alpha-D-Mannosidase of Saccharomyces cerevisiae. Characterization and modulation of activity.

A unique and interesting alpha-D-mannosidase (alpha-D-mannoside mannohydrolase EC 3.2.1.24) activity has been isolated from Saccharomyces cerevisiae. The enzyme was localized in a crude particulate fraction of the cell extract and was not solubilized by treatment with detergents or high ionic strength NaCl. The enzyme had a pH optimum of 6.3, Km 50 micron with p-nitrophenyl-alpha-D-mannopyranoside, and was competitively inhibited by D-mannose (Ki 20 mM). The enzyme is not affected by ethylenediaminetetraacetic acid, a number of different cations, or sulfhydryl reagents. It was inhibited by p-chloromercuriphenyl sulfonic acid and this inhibition is prevented by the addition of substrate. The cellular concentration of alpha-D-mannosidase is inversely proportional to growth rate, suggesting that the enzyme is under catabolite repression. The level of enzyme was found to increase approx. 8-fold during sporulation. This is apparently due to de novo synthesis, since inhibition of protein synthesis by cycloheximide prevents the increase in enzyme activity.     

Effect of estradiol on the amino acid-accepting activity of uterine tRNAs and their participation in protein synthesis

Estradiol (E2) induces a complementary increase in both the amount of mRNA and the rate of translation of the mRNA in the uterus of ovariectomized mature rats. The mechanism of the translational effect was evaluated by measuring the functional capacity of uterine tRNA isolated from control, E2 (1 h)- and E2 (14 h)-treated ovariectomized rats to support amino acid acceptor activity and uterine protein synthesis. The specific amino acid acceptor activity (SAA) of deacylated tRNA for 18 individual amino acids was determined using a tRNA-dependent rat liver tRNA synthetase preparation. The SAA was the same for all amino acids for uterine tRNA from control and E2 (1 h)-treated rats but was increased for uterine tRNA from E2 (14 h)-treated rats to levels that were 1.4-4.3 times the SAA of uterine tRNA from control rats. When uterine tRNA from control and E2 (14 h)-treated rats was incubated with purified tRNA nucleotidyltransferase, the SAA for all amino acids was increased an average of 1.6-fold for control tRNA and 0.3-fold for tRNA from E2 (14 h)-treated rats. The ability of uterine tRNA to support maximal rates of protein synthesis in tRNA-dependent uterine ribosome protein synthesis assay was increased by either in vivo treatment of the rats with estradiol or by in vitro repair of the 3'-CCA terminus of this tRNA by nucleotidyltransferase. These observations suggest that E2 may increase the rate of mRNA translation in the uterus, in part, by increasing the proportion of certain tRNAs with intact and functional 3'-CCA acceptor termini.     

Cobalt-ion chelate affinity chromatography for the purification of brain neutral alpha-D-mannosidase and its separation from acid alpha-D-mannosidase.

A method is described for the purification of neutral alpha-D-mannosidase and its separation from acid alpha-D-mannosidase from monkey brain by utilizing Co2+ chelate affinity chromatography. The neutral enzyme, which selectively bound to the metal-ion chelate column, was elutable by Tris at pH 7.5 and gave over 80-fold purification in a single step with 100% recovery.     

The metabolism of L-tryptophan by liver cells prepared from adrenalectomized and streptozotocin-diabetic rats.

1. The metabolism of L-tryptophan by liver cells prepared from fed normal, adrenalectomized and streptozotocin-diabetic rats was studied. 2. At physiological concentrations (0.1 mM), the rate of oxidation of tryptophan by tryptophan 2,3-dioxygenase was 3-fold greater in liver cells from diabetic rats than in those from fed rats. In liver cells from diabetic rats, oxidation of tryptophan to CO2 and metabolites of the glutarate pathway was increased 7-fold. Quinolinate synthesis was decreased by 50%. These findings are consistent with an increase in picolinate carboxylase activity. 3. Rates of metabolism of 0.1 mM-tryptophan by hepatocytes from fed and adrenalectomized rats were similar. 4. In all three types of cell preparation, fluxes through tryptophan 2,3-dioxygenase with 2.5 mM-tryptophan were 7-fold greater than those obtained with 0.1 mM-tryptophan. Tryptophan 2,3-dioxygenase and kynureninase fluxes in hepatocytes from fed and adrenalectomized rats were comparable, whereas those in liver cells from diabetic rats were increased 2.5-fold and 3.3-fold respectively. Picolinate carboxylase activities of liver cells from diabetic rats were 15-fold greater than those of cells from fed rats, but rates of quinolinate synthesis were unchanged. 5. It is concluded that: (i) adrenal corticosteroids are not required for the maintenance of basal activities of the kynurenine pathway, whereas (ii) chronic insulin deficiency produces changes in both the rate of oxidation and metabolic fate of tryptophan carbon.     

L-tryptophan administration promotes the reversion of pre-established chronic liver injury in rats treated with carbon tetrachloride

We examined the effect of L-tryptophan (Trp) administration on the reversion of CCl(4)-induced chronic liver injury after hepatotoxicant withdrawal in rats. When rats treated with CCl(4) twice a week for 6 weeks were released from CCl(4) treatment for 2 weeks, there was an incomplete reversion of liver injury. The reversion was enhanced by 2 weeks of daily intraperitoneal administration of Trp (50 mg/kg body weight), starting just after CCl(4) withdrawal. There were increases in the levels of thiobarbituric acid reactive substances, an index of lipid peroxidation, Ca(2+), triglycerides, and Trp, and decreases in tryptophan 2,3-dioxygenase activity and serum triglyceride concentrations in the liver of rats treated with CCl(4) for 6 weeks. Serum albumin concentrations and in vitro hepatic protein synthesis activity did not change in the CCl(4)-treated rats. The changes in the CCl(4)-treated rats were partially attenuated 2 weeks after CCl(4) withdrawal. The attenuation was enhanced by 2 weeks of daily Trp administration. The increases in hepatic thiobarbituric acid reactive substances and triglycerides and the decreases in hepatic tryptophan 2,3-dioxygenase activity and serum triglyceride concentrations observed 2 weeks after CCl(4) withdrawal were almost completely attenuated by Trp administration. In vitro hepatic protein synthesis in CCl(4)-treated and untreated rats was increased by 2 weeks of daily Trp administration. These results indicate that Trp administration promotes the reversion of pre-established chronic liver injury in rats treated with CCl(4,) and suggest that Trp exerts this effect by enhancing the improvement of several parameters of liver dysfunction associated with chronic liver injury and by stimulating hepatic protein synthesis.     

Effect of swainsonine on rat epididymal glycosidases

Each epididymis of control and swainsonine-fed rats (5 micrograms/ml drinking water) was divided into 5 segments, and tissue, spermatozoa and sperm-free supernatants were prepared from each segment. When levels of 3 lysosomal glycosidases and total protein were determined, the proximal cauda contained the greatest concentration of glycosidase. The specific-activity profile for beta-glucuronidase and beta-galactosidase was similar in swainsonine-fed and control rats. However, the concentration of alpha-D-mannosidase in tissue of all segments was significantly greater in swainsonine-fed rats than in age-matched controls. Enzyme activity for alpha-D-mannosidase after swainsonine treatment was significantly greater in spermatozoa from the caput, than in spermatozoa from the corpus and the cauda epididymidis. Since the alpha-D-mannosidase activity was optimal at pH 4.5 and studies with highly specific antibody to lysosomal alpha-D-mannosidase immunoprecipitated all of the alpha-D-mannosidase present in detergent extracts of epididymal tissue, spermatozoa, and sperm-free supernatant, the enzyme studied is of lysosomal origin.     

Purification and properties of major alpha-D-mannosidase in the luminal fluid of porcine epididymis.

A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0).Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH.These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit.     

Comparative study on the activity, pH optimum and thermal stability of some glycosidases and acid phosphatase from pig and mule leukocytes.

1. alpha-D-mannosidase, beta-D-galactosidase, alpha-L-fucosidase, beta-N-acetylgalactosaminidase, alpha-D-glucosidase and acid phosphatase activities were studied in circulating blood leukocytes from Sus scropha var. domestica L. (pig) and Equus asinus x caballus (mule) by spectrophotometric procedures using p-nitrophenyl derivatives as substrates and three different buffer solutions. 2. The highest specific activity corresponds to acid phosphatase. The specific activities of the glycosidases, all relatively close together in all cases, were low in comparison with that of phosphatase. 3. Generally, each of the above-mentioned enzymes shows one common peak for the pH optimum between 3.5 and 6.0, except alpha-D-glucosidase, which shows two peaks. 4. The pH optima values are generally similar in three buffer solutions employed. 5. Specific activities of the studied enzymes show a parallelism in leukocytes from both pig and mule. Also, this parallelism is observed in their pH optima values. 6. Thermal stability of alpha-D-mannosidase is high whereas that of acid phosphatase is low, in both materials. For other enzymes, differences in the thermal stability was observed according to their source.     

Interleukin-1 alpha stimulates prostaglandin biosynthesis in serum-activated mesangial cells by induction of a non-pancreatic (type II) phospholipase A2

Enhanced prostaglandin (PG) biosynthesis is a hallmark of inflammation, and interleukin-1 (IL), a proinflammatory cytokine, is a potent stimulus of PG production. We investigated the mechanisms of IL-1 alpha-enhanced PG synthesis in serum-stimulated mesangial cells. The rIL-1-stimulated increase in PGE2 synthesis was dose- and time-dependent and inhibited by both cycloheximide and actinomycin D. Phospholipase (PL) activity was increased 5- to 10-fold in acid extracts of rIL-1-treated cells as measured by arachidonate release from exogenous [14C]arachidonyl-phosphatidyl-ethanolamine. This induced phospholipase activity was Ca(2+)-dependent and inhibited by the PLA2 inhibitors, aristocholic acid, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacylbromide, but not by the 1,2-diacylglycerol lipase inhibitor RHC 80267. The rIL-1-stimulated PLA2 had an alkaline pH optimum, and phosphatidylethanolamine was preferred over phosphatidylcholine as substrate. The PLA2 activity increased by rIL-1 was inhibited in cells coincubated with cycloheximide and was measurable after 6 h. A sensitive and specific solution hybridization assay demonstrated a coordinate time-dependent induction of non-pancreatic PLA2 mRNA expression which was increased at least 6-fold by 24 h. In whole cells, IL-1 had no effect on basal [3H]arachidonic acid release but vasopressin (1 microM)-stimulated release was potentiated 2- to 3-fold, suggesting that IL-1 may prime cells for increased PG synthesis via increased PLA2 activity. Thus IL-1 directly stimulates, as well as primes cells for, enhanced PG synthesis, in part, by increasing PLA2 activity through new synthesis of a non-pancreatic (Type II) PLA2.     

Potato sucrose synthetase: purification, properties, and changes in activity associated with maturation

Sucrose synthetase activity is high in young potato tubers but decreases markedly during maturation. The activity decreases rapidly after the tubers are harvested and remains low regardless of storage temperature. This enzyme was purified 34-fold from freshly harvested immature potatoes. It catalyzes both cleavage and synthesis of sucrose but the 2 activities differ in a number of ways. The pH optima are 6.6 and 8.8 for sucrose cleavage and synthesis. respectively. Sucrose cleavage is activated 4-fold by mercaptoethanol and is inhibited by Mn²⁺. In contrast, sucrose synthesis is activated only slightly by either mercaptoethanol or Mn²⁺ alone but 2-fold in the presence of both reagents. However, it was not possible to resolve the 2 activities, their stabilities to partial thermal inactivation are identical, and their ratios are constant over a wide range of activities.     

Early effect of myo-inositol deficiency on fatty acid synthetic enzymes of rat liver

Young rats (100 g) were fed either a purified myo-inositol-deficient balanced diet or a control diet containing 0.5% by weight myo-inositol, ad libitum, for up to 2 weeks following a 48 h fast. Weight gain was the same for animals in both groups. Liver triacylglycerol levels in the deficient animals were 1.8-, 3.5- and 3.0-fold higher than the corresponding levels in the control animals after 4, 8 and 14 days of feeding, respectively. In the myo-inositol-deficient group the specific activities of liver fatty acid synthetase and acetyl-CoA carboxylase were elevated 1.5-2.0-fold over controls, reaching a maximum after 3-4 days of feeding. Subsequently, activities declined to control levels. Rates of fatty acid synthetase synthesis in the deficient group, as measured by [3H]leucine incorporation into immunoprecipitable fatty acid synthetase polypeptide, were significantly higher (1.5-2.0-fold) than controls after 12-18 h of feeding and then declined to control levels by 1 day. No difference was noted between groups in either the rate of total, soluble liver protein synthesis or the half-life of fatty acid synthetase over this time period. These results suggest that the liver lipodystrophy observed during myo-inositol deficiency in rats may be due in part to elevated levels of lipogenic enzymes in this tissue in the early stage of the deficiency.     

Antioxidant effect of Ajuga iva aqueous extract in streptozotocin-induced diabetic rats

The purpose of this study was to investigate the possible antioxidant effect of an aqueous extract of Ajuga iva (Ai) in streptozotocin (STZ)-induced diabetic rats. Twelve diabetic rats were divided into two groups fed a casein diet supplemented or not with Ai (0.5%), for 4 weeks. In vitro, the Ai extract possessed a very high antioxidant effect (1 mg/ml was similar to those of trolox 300 mmol/l). The results indicated that plasma thiobarbituric acid reactive substances (TBARS) values were reduced by 41% in Ai-treated compared with untreated diabetic rats. TBARS concentrations were lower 1.5-fold in liver, 1.8-fold in heart, 1.9-fold in muscle and 2.1-fold in brain in Ai-treated than untreated group. In erythrocytes, Ai treatment increased significantly the activities of glutathione peroxidase (GSH-Px) (+25%) and glutathione reductase (GSSH-Red) (+22%). Superoxide dismutase activity was increased in muscle (+22%), while GSH-Px activity was significantly higher in liver (+28%), heart (+40%) and kidney (+45%) in Ai-treated compared with untreated group. Liver and muscle GSSH-Red activity was, respectively, 1.6- and 1.5-fold higher in Ai-treated than untreated diabetic group. Catalase activity was significantly increased in heart (+36%) and brain (+32%) in Ai-treated than untreated group. Ai treatment decreased plasma nitric oxide (?33%), carbonyls (?44%) and carotenoids (?68%) concentrations. In conclusion, this study indicates that Ajuga iva aqueous extract improves the antioxidant status by reducing lipid peroxidation and enhancing the antioxidant enzymes activities in plasma, erythrocytes and tissues of diabetic rats.     

Effects of mitogenic stimulation on lymphocyte alpha-D-mannosidases

Three types of alpha-D-mannosidase are present in human and murine lymphocytes. Their levels increased substantially when the cells were activated by T-cell mitogens, concanavalin A (Con A) and phytohaemagglutinin (PHA), and in the murine cells also by lipopolysaccharide (LPS), a B-cell mitogen. The intracellular localization of the alpha-D-mannosidases in the non-stimulated and activated murine cells was investigated by fractionation of lymphocyte lysates on colloidal silica (Percoll) and discontinuous sucrose gradients. In both types of cell, an enzyme having optimal activity at neutral pH was obtained in the cytosolic fraction and another alpha-D-mannosidase most active at an intermediate pH was obtained partly in membrane-bound form. In contrast, an acidic alpha-D-mannosidase, which was particularly elevated in the activated murine spleen cells, had a distribution in these lymphoblasts which was markedly different from that in non-stimulated lymphocytes. In the latter, the major proportion of the activity was obtained in a cytosolic fraction and the remainder in a particulate fraction of light density, whereas the enzyme in activated lymphocytes was distributed between vesicles of light and heavy density comparable with lysosomal organelles. Moreover, the acidic alpha-D-mannosidase still remained membrane bound even when cell lysates were prepared under hypotonic conditions which disrupt lysosome integrity. These results suggest that lymphocyte activation involves either stabilization of fragile lysosomes present in resting cells or de novo synthesis of lysosome-like structures. The acidic alpha-D-mannosidase present within isolated, intact lysosomes was found to be in a form, A, whereas a different form, B, was most prominent in whole-cell extracts of both types of lymphocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

The multiple forms of alpha-D-mannosidase in human plasma.

The acidic alpha-D-mannosidase in human plasma closely resembles liver acidic alpha-D-mannosidase in its affinity for concanavalin A-Sepharose, molecular weight and resolution into multiple components on DEAE-cellulose. A combination of chromatography on concanavalin A-Sepharose and gel filtration on Sephadex G-200 and Sepharose 6B suggests that four forms of intermediate alpha-D-mannosidase, which differ either in their molecular weight of affinity for concanavalin A, exist in human plasma. A practical classification and nomenclature for the multiple forms of intermediate alpha-D-mannosidase in plasma based on molecular weight and affinity for concanavalin A is proposed. Multiple forms of intermediate alpha-D-mannosidase were also observed by ion-exchange chromatography on DEAE-cellulose, but there was not a simple correlation between these forms and those obtained with the other separation procedures. The form of intermediate alpha-D-mannosidase least abundant in plasma, approx. 7% of the activity, has very similar properties to the neutral alpha-D-mannosidase in human liver. In contrast, the other three forms of intermediate alpha-D-mannosidase, which account for over 90% of the activity, do not appear to be present in liver, except perhaps in trace amounts.     

Regulation of lipid flux between liver and adipose tissue during transient hepatic steatosis in carnitine-depleted rats

Rats with carnitine deficiency due to trimethylhydrazinium propionate (mildronate) administered at 80 mg/100 g body weight per day for 10 days developed liver steatosis only upon fasting. This study aimed to determine whether the transient steatosis resulted from triglyceride accumulation due to the amount of fatty acids preserved through impaired fatty acid oxidation and/or from up-regulation of lipid exchange between liver and adipose tissue. In liver, mildronate decreased the carnitine content by approximately 13-fold and, in fasted rats, lowered the palmitate oxidation rate by 50% in the perfused organ, increased 9-fold the triglyceride content, and doubled the hepatic very low density lipoprotein secretion rate. Concomitantly, triglyceridemia was 13-fold greater than in controls. Hepatic carnitine palmitoyltransferase I activity and palmitate oxidation capacities measured in vitro were increased after treatment. Gene expression of hepatic proteins involved in fatty acid oxidation, triglyceride formation, and lipid uptake were all increased and were associated with increased hepatic free fatty acid content in treated rats. In periepididymal adipose tissue, mildronate markedly increased lipoprotein lipase and hormone-sensitive lipase activities in fed and fasted rats, respectively. On refeeding, carnitine-depleted rats exhibited a rapid decrease in blood triglycerides and free fatty acids, then after approximately 2 h, a marked drop of liver triglycerides and a progressive decrease in liver free fatty acids. Data show that up-regulation of liver activities, peripheral lipolysis, and lipoprotein lipase activity were likely essential factors for excess fat deposit and release alternately occurring in liver and adipose tissue of carnitine-depleted rats during the fed/fasted transition.     

Rat epididymal alpha-D-mannosidase: purification, carbohydrate composition, substrate specificity, and antibody production

Two alpha-D-mannosidases have previously been identified in rat epididymis. This communication reports the purification and characterization of the "acid" alpha-D-mannosidase. The enzyme was purified over 1000-fold to near homogeneity by acetone and (NH4)2SO4 precipitation followed by ion-exchange and hydroxylapatite chromatography. The molecular weight of the enzyme was estimated to be 220,000 by gel filtration. Polyacrylamide gel electrophoresis of the native enzyme under two conditions of buffer and pH showed a single band when stained for protein while electrophoresis under denaturing conditions resulted in bands of apparent Mr 60,000 and 31,000. The enzyme is a glycoprotein containing about 5.6% hexose. In addition to mannose (3.1%) and glucosamine (2.0%), the enzyme also contained small amounts of glucose, fucose, and galactose. Chemical analysis indicated the absence of sialic acid. The substrate specificity of the purified enzyme was investigated using linear and branched mannose-containing oligosaccharides. The enzyme cleaved linear oligosaccharides [Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc and Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc] very efficiently. However, little or no activity was observed toward high mannose oligosaccharides (Man9GlcNAc through Man5GlcNAc) or the branched trimannosyl derivative Man3GlcNAc. This specificity is very similar to that observed with rat kidney lysosomal alpha-D-mannosidase. Additional evidence that the epididymal enzyme is essentially a lysosomal alpha-D-mannosidase is the fact that polyclonal antibody prepared against the purified epididymal enzyme cross-reacted with lysosomal alpha-D-mannosidase from several rat tissues and with acidic alpha-D-mannosidase of a human cell line, results suggesting that the antibody will be useful in studying the biosynthesis and turnover of lysosomal alpha-D-mannosidases in at least two species.     

Effect of triton WR-1339 on peroxisomal enzymes of rat liver

The effect of Triton WR-1339 on peroxisomal enzymes of rat liver was studied. The dose vs. response relationships of peroxisomal enzyme activities to Triton WR-1339 were first examined 3.5 days after injection. Catalase activity was reduced to 50% of that of the control at a dose of 200 mg per 100 g body weight; it was found that the decrease depended on the dose of this compound. Urate oxidase activity was not significantly affected. D-Amino acid oxidase activity showed intermediate behavior. The activities of these enzymes were found to be reduced more markedly at 2 days than at 3.5 days after injection, and subsequently the levels of the activities recovered. At 2 days after injection of a dose of 200 mg per 100 g body weight, the activities of catalase, D-amino acid oxidase and urate oxidase had decreased to 40, 60 and 60%,respectively, of the control values.It was found that the decreases in the activities of these enzymes caused by Triton WR-1339 had occurred in the large granule fraction, but not in the cytoplasm.Measurement of the specific activity, Ouchterlony gel diffusion and quantitative immunoprecipitation suggested that there was a similarity between the Triton WR-1339-treated and untreated rats in the nature of purified catalases.These results suggest that Triton WR-1339 depresses the activities of liver peroxisomal enzymes, especially the catalase activity.     

Glycosphingolipid glycosyl hydrolases and glycosidases of synchronized human KB cells.

KB cells were synchronized by a double thymidine block procedure. An investigation was made of the activities of alpha-L-fucosidase (EC 3.2.1.51), alpha-D-galactosidase (EC 3.2.1.22), beta-D-galactosidase (ec 3.2.1.23), alpha-D-glucosidase (EC 3.2.1.20), beta-D-glucosidase (EC 3.2.1.21), alpha-D-mannosidase (EC 3.2.1.24), beta-D-N-acetylgalactosaminidase (EC 3.2.1.53), and beta-D-N-acetylglucosaminidase (EC 3.2.1.52) from synchronized cultures, using appropriate artificial substrates. Ceramide glucosidase (EC 3.2.1.45) and ceramide trihexosidase levels (EC 3.2.1.47) were also investigated at various stages in the cell cycle, using appropriate glycosphingolipid substrates. Whereas each of these enzymes exhibited some activity throughout the cell cycle, peak activity (2- to 6-fold increase) occurred late in the S phase. Two molecular forms of ceramide glucosidase (optimal activity at pH 4.0 and pH 6.0) and two forms of ceramide trihexosidase (pH 4.0 and pH 7.5) were identified. Peak levels of the forms that preferred the relatively acid pH occurred earlier in the S phase of the cell cycle than those of the forms that were more active at the higher pH. The possibility that the forms with optimal activity at pH 4 are precursors of those with optimal activity at pH 6 to 7.5 is discussed. Precipitation of beta-galactosidase of synchronized KB cells with specific antibody revealed that changes in the activity of this enzyme during the cell cycle were the result of fluctuations in the amount of the enzyme.