NATURAL
LIMITATIONS
TO
It is evident
that the lysosomal
enzymes
extracellular
structures,
and one may question
of these
enzymes
at unwanted
sites.
In the
skeletal
tissues
in high
is diminished,
it is not important
activity,
it is probable
LYSOSOMAL
are
capable
what
FUNCTION
of causing
limitation
case
of the
that
action
if not abolished,
by unfavourable
that, so far as is known
at present,
zone
ENZYME
is
considerable
placed
upon
proteases,
acid
from
away
conditions
cathepsin
the
damage
spurious
which
immediate
are
to
action
present
in
pericellular
of pH.
Perhaps,
D is unique
in the
therefore,
group
of
matrix-degrading
enzymes
in having
no naturally
occurring
inhibitors.
Collagenase
of human
skin is readily
inhibited
by serum,
probably
by both
a1-antitrypsin
and
a2-macroglobulin.
Rheumatoid
synovial
collagenase
is inhibited
by normal
synovial
fluid,
but the granulocyte
collagenase
inhibited
presence
VOL.
55 B,
to serum
is resistant
readily
by
of a potent
NO.
1,
inhibition
(Lazarus
serum
and tissue extracts:
inhibitor
in synovial
fluid.
FEBRUARY
1973
1972).
thus Wood
Cathepsin
The
et
neutral
a!. (1971)
B! is also
CPGases
are
usually
have
reported
the
inhibited
by serum
94
J. T. DINGLE
components.
this
Starkey
enzyme
and
Barrett
in a 1 :1 molar
ratio.
binding
enzyme
even
when
was
bound
substrates.
firmly
They
manner
and
skeletal
were
have
The fact
matrix-degrading
dependent
rise
low
can
molecular
readily
locally
to an
can
other
degradation
We
already
effect
tissue
a novel
enzyme’s
that
study
being
certain
n2-macroglobulin
they
active
particularly
that
theory
shown
interesting
macroglobulin,
the
further
and
small
enzymes
would
binding
to be able
reach
the
ofthe
matrix
of skeletal
tissues.
In conclusion,
the emphasis
of matrix
macromolecules
should
50,000.
matrix.
of the
the cell
either
to allow
to be true
implications
in determining
is about
enzymes,
for
the
to their
latter
diffusion
weight
results.
most
of the
enzymes
in
of cartilage,
have
a relatively
point
of material
that
which
are produced
substrate
; the inhibitors
out
of the
large
IgG.
in a similar
their
particularly
in ofthe
3H labelled
molecular
react
enzymes
diffuse
the
activity
against
of these
the cut-off
therefore,
that
it retained
to explain
matrix,
matrix-degrading
This
The
from
until
occurs
this
seems
to have important
degrading
enzymes
and
of the
to diffuse
enzymes
demonstrated
properties
Most
inhibits
showed
that
against
other
of enzyme-globulin
physical
situation.
weight,
probably
below
diffuse
through
cartilage
sufficient
have
the
to predict
However,
cannot
have
a very
inhibitors
exist in the serum
or synovial
fluid
might
seem at first sight to diminish
the role
interesting
be shown
hand
to the
potent
enzymes
catabolism.
give
In
upon
able
produced
that
(unpublished)
This
on the
matrix,
or until
inhibitor
molecules.
selective
“filtration”
both
in limiting
naturally
the site
the natural
history
of some
of the
of action
pathological
of
lesions
in skeletal
tissues
balance
can lead
factors
Much
Tissue
that
between
synthesis
and
to pathological
changes
control
this
of the work reported
Physiology
Department,
Hembry,
R. I. G.
privilege
of working
placed
on the role of the lysosomal
enzymes
in the
not distract
attention
from the concept
of a dynamic
Morrison,
with
balance
is a goal
degradation.
Disturbance
instead
of normal
turnover.
of those
interested
in the
turnover
balance
of either
aspect
Determination
biology
of skeletal
of this
of the
tissues.
in this article
has been carried
out in collaboration
with my colleagues
including
Drs A. J. Barrett,
J. J. Reynolds,
P. D. Weston,
A. R. Poole,
of the
R. M.
G.
for
S. Lazarus,
P. Starkey
and
M.
Burleigh.
I should
like
to thank
them
the
them.
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