56 1
603rd MEETING, LIVERPOOL
Some modern mass spectrometric methods for steroids and terpenoids
MALCOLM E. ROSE
Department of Biochemistry, University of Liverpool,
P.O.Box 147, Liverpool L69 JBX, U.K.
Introduction
Mass spectrometry has been applied to isoprenoid molecules
for many years, resulting in well established analytical procedures such as combined gas chromatography-mass spectrometry (Rose & Johnstone, 1982; Rose, 1983). Such methods are
not discussed here. Obviously, this does not reflect their value:
g.c.-m.s. is an excellent tool for detection, identification and
quantification of isoprenoids as exemplified by the recent
discovery of a fourth juvenile hormone, J H O (Bergot et al.,
1980), identification of the first naturally occurring 1p-hydroxygibberellins (Gaskin et al., 1980; Kirkwood & MacMillan,
1982), and quantification of abscisic acid (Netting et al., 1982).
Such techniques are not covered because their capabilities are
well known. Instead, this paper examines some areas in which
mass spectrometry has enjoyed less success and recent attempts
to improve its applicability. The topics reviewed are metastable
ion techniques for structure elucidation of isomeric compounds
and for increasing the specificity of detection in quantitative
studies, analysis of involatile compounds using mild methods of
ionization and the relative merits of off-line and on-line liquid
chromatography-mass spectrometry for characterizing mixtures of involatile materials.
Stereochemical problems
Routine mass spectrometry is frequently insensitive to subtle
differences in structure, with many isomeric substances behaving identically so that normal mass spectra do not differentiate
them. There have been many reports that the various metastable
ion techniques, or so-called mass spectrometry-mass spectrometry, are more diagnostic of fine differences in structure and
are preferred for analysis of isomers (McLafferty, 1980; Rose &
Johnstone, 1982).
Routine mass spectra consist almost entirely of 'normal ions'.
These are molecular ions that are stable within the lifetime of the
mass spectrometric experiment (about lo-' s) and normal
fragment ions which are the result of fragmentations occurring
in the ion source (within about 10-6s). Between these extremes,
there exist metastable ions which decompose unimolecularly
during flight through the analyser part of a mass spectrometer
(rate constants, 105-106s-'). There is a variety of scanning
methods and instruments designed to study the behaviour of
metastable ions selectively, i.e. in the absence of normal ions
(Boyd & Beynon, 1977; McLafferty, 1980; Rose & Johnstone,
1982). In effect, it is possible to focus on one particular ion of
interest, defocus all other ions (these can be from the same
compound or from other components of a mixture) and acquire a
'mass spectrum of a metastable ion'. The same methods are also
used to examine the behaviour of selected ions when induced to
fragment by collision with a neutral gas during flight through the
mass spectrometer. The ions of interest are not necessarily
metastable ions but the resulting mass spectra (collisional
activation mass spectra) are of the same form as metastable ion
mass spectra and are similarly useful for distinguishing isomers
(Bente & McLafferty, 1980).
The basis for the extra structural discrimination by metastable
ion methods is not yet clear but has been discussed (Rose,
1982). The methods allow the mass spectrometrist to ignore
(defocus) the vast majority of ions in the routine spectra which
are common to different isomers and observe selectively those
ions which are most likely to differentiate different structures.
Abbreviations used: g.c.-m.s., gas chromatography-mass spectrometry: FD, field desorption: FAB, fast atom bombardment; h.p.1.c.m.s., high performance liquid chromatography-mass spectrometry.
VOl. 11
Such an approach was used to distinguish steroids with different
side-chains by focusing initially on ions containing or corresponding to the side-chains and recording their metastable ion
mass spectra (Bozorgzadeh et al., 1979). Using this methodology, whereby ions from the common steroid nucleus are
defocused, isomeric side-chains can be differentiated, as for
example, fucosterol and isofucosterol (M. E. Rose & L. J. Goad,
unpublished work).
Differentiation of carotene isomers is often difficult by routine
electron impact mass spectrometry, especially if the samples are
impure and in small amounts. On the other hand, the metastable
ion method of linked scanning allows the six carotene isomers to
be differentiated readily even in the presence of gross contamination (Rose, 1982). In the reported method, the metastable
molecular ion of each isomer at m/z 536 was allowed to
fragment unimolecularly and the product ions recorded. Each
isomer afforded a unique and predictable spectrum. The
essential features of the spectra are given in Table 1, normalized
to the height of the [M-921+' peak which corresponds to
elimination of toluene. This peak is common to each isomer
because the neutral molecule originates from the common
polyene chain. The other products are highly characteristic of
the structure of the end-groups as shown in Fig. 1, making the
metastable ion method a surer means of differentiating the
carotene isomers than routine mass spectrometry.
In the steroid field, A/B and C/D ring junction stereochemistry of alcohols, ketones and hydrocarbons has been
determined by the metastable ion technique of mass-analysed
ion kinetic energy spectroscopy (Larka et al., 1981a; Gonzalez
& Griitzmacher, 1982). Differences in the linked scan spectra of
the tert-butyldimethylsilyl ethers of androstane-3,17-dioIs reflect
the isomeric structures (Gaskell et al., 19796). Metastable ion
methods can be combined with any method of ionization: for
example, chemical ionization for the study of isomeric steroids
(Kruger et al., 1979) and FAB mass spectrometry for
differentiation of isomeric steroid sulphates (Gaskell et al.,
1983). Also, the translational energy released in the decompositions of metastable ions provide a criterion for distinction of steroid epimers (Larka et al., 1981 b). The
differences in the energy for M+' -+ [ M - CH,']+ decompositions
were explicable in terms of differences in strain energy between
configurational isomers. Finally, the metastable molecular ions
of 14a- and 14p-equilenin can be distinguished by studying their
unimolecular fragmentations occurring consecutively in different parts of a mass spectrometer (Proctor et al., 1982).
Generally, metastable ion methods are of most value when
sample size is limited (so that spectroscopic methods, such as
n.m.r., that are less sensitive but more diagnostic of isomeric
structures than mass spectrometry are not suitable) and with
impure samples. To analyse mixtures, it is necessary that the
compound of interest afford at least one unique peak (i.e.
without contribution from the chemical matrix), otherwise the
resulting metastable ion spectrum will be composite. This effect
largely rules out analysis of mixtures of isomers. To perform
such an analysis, the combination of chromatography and
metastable ion methods is preferred (Rose, 1983). To date, this
technique has not been applied to terpenoid or steroidal
compounds and thus is outside the scope of this review.
Specijicity of quantijication
Because mass spectrometry, unlike spectroscopic methods,
does not examine a well-defined property of a molecule,
reproducibility has always caused some difficulty, particularly
for quantitative studies. However, the use of internal standards,
chemically similar to the analyte, now permits quantification by
such techniques as selected ion monitoring during g.c.-m.s.
(Rose & Johnstone, 1982). For assays of substances in complex
562
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. Metastable ion spectra of the carotene isomers
Results of linked scanning at constant magnetic field to electric sector voltage for all product ions of metastable molecular ions
( m / r 536) of the carotene isomers
Relative abundance of products of metastable molecular ions (%)
7
Compound*
[ M - 56]+'
[M- 691'
[M-921t'
0
0
100
/%Carotene(I/ I)
11
0
100
a-Carotene ( 1/2)
44
0
100
&-Carotene(2/2)
0
27
100
y-Carotene ( I D )
&Carotene (2/3)
21
52
100
0
1 I4
100
Lycopene (3/3)
* Figures in parentheses refer to the identity of the end groups of each carotene (see Fig. I).
-137
[M- 1231+
0
2
3
0
8
0
[M-1371:
2
0
0
0
0
6
-
-56
-137
-123
1
2
3
Fig. 1. Expected fragmentations of the carotene end-groups
The mass of the neutral species ejected is marked by the line that points to the fragment
that retains the charge.
biological samples, the specificity of detection must be very high Involatile and thermally labile compounds
to ensure that any response is due to the compound under test.
For electron impact and chemical ionization, the sample must
One means of attaining very high specificity is to monitor not a be presented to the mass spectrometer in the gas phase. This
small number of normal ions (selected ion monitoring) but ions requirement does not constitute a difficulty for volatile terthat are a result of decomposition of metastable ions during penoids and steroids but excludes analysis of involatile,
flight through the instrument (selected metastable peak monitor- thermally labile and/or high-mass compounds such as steroid
ing). The high specificity thus attained makes for very low noise sulphates, phosphates and complex glycosides. There are,
levels and hence good signal-to-noise ratios (i.e. the effective however, several methods for obtaining mass spectra of samples
sensitivity is high). For example, when applied to quantification directly from the sol3 or xquyd phase at ambient temperature or
of testosterone, the detection limit was 30pg (Gaskell et al., with mild heating (Rose & Johnstone, 1982). Such methods
1 9 7 9 ~ ) Selected
.
metastable peak monitoring has also been used have provided useful data as part of strategies for elucidation of
successfully to improve the specificity of detection of stearanes complex isoprenoid structures. The two methods to be described
in geochemical samples when routine selected ion monitoring here are field desorption (FD) and fast atom bombardment
afforded complex, incompletely resolved chromatograms even (FAB) mass spectrometry.
when using a capillary g.c. column (Warburton & Zumberge,
For the F D method, the sample is deposited on an activated
1983).
anode (emitter) to which a very high positive potential is
Selected metastable peak monitoring, developed largely by subsequently applied. Under the influence of the potential
Gaskell and co-workers, has been utilized extensively for assay gradient, the molecular orbitals of a sample molecule are
of steroids but few other compounds. Measurement of A'- distorted and a valence electron migrates (tunnels) from the
tetrahydrocannabinol in plasma by this method was so specific molecule to the anode. Being positively charged, the molecular
that no clean-up of the sample was required prior to g.c.-m.s.
ion is accelerated from the anode. Due to the high density of
and 5pg.ml-' was detectable (Harvey et al., 1980, 1982). The sample molecules in the region of the emitter, ion-molecule
technique is normally, but not always, used in conjunction with reactions normally occur, yielding [ M + XI+ ions where X = H,
g.c.-m.s. For example, fatty acid esters of lanosterol and Na, etc., and some fragment ions (Schulten, 1979).
dihydrolanosterol have been identified and quantified in wool
FAB mass spectrometry relies on a totally different principle
wax by selected metastable peak monitoring using temperature- (Barber et al., 1981). As shown in Fig. 2, rapidly moving atoms
programming of a direct insertion probe (Patouraux et al., impinge on the sample as a solution in glycerol, a-thioglycerol,
1981).
poly(ethy1ene glycol) or other solvent. Impact results in
Selected metastable peak monitoring is best applied in those momentum transfer from atom to sample molecule, sufficient to
cases where tolerance to contamination is important and routine bring about ionization (if the sample is non-ionic) and
selected ion monitoring does not give the required specificity desorption of ions. If the sample is a salt, then the energy
(i.e. when identification of analyte is in doubt and/or interfering imparted from the kinetic energy of the fast atoms serves only to
peaks prevent quantification). The method has been reviewed by overcome the electrostatic attraction between counter-ions and
to desorb those ions into the gas phase. As with FD, [ M + XI+
Rose (1983).
1983
603rd MEETING, LIVERPOOL
563
lone into
It would be invidious to compare FD and FAB mass
spectrometry for the analysis of terpenoid and steroid conjugates, because application of the latter is new. However, the FAB
technique is generally simpler in practice, yielding results that
are more reproducible.
mass spectrometer
a
I
I
Stainleas-eteel
eupport
Fast PtOmS
/
+7-
Probe
I
Fig. 2. Schematic arrangement of the ionization region for fast
atom bombardment mass spectrometry
and [M-XI- ions are usually formed in abundance ( X = H ,
Na, etc.) along with fragment ions containing structural
information. The instrument can be set to measure positive ion
or negative ion spectra. Generally, it is best to select that mode
in which the sample molecules are able most readily to
accommodate the given charge, i.e. basic compounds afford
particularly diagnostic positive ion FAB spectra and acidic
compounds yield useful negative ion FAB mass spectra with
relatively stable [M-HI- ions (Williams et al., 1982). However, each mode appears to be sensitive to different structural
aspects (the groups best able to accommodate the charge) so to
maximize structural information it is prudent to utilize both
positive ion and negative ion techniques (Williams et al., 1982;
Rose et al., 1983~).
Terpenoid glycosides are, for electron impact and chemical
ionization mass spectrometry, intractable compounds but they
do yield to both FD and FAB techniques. Excellent FD mass
spectra of intact and underivatized saponins have been obtained
(see, for example, Encarnacion et al., 1981; Schulten et al.,
1982). Information on molecular weight and sugar sequence is
clear in FD spectra. Similar results have now been reported
using negative ion FAB mass spectra (Rose et al., 19836).
Molecular weights of saponins are characterized by abundant
[M-HI- ions and cleavage either side of each glycosidic oxygen
atom yields fragment ions indicative of sugar sequence including
any branching. The method was illustrated by reference to the
spectrum of digitonin (Rose et al., 19836). Unfortunately, these
mass spectrometric methods do not yield unequivocal information on sugar linkages or configuration, so these must be
established by further analyses. Some data on configuration may
be obtained by recording a second negative ion FAB spectrum
after performing a derivatization in situ of the sugar residues
with a boronic acid, a reaction that is highly sensitive to relative
orientation of hydroxy groups (Rose et al., 1983~).
Simpler steroid conjugates have also been analysed by FAB
mass spectrometry. Phosphate (Isaac et al., 1982), sulphate and
glucuFonide (&hr et al., 1982; Shackleton & Straub, 1982;
Gaskell et al., 1983; Rose et aL, 1983b), and glycoside (Rose et
al., 19836) conjugates of sterols have been characterized by the
FAB method, particularly in the negative ion mode. The
sulphate and phosphate moieties afford abundant negative ions
following FAB, allowing their ready differentiation by observing either SO,-’ at m/z 80 or PO,- at m/z 79 (Rose et al.,
19836; R. E. Isaac, M. E. Rose, H. H. Rees & T. W. Goodwin,
unpublished work). Again using negative ion FAB mass
spectrometry, quantitative assay of dehydroepiandrosterone
sulphate has been reported (Gaskell et al., 1983). It was also
possible to differentiate between this conjugate and the isomeric
testosterone sulphate by a metastable ion technique used in
conjunction with the FAB method (Gaskell et al., 1983).
VOl. 11
Mixtures of involatile compounds
Mass spectra of mixtures are usually too complex to be of
much use. It is therefore necessary to separate the components
prior to mass spectrometric analysis. Gas chromatography is
restricted to compounds that are volatile and thermally stable or
that can be made so by derivatization. No such limitation
applies to h.p.1.c. The potentially powerful on-line h.p.1.c.-m.s.
combination, in which the h.p.1.c. effluent is led directly into the
mass spectrometer, has been reviewed extensively, for example,
by Games (1981) and Rose (1983) and applied to many lipid
analyses (Privett & Erdahl, 1981; Sugnaux & Djerassi, 1982).
The brief discussion here concerns the synergistic combination
of h.p.1.c. with F D or FAB mass spectrometry for characterization of mixtures of intact and underivatized compounds that are
involatile and/or thermally labile.
The nature of the F D ionization process makes impossible the
on-line combination of h.p.1.c.-FD m.s.. Instead, eluates are
collected from the h.p.1.c. column and introduced manually into
the mass spectrometer (Schulten, 1982). This off-line procedure
has distinct advantages, such as cheapness and the imposition of
no restraints on the h.p.1.c. conditions, but is disadvantageous in
the sense that it is laborious and even not possible with complex
chromatograms with partially unresolved peaks. The method
has been utilized extensively for analysis of saponins (see, for
example, Schulten & Soldati, 1981).
One of the most successful h.p.1.c.-m.s. interfaces in
operation is based on an endless moving belt (Fig. 3). The
effluent is deposited on the belt and mechanically transported
into the ion source of a mass spectrometer. Ionization of the
solutes on the belt by electron impact, chemical ionization and
bombardment with ions has been reported (Rose, 1983). It is
also possible to bombard the sample on the belt with fast atoms.
This is one way in which on-line h.p.1.c.-FAB m.s. of eluates
may be performed (see Fig. 3). The system is, at the time of
writing, under development and constitutes a potentially
powerful method providing a fast, convenient analysis of even
complex h.p.1.c. chromatograms. Repetitive recording of FAB
mass spectra with data system control will provide access to
automatic data processing and evaluation, the same as that used
to great effect in g.c.-m.s. for deconvoluting ‘background ions’
and unresolved eluates, for identifying substances from their
spectra and so on (Rose & Johnstone, 1982). On-line h.p.1.c.FAB m.s. will also have disadvantages. The moving belt
interface is expensive and mechanically complex and its
tolerance to h.p.1.c. conditions has yet to be established. For
example, inorganic salts added as buffers may cause difficulties
and need to be removed by an on-line method (e.g. by affinity
chromatography with immobilized complexing agents such as
crown ethers, or ion-exchange chromatography). The choice
between off-line and on-line combinations is not simple; their
essential features are compared in Table 2.
Concluding remarks
This paper has reviewed some problems which, for a mass
spectrometrist, are difficult and reported how far modern mass
spectrometric methods have gone towards solving those problems. Mass spectrometry remains a rather poor tool for
characterizing many isomeric compounds, relative to n.m.r.
spectroscopy for instance. but its detection limits are much
lower and it has the capability of analysing impure samples. For
quantitative assays, the various selected ion or metastable peak
monitoring methods offer unparalleled sensitivity and can be
designed to be highly specific too. The newer milder methods of
ionization such as F D and FAB mass spectrometry have
564
BIOCHEMICAL SOCIETY TRANSACTIONS
H.p.l.c. column effluent
I
FA8
ion source
I I
’ 1‘
TO
pump
I
I
-1
(if required)
~opump ~opump
Clean-up
heater
Fig. 3. Schematic representation of a moving belt interfacefor on-line h.p.1.c.-FAB m.s.
Table 2. Comparison of off-lineand on-line h.p.1.c.-m.s. systems
Slow
Not suitable
On-line h.p.1.c.-FAB m.s.
Yes (flow rate and type
of solvent restricted:
involatile buffers
probably deleterious)
Yes (ionization method
must be compatible
with efRuent properties
after passage through
interface)
Fast
Suitable
Cheaper
Simpler
Dearer
Complex
Currently available
Under development
Off-line h.p.1.c.-m.s.
No (sample can be
manipulated prior
to mass spectral
analysis)
Constraints on mass No (any method of
ionization can be
spectrometric
applied)
conditions
Constraints on
h.p.1.c. conditions
Analysis time
Complex h.p.1.c.
chromatograms
Expense
Mechanical
complexity
Availability
increased the molecular weight limit for sample molecules well
into thousands of atomic mass units, making complex terpenoid
conjugates like saponins amenable to analysis. On the other
hand, it is easy for the mass spectrometrist to forget that there
are very many important biomolecules that still cannot be
examined intact (e.g. proteins, polysaccharides, nucleotides). In
the analysis of most mixtures, the mass spectrometrist relies
heavily on prior chromatography (g.c.-m.s. and h.p.1.c.-m.s.).
Relatively few real analytical problems involving mixtures are
solved by mass spectrometers not coupled to chromatographs.
The off-line combination of h.p.1.c. for isolating, separating and
purifying, and mass spectrometry for identifying, non-volatile
substances such as glycosidic conjugates of terpenoids and
steroids has met with considerable success whilst on-line
h.p.1.c.-m.s. interfaces require further improvement before their
application can become as widespread as that of g.c.-m.s.
The author thanks the SERC for grants to purchase mass
spectrometric equipment, Professor T. W. Goodwin and Drs. L. J.
Goad, G. Britton and P. D. G. Dean for useful discussions, and Dr. S.
Pollard, Mr. M. C. Prescott and Mr. C. Longstaff for their valuable
contributions to the author’s work described herein.
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565
603rd MEETING, LIVERPOOL
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Biochem. J. 201,105-1 17
The biosynthesis of plant steryl glycosides and saponins
Z. A. WOJCIECHOWSKI
Department of Biochemistry, Warsaw University,
Al. Zwirki i Wigury 93,02489 Warsaw. Poland
A great variety of triterpenoid glycosides occur in plants. Three
groups of these compounds are recognized on the basis of their
aglycone structure: triterpene glycosides (saponins), steryl
glycosides, and steroid glycosides (cardiac glycosides, steroidal
glycoalkaloids, etc.). All these substances have attracted the
attention of natural product chemists and plant biochemists for
many years. The chemical structures of hundreds of triterpenoid
glycosides have been elucidated and the biosynthetic pathways
leading to the formation of various triterpenoid aglycones are
relatively well understood. However, much less is known about
the biosynthesis of the sugar chains of these glycosides. This
paper will briefly review recent studies on plant enzymes
catalysing glycosylation of triterpenes and sterols. Two important aspects will be discussed in more detail; (i) the specificity of
glycosyltransferases participating in the glycosylation reactions
and (ii) the subcellular localization of these enzymes.
Enzymic glucosylation ofplant sterols
Unlike other triterpenoid glycosides, which are typical secondary metabolites occurring only in some plant species, steryl
glycosides seem to be present in all tissues of all higher plants.
More recently they have also been found in some algae, fungi
and slime moulds. Very little is known about the physiological
function of steryl glycosides. The accumulating evidence
suggests that these compounds are components of plant
membrane structures together with free sterols. It seems possible
that reversible processes of glycosylation and deglycosylation of
sterols present in plant cell membranes may have a regulatory
effect on membrane organization and function due to changes in
lipid-lipid and/or lipid-protein interactions (Forsee et al., 1974;
Wojciechowski, 1980). The sterol moiety of plant steryl
glycosides consists of typical phytosterols. The sugar part
consists usually bf a single molecule of D-glucose attached to the
~
OH
2
Fig. 1. Typicalplant steryl glycosides
Sitosteryl glucoside (1) and 6’-O-palmitoyl sitosteryl glucoside (2).
VOl. 1 I
C-3 hydroxy group of the sterol. In single cases steryl
monogalactoside, monomannoside, monoxyloside, gentiobioside
and celobioside have also been isolated from plant tissues (for
review see Eichenberger, 1977). In photosynthesizing organisms
a portion of the steryl glycosides occur in an acylated form, i.e.
as steryl 6’-O-acyl-/3-~-monoglucoside
(see Fig. 1) in which the
carbohydrate moiety is additionally esterified at the C-6 position
with a long-chain fatty acid. The most frequent fatty acid
moieties are palmitate, stearate, oleate, linoleate and linolenate.
The enzymic glucosylation of phytosterols, with the
utilization of UDP-glucose as a source of the carbohydrate
moiety, was first demonstrated with a cell-free particulate
preparation from immature soybean (Hou et al., 1967) and
since then confirmed by experiments with homogenates and
various subcellular fractions from many higher plants (for
reviews see Eichenberger, 1977; Wojciechowski, 1980). The
glucosyltransferase present in higher plants is tightly membranebound but can be solubilized with use of detergents such as
Triton X-100 or deoxycholate and partially purified by usual
protein fractionation techniques such as gel permeation
chromatography, ion-exchange chromatography or electrophoresis (Yoshikawa 8c Furuya, 1979; Wojciechowski et al.,
1979). An approx. 7O-fold purification was obtained for the
enzymes from Calendula oficinalis seedlings (Wojciechowski 8c
Van Uon, 1975) or from Digitalis purpurea cell cultures
(Yoshikawa & Furuya, 1979). Glucosyltransferase from white
mustard (Sinapis aha) seedlings, which was partially purified
by gel filtration on Sephadex G-150 and extensively studied in
our laboratory, has an M , of approx. 140000 (Wojciechowski et
al., 1979). For this enzyme the apparent K, values for
UDP-glucose and sitosterol are 8.0 x 1 0 - 5 ~and 5.0 x 1 0 - 6 ~ ,
respectively. More recent studies indicate that similar glucosyltransferases catalysing the formation of steryl P-D-monoglucosides are present also in green algae (Hopp et al., 1978)
and in a number of non-photosynthesizing organisms such as
fungi (Parodi, 1978; Wojciechowski & Zimowski, 1979; tyinik
8c Wojciechowski, 1980) and slime moulds (Wojciechowski et
al., 1977, 1979). Microplasmodia of the slime mould Physarurn
polycephalurn seem to be a particularly rich source of the
enzyme. This glucosyltransferase was purified about 28-fold and
characterized in our laboratory (Wojciechowski et al., 1979). It
differs in many respects from the glucosyltransferase isolated
from higher plants. Glucosyltransferase of Ph. polycephalurn is
weakly bound to cell membranes and can be easily solubilized,
without the use of detergents, by acetone treatment of
particulate fractions and buffer extraction. In contrast to the
enzyme isolated from higher plants it is not stimulated by
divalent metal ions and low concentrations of detergents. Its M ,
is 67000, i.e. about half that of the glucosyltransferase present in
white mustard seedlings. The apparent K, values are
1.8 x lOV4mfor UDP-glucose and 5.6 x 1 O P 6for
~ sitosterol.
As concerns the possible functions of sterol glucosylation in
plant cells an important question arises concerning the possibility that glucosyltransferases are not really specific for
sterols, i.e. is the main function of these enzymes in plant cells