Effect of eatradiol and progesterone on UDPgalactose pyrophosphatase activity in the endometrium of ovariectomized rats

Rat endometrium was found to contain a UDPgalactose pyrophosphatase for the hydrolysis of UDPgalactose into galactose 1-phosphate and UMP. The administration of 17β-estradiol to ovariectomized rats resulted in a significant decrease in the activity of the enzyme in endometrium while have little effect on that in myometrium. The response was linear with the dose of estradiol and as little as 0.07 μg per 100 g body weight produced maximum inhibition of the enzyme. Progesterone on its own had little effect on the enzyme activity but in combination with estradiol, it effectively prevented the inhibitory effect of estradiol. This inhibitory effect of estradiol on the activity of UDPgalactose pyrophosphatase may function in the regulation of glycoprotein biosynthesis in endometrium.     

The distribution and partial characterization of UDPgalactose: N-acetylgalactosamine mucin galactosyltransferase activity from rat small intestine and its sensitivity to Zn2+

UDPgalactose: N-acetylgalactosamine mucin galactosyltransferase activity of the rat intestine was studied and purified using asialo-ovine submaxillary mucin as the acceptor substrate and inhibitors to suppress UDPgalactose breakdown by pyrophosphatase activities particularly prevalent in the duodenal-jejunal regions. Despite adequate suppression of UDPgalactose breakdown, significant intestinal region differences of mucin galactosyltransferase activity were observed. Elevations of activity were observed in the duodenum and distal ileum of the small intestine and the cecum and proximal colon; these elevations in activity correspond to areas of increased mucin production. Similarly, mucin galactosyltransferase activity of duodenal cells isolated along a crypt-to-villus axis showed a moderate increase (67.7%) in activity associated with cells in the crypt region. Small intestine mucin galactosyltransferase activity was purified 800-fold using a series of ion exchange (DEAE-Sepharose), gel filtration (S-200 Sephacryl) and affinity chromatographic steps to isolate the mucin galactosyltransferase activity from a Triton X-100/Nonidet P-40 extract of homogenized cells obtained by scraping everted intestines. The partially purified enzyme showed two distinct protein bands of 81.5 and 50 kDa and a faint band at 53.3 kDa. Kinetic analysis gave an apparent Km of 152 microM for UDPgalactose. The enzyme showed optimal activity with Mn2+ (20 mM) and partial activities using a number of other divalent cations. Higher concentrations of Mn2+ were slightly inhibitory. Mucin galactosyltransferase activity was inhibited by more then 90% in the presence of Zn2+ (4 mM) and this inhibition could not be reversed by additional Mn2+. Addition of Zn2+ (4 mM) to assays containing Mn2+ (20 mM) did not cause appreciable UDPgalactose breakdown, as measured by high-voltage paper electrophoresis, suggesting that Zn2+ inhibition is not a result of pyrophosphatase activation. In addition, Zn2+ does not appear to activate a protease or glycosidase activity in the partially purified enzyme preparation which could hydrolyze the galactosylated product prior to isolation.     

Distribution, purification and characterization of rat intestinal UDPgalactose: N-acetylglucosaminyl(beta 1----4)galactosyltransferase

Rat intestinal UDPgalactose: N-acetylglucosaminyl(beta 1----4)galactosyltransferase activity was studied as to its intestinal and villus-to-crypt distribution, and then purified and characterized. Rapid UDPgalactose hydrolysis was noted in the duodenum and jejunum; little to no breakdown was detected in the distal ileum, cecum and proximal colon. Product analysis suggested that UDPgalactose hydrolysis was due to nucleotide-sugar pyrophosphatase and galactose-1-phosphate phosphatase activities; ileum appeared to have little of the first activity and none of the latter. An aboral gradient of galactosyltransferase activity was noted, activity being 3-4-fold higher in the ileum, cecum and proximal colon. Total homogenate exogenous acceptor galactosyltransferase activities showed no villus-crypt differences but activity measured with intact isolated cells demonstrated higher activity with crypt cells; this was particularly evident in the ileum. Galactosyltransferase activity was purified from ileal-colonic mucosa. An over 4000-fold purification with 75 percent yield was achieved. Only one band of approx. 70-75 kDa was noted on sodium dodecyl sulfate polyacrylamide electrophoresis. As with other eukaryotic galactosyltransferase activities, there was an absolute requirement for Mn2+; the concentration required for half maximal activity was only 2.5 microM and higher concentrations did not inhibit. The Km for UDPgalactose was 30 microM.     

Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.     

Distribution,purification and characterization of rat intestinal UDPgalactose: N-acetylglucosaminyl(β1 → 4)galactosyltransferase

Rat intestinal UDPgalactose:N-acetylglucosaminyl(β1 → 4)galactosyltransferase activity was studied as to its intestinal and villus-to-crypt distribution, and then purified and characterized. Rapid UDPgalactose hydrolysis was noted in the duodenum and jejunum; little to no breakdown was detected in the distal ileum, cecum and proximal colon. Product analysis suggested that UDPgalactose hydrolysis was due to nucleotide-sugar pyrophosphatase and galactose-1-phosphate phosphatase activities; ileum appeared to have little of the first activity and none of the latter. An aboral gradient of galactosyltransferase activity was noted, activity being 3–4-fold higher in the ileum, clecum and proximal colon. Total homogenate exogenous acceptor galactosyltransferase activities showed no villus-crypt differences but activity measured with intact isolated cells demonstrated higher activity with crypt cells; this was particularly evident in the ileum. Galactosyltransferase activity was purified from ileal-colonic mucosa. An over 4000-fold purification with 75 percent yield was achieved. Only one band of approx. 70–75 kDa was noted on sodium dodecyl sulfate polyacrylamide electrophoresis. As with other eukaryotic galactosyltransferase activities, there was an absolute requirement for Mn²⁺; the concentration required for half maximal activity was only 2.5 μM and higher concentrations did not inhibit. The K_m for UDPgalactose was 30 μM.     

Galactosyltransferase activity and cell growth: uridine diphosphate (UDP)galactose inhibition of murine leukemic L1210 cells

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS-DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose-1-phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat-inactivated calf serum (HICS)-DMEM, 5 mM UDPgalactose inhibited L1210 cell growth in HICS-DMEM to the same degree as that observed in CS-DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS-DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2-M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.     

Inhibitory effects of estradiol on glycogen synthesis in primary cell cultures of human endometrium

The effect of estradiol on glycogen synthesis was examined in a primary cell culture system of the human endometrium. Estradiol inhibited glycogen synthesis in a dose dependent manner with a minimum effective dose of 10(-9)M. Progesterone-induced glycogen synthesis was completely abolished by the simultaneous addition of estradiol. Estradiol did not affect the activity of glycogen phosphorylase in endometrial cells, but the activity of glycogen synthetase was decreased by estradiol irrespective of the presence of progesterone. The inhibitory effect of estradiol on the glycogen synthesis of endometrial cells seemed to be mediated by estradiol receptor, because LY156758, an antiestrogenic drug at receptor site, reversed the inhibitory effect of estradiol.     

Effect of ovarian hormones on glycosyltransferase activities in the endometrium of ovariectomized rats.

Some of the properties of galactosyl- and sialyltransferases present in rat endometrial tissue were investigated. The enzyme activities were found to be partly membrane-bound and partly in soluble form. The galactose enzyme was also present in uterine secretions. The specific activities of both galactosyl- and sialyltransferases were greatly enhanced in endometrium of ovariectomized rats following 17 β-estradiol injections, although the enzyme activities in the liver remained unaffected. Mixing experiments with the homogenates of endometrium from control and estradiol-treated rats failed to suggest the presence of any “activator” or “inhibitor” of the enzymes. Diethylstilbesterol, estrone, and estriol also stimulated galactosyl- and sialyltransferase activities, whereas testosterone stimulated sialyltransferase only. Prolactin administration had no effect on either of the enzymes. The effect of estradiol on both enzymes was shown to be dose-dependent and the specific activities of the enzymes started to increase about 6 hr after hormone administration, reaching a peak around 48 h. Progesterone, on its own, had no effect on the galactosyltransferase in ovariectomized rat endometrium but effectively prevented the stimulatory effect of estradiol. When estradiol-primed rats were treated with progesterone, it was found that very small doses of progesterone resulted in decrease of galactosyltransferase activity. In such animals sialyltransferase activity was stimulated by a low concentration of progesterone which was followed by inhibition at higher concentrations. These effects of ovarian hormones on glycosyltransferase activities in endometrium are compatible with earlier reports on the effects of these hormones on glycoprotein and glycosaminoglycan levels in rabbit uterus after ovariectomy (7). Regulation of glycosyltransferase activities in endometrium induced by estradiol and progesterone may bear some relationship to the “receptive” state of the uterus for blastocyst implantation.     

Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes.

Rat liver microsomes showed very active uridine diphosphate-galactose pyrophosphatase activity leading to the hydrolysis of uridine diphosphate-galactose into galactose1-phosphate and finally into galactose. The activity was observed in presence of buffers with wide ranges of pH. Different concentrations of divalent cations, such as Mn²⁺, Mg²⁺, and Ca²⁺ had no significant effect on the enzyme activity. A number of nucleotides and their derivatives inhibited the pyrophosphatase activity. Of these, different concentrations of uridine monophosphate, cytidine 5′-phosphate and cytidine 5′-diphosphate have slight or no effect; cytidine 5′-triphosphate, adenosine 5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-diphosphate-glucose and guanosine 5′-diphosphate-glucose showed strong inhibitory effect whereas cytidine 5′-diphosphate-choline showed a moderate effect on the pyrophosphatase. All these nucleotides also showed variable stimulatory effects on uridine diphosphate-galactose:glycoprotein galactosyltransferase activity in the microsomes which could be partly related to their inhibitory effects on uridine diphosphate-galactose pyrophosphatase. Among them uridine monophosphate, cytidine 5′-phosphate, and cytidine 5′-diphosphate stimulated galactosyltransferase activity without showing appreciable inhibition of pyrophosphatase, cytidine 5′-diphosphate-choline, although did not inhibit pyrophosphatase as effectively as cytidine 5′-triphosphate, guanosine 5′-triphosphate, adenosine 5′-triphosphate, cytidine 5′-diphosphate-glucose, and guanosine 5′-diphosphate-glucose but stimulated galactosyltransferase activity as well as those. The fact that cytidine 5′-diphosphate-choline stimulated galactosyltransferase more effectively than cytidine 5′-phosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate suggested an additional role of the choline moiety in the system. It has been also shown that cytidine 5′-diphosphate-choline can affect the saturation of galactosyltransferase enzyme at a much lower concentration of uridine diphosphate-galactose. Most of the pyrophosphatase and galactosyltransferase activities were solubilized by deoxycholate and the membrane pellets remaining after solubilization still retained some galactosyltransferase activity which was stimulated by cytidine 5′-diphosphate-choline. In different membrane fractions a concerted effect of both uridine diphosphate-galactose pyrophosphatase and glycoprotein:galactosyltransferase enzymes on the substrate uridine diphosphate-galactose is indicated and their eventual controlling effects on the glycopolymer synthesis in vitro or in vivo need careful evaluation.     

Effect of estradiol and progesterone on glucosamine 6-phosphate synthase in the uterus of rat

Injection of estradiol to ovariectomised rats caused significant increase in the activity of glucosamine 6-phosphate synthase of uterus. Progesterone did not cause any increase in the activity of the enzyme; however, it antagomised the effect of estradiol. It was observed that the enzyme is predominantly localised in the endometrium of uterus.     

Isolation of a Golgi apparatus-rich fraction from rat liver. IV. Thiamine pyrophosphatase

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.     

Effect of nucleotides on UDP-N-acetylglucosamine pyrophosphatase and N-acetylglucosaminyltransferase activities in microsomal membranes.

Rat liver microsomes solubilized by incubating with lysolecithin or Triton X-100 showed very active UDP-N-acetylglucosamine pyrophosphatase activity leading to the hydrolysis of the substrate into N-acetylglucosamine-P and N-acetylglucosamine. ATP, GTP, CDPcholine, and CDPglucose exerted a considerable inhibitory effect on the solubilized membrane pyrophosphatase activity. CDPcholine and CDPglucose, in addition, appeared to stimulate the transfer of N-acetylglucosamine into endogenous and exogenous acceptor proteins. Evidence is also presented of an inhibitory effect of ATP (and to some extent GTP) on N-acetylglucosaminyltransferase activity. This inhibitory effect of ATP and GTP became clearly evident when the pyrophosphatase activity in the membranes was virtually eliminated in the presence of CDP-choline and CDPglucose. The effect of ATP and GTP on the solubilized membrane enzymes indicated that the inhibition of pyrophosphatase activity alone did not determine the rate of transfer of sugar to protein. The results also suggested that the UDP-N-acetylglucosamine pyrophosphatase and N-acetylglucosaminyltransferase activities were controlled independently and the effect of each nucleotide on these enzymes should, therefore, be carefully evaluated to understood its role in glycopolymer biosynthesis. Also, a possible role of choline and its derivatives in glycoprotein synthesis is discussed.     

Evidence for a membrane-bound pyrophosphatase in Dictyostelium discoideum

Plasma membrane enriched fractions of Dictyostelium discoideum contain a Des-insensitive ATPase activity that can be fractionated by DEAE-Sephacel into a major vanadate-sensitive activity and a minor vanadate-insensitive activity. The vanadate-insensitive activity hydrolyzed pyrophosphate considerably more rapidly than ATP or any other substrate tested, and the enzyme was therefore designated a pyrophosphatase. The enzyme had no activity on AMP or p-nitrophenyl phosphate. The pyrophosphatase activity was maximal at alkaline pH values and stimulated by Mg2+ but not by Ca2+, properties of the enzyme that are very similar to those of the previously characterized pyrophosphatases of the plant tonoplast membrane. The pyrophosphatase activity of total membrane extracts changed very little during Dictyostelium differentiation.     

H(+)-translocating inorganic pyrophosphatase of plant vacuoles. Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases.

The effects of divalent cations, especially Ca2+ and Mg2+, on the proton-translocating inorganic pyrophosphatase purified from mung bean vacuoles were investigated to compare the enzyme with other pyrophosphatases. The pyrophosphatase was irreversibly inactivated by incubation in the absence of Mg2+. The removal of Mg2+ from the enzyme increased susceptibility to proteolysis by trypsin. Vacuolar pyrophosphatase required free Mg2+ as an essential cofactor (K0.5 = 42 microM). Binding of Mg2+ stabilizes and activates the enzyme. The formation of MgPPi is also an important role of magnesium ion. Apparent Km of the enzyme for MgPPi was about 130 microM. CaCl2 decreased the enzyme activity to less than 60% at 40 microM, and the inhibition was reversed by EGTA. Pyrophosphatase activity was measured under different conditions of Mg2+ and Ca2+ concentrations at pH 7.2. The rate of inhibition depended on the concentration of CaPPi, and the approximate Ki for CaPPi was 17 microM. A high concentration of free Ca2+ did not inhibit the enzyme at a low concentration of CaPPi. It appears that for Ca2+, at least, the inhibitory form is the Ca2(+)-PPi complex. Cd2+, Co2+ and Cu2+ also inhibited the enzyme. The antibody against the vacuolar pyrophosphatase did not react with rat liver mitochondrial or yeast cytosolic pyrophosphatases. Also, the antibody to the yeast enzyme did not react with the vacuolar enzyme. Thus, the catalytic properties of the vacuolar pyrophosphatase, such as Mg2+ requirement and sensitivity to Ca2+, are common to the other pyrophosphatases, but the vacuolar enzyme differs from them in subunit mass and immunoreactivity.     

Identification of an extracellular nucleotide pyrophosphatase in the culture media of Streptomyces mediterranei ME/R 17]

An exocellular pyrophosphatase, active on the nucleotide precursors of peptidoglycans, has been found in the culture medium of Streptomyces mediterranei ME/R 17. This enzyme was separated from the DD-carboxypeptidase by batchwise adsorption on DEAE cellulose. The pyrophosphatase had no strict substrate requirements, it hydrolyzed various UDP-sugar substrates: UDP-GlcNAc, UDP-Mur NAc and UDP-MurNAc peptides, giving rise to the corresponding sugar phosphate and to UMP. The enzyme preparation also contained a 5'-nucleotidase activity and UMP was further split to give uridine. This nucleotidase activity was inhibited by potassium tetraborate. Both cytoplasmic and particulate preparations from cells of S. mediterranei also contained a pyrophosphatase activity while only the particulate fractions showed the DD-carboxypeptidase activity. The pyrophosphatase excretion was tested during the grwoth cycle. The activity of the enzyme showed a constant increase throughout the exponential growth and a stronger increase in the late exponential phase. Such a result could be correlated with a consumption of the nutrients in the culture medium, in fact a relatively poor culture medium had a strong positive effect upon the production of the exocellular pyrophosphatase.     

Association of inorganic pyrophosphatase activity with normal calcification of rat costal cartilage in vivo

1. Dialysed extracts of rat costal cartilage were shown to possess an enzyme that hydrolyses inorganic pyrophosphate. 2. Inorganic pyrophosphatase activity assayed in the presence of 2mm substrate was maximal at pH6.8. 3. Mg(2+) was essential for activity, which was greatest with 10mm or higher concentrations of Mg(2+). 4. Extracts prepared from cartilage taken from suckling rats (<20g.) showed little or no hydrolytic activity, but as rat weight increased inorganic pyrophosphatase activity was detected, increased to a maximum in tissue from animals weighing about 40g., and then rapidly declined. 5. The increase in inorganic pyrophosphatase activity was associated with an increase in the uptake of (45)Ca by the cartilage in vivo. 6. Accumulation of calcium, inorganic phosphate and magnesium occurred when inorganic pyrophosphatase activity was at its maximum. 7. Alkaline phosphatase activity, measured in the same extracts used to determine pyrophosphatase activity, was highest in the tissues of the animals weighing <20g., and decreased as inorganic pyrophosphatase activity increased to its maximum. 8. There was no direct relationship between alkaline phosphatase activity and the onset of calcification.     

Participation of the estradiol receptor in the hormonal inhibition of cyclic nucleotide phosphodiesterase in the uterus

The role of estradiol receptor was studied in the inhibitory effect of hormone on the cyclic nucleotide phosphodiesterase from immature Wistar rat uterus. It was shown that the preparative separation of the enzyme and hormone receptor by ultracentrifugation in isokinetic sucrose density gradient results in a 2.5-3-fold decrease of the estradiol effect on phosphodiesterase. This effect is completely restored after adding the separated estradiol receptor to the phosphodiesterase devoid of it. The effect of estradiol on the phosphodiesterase activity depends on a degree of receptor component aggregation: the action of estradiol on the enzyme intensities after transformation of receptor into the dissociated form (4S) and removes in the presence of the receptor component associated form (8S).     

Maize leaf proteases and their effect on endogenous inorganic pyrophosphatase

Several different proteolytic enzymes are present in leaf and root tissue of maize seedlings. The activity of these enzymes diminishes to a basal level by the time seedling height reaches 20–30 cm. We have partially characterized an endopeptidase with trypsin-like specificity and two aminopeptidases, all from leaf tissue, and compared them to previously reported proteases from maize. Both the endopeptidase and the aminopeptidases degrade the maize leaf enzyme, inorganic pyrophosphatase. Modification of the pyrophosphatase by the peptidases results in the formation of catalytically active, electrophoretically distinct products. The aminopeptidases have little effect on several other maize leaf enzymes, but also modify yeast inorganic pyrophosphatase.     

The effect of steroids and nucleotides on solubilized bilirubin uridine diphosphate glucuronyltransferase

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis-Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The K(m) for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca(2+) were inhibitory in the absence of Mg(2+) but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3beta-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.     

The effect of cycloheximide on galactosyltransferase of rat liver Golgi membranes.

An inhibitory effect of cycloheximide on the initial rate of galactosyltransferase of rat liver Golgi membranes has been demonstrated. Cycloheximide was effective in inhibiting the activity of the enzyme when added directly to the assay medium or after pre-incubation of the membranes with the drug. The inhibition observed with different concentrations of nucleotide sugar was shown to be competitive at higher concentrations of the nucleotide sugar (0.10-1.0 mumol). The inhibition observed with different concentrations of acceptor, N-acetylglucosamine was complex and could not be analysed further with the present data. Washing the Golgi membranes previously incubated with cycloheximide with water failed to reverse the inhibition. Washing with UDPgalactose partially reversed the inhibition only. These results, together with the observation that serum galactosyltransferase was not inhibited by cycloheximide supported the view that the cycloheximide effect may be primarily on the membrane system.