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Two methods for determining the quantitative localization of sucrose-P synthase in plant tissues were evaluated. The single-cell method (rapid freezing, freeze-drying, microdissection, micro-analysis) was validated in several ways, including comparative biochemistry, comparative histochemistry, and kinetics. In contrast, bulk isolation of cells by protoplast-forming methods resulted in loss of sucrose-P synthase activity. This latter approach is widely used and, as far as we are aware, can be successfully used for measurement of other enzymes. Thus, our observations form the basis for a specific caution against the use of protoplast-forming methods in an assay protocol for sucrose-P synthase.

Crude membrane preparations of fungi contain the enzyme glucan synthase (EC 2.4.1.34) which produces a polymer of glucose linked through 1,3-beta-glycosidic bonds. This polymer is a major structural element of the fungal cell wall. Preparations of glucan synthase are contaminated with the enzyme glycogen synthase (EC 2.4.1.11). Glycogen synthase forms the storage carbohydrate glycogen, a polymer of glucose consisting of mainly 1,4-alpha-glycosidic linkage. Both enzymes utilize uridine diphosphoglucose as substrate. Discrimination of glucan synthase from glycogen synthase activity has relied upon the inclusion of glycogen-degrading enzymes in the crude reactions. The polysaccharide reaction products of glucan synthase assays have been characterized by their susceptibility to enzymatic degradation by various glucanohydrolases. These degradative enzymes are impure and inclusion of appropriate control polysaccharides often leads to ambiguous results. A method for comparative qualitative analysis of polysaccharides formed in fungal glucan synthase reactions has been developed using high-performance anion-exchange chromatography. Using this method, polymers of glucose with 1,3-beta-glycosidic linkage and 1,4-alpha linkage can be readily distinguished. This method has been applied to map oligosaccharides derived by partial acid hydrolysis from fungal glucan synthase reaction products from Candida albicans protoplasts prepared by two different methods.

An 87-kDa glucan-binding protein (GBP) of Streptococcus sobrinus B13 (serotype d) was isolated and purified from extracellular culture supernatant by using affinity chromatography on Sephadex G-50 and elution with a guanidine HCl gradient. Western blot (immunoblot) analysis showed it to be antigenically related, but not completely identical, to the 74-kDa GBP of Streptococcus mutans Ingbritt. The 87-kDa GBP has no glucosyltransferase activity. A possible role for this GBP in the cariogenicity of S. sobrinus B13 is suggested.

Alkaliphilic bacterial strains producing the enzyme cyclodextrin glucanotransferase were cultivated on solid agar medium containing an indicator system detecting the enzyme. The growth of the colony and the surrounding diffusion zone, due to the enzyme, were measured by the image analysis during the cultivation. It was possible to differentiate between relatively similar clones by observing quantitatively the changes at and around the colony. Optimal experimental conditions for such measurements are discussed. The image analysis technique provides a potential tool for characterizing microbes grown on solid media.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.

This study was conducted to evaluate UDP-glucuronosyl-transferase (UDP-GT) activity, UDP-glucuronic acid (UDP-GA) concentration, and UDP-glucose (UDPG) concentration in the rat intestine and liver following oral administration of butylated hydroxyanisole (BHA), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC), phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or trans-stilbene oxide (TSO). Microsomal UDP-GT activity was assayed in vitro with acetaminophen (AA), harmol (HA), and 1-naphthol (NA) as the aglycones. Intestinal HA and AA glucuronidation were enhanced by BHA, BaP, and TSO, whereas 3MC, PB, PCN, and TCDD augmented hepatic HA-glucuronide formation and BHA, PB, PCN, TCDD, and TSO significantly increased hepatic AA glucuronidation. All inducing agents except PB and PCN markedly increased both intestinal and hepatic NA glucuronidation. PB, PCN, and TCDD paradoxically decreased intestinal glucuronidation of AA and HA. A similar effect upon hepatic glucuronidation was not observed with any of the agents studied. Hepatic UDP-GA concentration was increased significantly by all inducers studied except PCN and TCDD, whereas hepatic UDPG concentration was increased only by BHA. In the intestine, significant increases in UDP-GA concentration were produced only by BHA and BaP, which also elevated intestinal UDPG. These results demonstrate that microsomal enzyme inducers evoke different effects upon intestinal and hepatic glucuronidation. These differences are manifested with regard to induced changes in UDP-GT activity as well as treatment-induced alterations in UDP-GA content. Thus, the present study further underscores the marked variance of intestinal and hepatic xenobiotic glucuronidation.

alpha-Fetoproteins (AFPs) were purified from 2 hepatoma cell lines (Hep G2 and HuH-7) and a hepatoblastoma cell line (HuH-6), and the structures of pyridylaminated (PA) derivatives of their sugar chains were analyzed by HPLC. Simultaneously, the activities of alpha 1-6 fucosyltransferase (alpha 1-6FT) and N-acetylglucosaminyltransferase III (GnT-III), IV (GnT-IV) and V (GnT-V) were assayed in these cell lines. For all 3 cell lines the major sugar chain detected was a fucosylated biantennary structure. Hep G2 cells contained a high level of GnT-V, which catalyzes the formation of a tri'-antennary structure, and in fact a substantial percentage of the AFP sugar chains in these cells had the tri'-antennary structure. alpha 1-6FT was also high, and fucosylated tri' structures were detected, which suggests that high activities of transferases affect the AFP sugar chains. In HuH-6 cells, GnT-III, which catalyzes the formation of bisecting GlcNAc, was elevated. Correspondingly, a fucosylated, bisected biantennary structure was found as a major sugar chain. In the HuH-7 cell line, the contents of bisecting GlcNAc and tri' structure were low and neither GnT-III nor GnT-V was elevated. These data indicate that the sugar structures of AFP in these cell lines correlate well with the activities of alpha 1-6 FT, GnT-III and GnT-V.

We recently reported [Huber, Huber & Nielsen (1989) Arch. Biochem. Biophys. 270, 681-690] that spinach (Spinacia oleracea L.) sucrose-phosphate synthase (SPS; EC 2.4.1.14) was phosphorylated in vivo when leaves were fed [32P]Pi. In vitro the enzyme was phosphorylated and inactivated by using [gamma-32P]ATP. We now report that SPS is phosphorylated both in vivo and in vitro on serine residues. The protein is phosphorylated at multiple sites both in vivo and in vitro as indicated by two-dimensional peptide maps of the immunopurified SPS protein. After being fed with radiolabel, leaves were illuminated or given mannose (which activates the enzyme), in the presence or absence of okadaic acid. Feeding okadaic acid to leaves decreased the SPS activation state in the dark and light and in leaves fed mannose. Across all the treatments, the activation state of SPS in situ was inversely related to the labelling of two phosphopeptides (designated phosphopeptides 5 and 7). These two phosphopeptides are phosphorylated when SPS is inactivated in vitro with [gamma-32P]ATP, and thus are designated as regulatory (inhibitory) sites [Huber & Huber (1991) Biochim. Biophys. Acta 1091, 393-400]. Okadaic acid increased the total 32P-labelling of SPS and in particular increased labelling of the two regulatory sites, which explains the decline in activation state. In the presence of okadaic acid, two cryptic phosphorylation sites became labelled in vivo that were not apparent in the absence of the inhibitor. Overall, the results suggest that light/dark regulation of SPS activity occurs as a result of regulatory serine phosphorylation. Multiple sites are phosphorylated in vivo, but two sites in particular appear to regulate activity and dephosphorylation of these sites in vivo is sensitive to okadaic acid.

We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransferase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction product, [14C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [14C]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn2+, Mg2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Apparent Km values of 8.7 microM for UDP-glucose and 292 microM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.

The tetrasaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-alpha-D- Manp-(1----6)-beta-D-Manp-OR (2) and the pentasaccharide beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp-OR (3), where R = (CH2)8COOMe, have been prepared by using combined chemical and enzymic procedures. Structure 2 is a substrate for UDP-GlcpNAc:beta-D-Galp-(1----4)- beta-D-GlcpNAc (GlcNAc to Gal) beta(1----3)-N-acetylglucosaminyltransferase, and 3 would be the product of its action. Antibodies raised against 3 are intended for use in an ELISA assay that would quantitate the enzymic conversion of immobilized 2 into 3.

Streptococcus gordonii exhibits a phase variation involving expression of high (Spp+) or low (Spp-) glucosyltransferase activity. The related bacterial accumulation on hydroxyapatite (HA) and saliva-coated HA surfaces was examined and found to be significant. Spp+ cells growing anaerobically in a defined medium utilize about 30% of the glucose available from sucrose to make insoluble glucans. These glucans formed cohesive masses on HA beads, which contained 80 to 90% of the total bacteria. The bacterial polymer mass had a volume of about 40 microns3 and contained more than 5 x 10(10) viable cells per cm3. In the absence of sucrose, the beads were saturated by 1 x 10(8) to 2 x 10(8) Spp+ cells. Spp- bacteria, which make 30-fold less glucan than do Spp+ bacteria, did not accumulate on surfaces in numbers significantly above the saturation level of 1 x 10(8) to 2 x 10(8) cells in the presence or absence of sucrose. Insoluble glucan synthesized by Spp+ cells from sucrose also enabled these bacteria to accumulate on saliva-coated HA seven times more effectively than the Spp- cells and 10 times more effectively than the Spp+ cells grown in medium without sucrose.

The occurrence of N-acetylglucosaminyltransferase, the initial step in the synthesis of the carbohydrate moiety of N-linked glycoproteins, is demonstrated in the microsomal fraction of the nematode Ascaris suum. Phosphatidylglycerol stimulated enzyme activity three- to six-fold without affecting the Km values of either substrates, uridinediphospho-N-acetylglucosamine or dolichylphosphate. The Km values were determined to be about 12 microM and 100 micrograms ml-1, respectively. The enzyme activity was strongly inhibited by tunicamycin acting as a competitive inhibitor with respect to the substrate uridinediphospho-N-glucosamine.

The gtf gene coding for glucosyltransferase (GTF), which produces a water-soluble glucan, was cloned from Streptococcus sobrinus OMZ176 (serotype d) into plasmid vector pBR322. This gene was expressed in Escherichia coli, and the product was purified to near homogeneity. The antigenicity of recombinant GTF (rGTF) was examined with the antisera raised against purified GTF P1, P2, P3, and P4 obtained from S. sobrinus AHT (serotype g). The rGTF reacted only with anti-GTF P1 serum in a Western blot (immunoblot) analysis. The rGTF closely resembled GTF P1 in its molecular mass, Km value for sucrose, optimal pH, primer dependency, and immunological properties. The high-molecular-weight, water-soluble glucan produced by the rGTF also resembled that of GTF P1, which is the most efficient primer donor for primer-dependent, water-insoluble glucan synthesis. Properties of the rGTF were also compared with those of rGTFS, which was purified from E. coli carrying the gtfS gene isolated from Streptococcus downei (previously S. sobrinus serotype h) MFe28. Both rGTF and rGTFS synthesized water-soluble glucan from sucrose without primer dextran, but their characteristics in Km values for sucrose, optimal pHs, and polymer sizes of the glucan were different. Furthermore, the gtf gene did not hybridize with the gtfS gene in a Southern blot analysis. These results showed that rGTF is similar to S. sobrinus AHT GTF P1 but distinct from rGTFS that has been previously purified from E. coli carrying the gtfS gene.

Because numerous attempts to detect an activity for a cellulose synthase in plants have failed, we have taken a different approach toward detecting polypeptides involved in this process. The uniqueness of the structure and function of cyclic diguanylic acid (c-di-GMP) as an activator of the cellulose synthase of the bacterium Acetobacter xylinum makes it an attractive probe to use in a search for a c-di-GMP receptor that might be involved in the process in plants. Direct photolabeling with 32P-c-di-GMP has been used, therefore, to identify in plants two membrane polypeptides of 83 and 48 kD derived from cotton fibers that possess properties consistent with their being components of a c-di-GMP-dependent cellulose synthase. Based upon several criteria, the 48-kD species is proposed to be derived by proteolytic cleavage of the 83-kD polypeptide. Both polypeptides bind c-di-GMP with high affinity and specificity and show antigenic relatedness to the bacterial cellulose synthase, and the N-terminal sequence of the 48-kD polypeptide also indicates relatedness to the bacterial synthase. Ability to detect both cotton fiber polypeptides by photolabeling increases markedly in extracts derived from fibers entering the active phase of secondary wall cellulose synthesis. These results provide a basis for future work aimed at identifying and characterizing genes involved in cellulose synthesis in plants.

Genetic polymorphisms in genes coding for the glucosyltransferases were detected among Streptococcus mutans serotype c strains by Southern blot analysis with DNA probes located within the gtfB gene (H. Aoki, T. Shiroza, M. Hayakawa, S. Sato, and H. K. Kuramitsu, Infect. Immun. 53:587-594, 1986). Restriction endonucleases were used to examine genomic DNAs isolated from serotype a to h strains. The variations were readily detected among 33 strains of serotype c by EcoRI and PstI restriction enzyme digestions. Serotypes e and f, which are genetically similar to serotype c, also had comparable polymorphism; however, serotypes a, b, d, g, and h did not hybridize to the same DNA probes in parallel experiments. Further analysis of enzymatic activities for glucan synthesis and sucrose-dependent adherence revealed no significant differences among the serotype c strains. Our results suggested that genetic polymorphisms existing in S. mutans serotype c strains may reflect a complexity in genes coding for the glucosyltransferases, which are produced ubiquitously in members of the S. mutans group.

Blood group antigen expression in the distal human colon is related to the development of the organ and is modified by malignant transformation. To elucidate the biochemical basis for these changes, we have (a) analysed the activity of glycosyltransferases coded for by the H, Se, Le, X, and A genes, in tissue biopsy specimens from normal and malignant proximal and distal human colon; (b) characterised the glycosphingolipids expressed in the various regions of normal and malignant colon by immunostaining of high performance thin layer chromatography plates; and (c) located the antigens on tissue sections from the same subjects by immunohistochemistry. In both secretors and non-secretors we found a significantly higher activity of alpha-2-L-fucosyltransferases in carcinomatous rectal tissue than in tissue from normal subjects, whereas the other transferase activities studied showed no significant differences. The acceptor substrate specificity suggested that both the Se and the H gene dependent alpha-2-L-fucosyltransferases are increased in carcinomas. In non-malignant tissue the only enzyme which showed appreciably higher activity in caecum than in rectum was alpha-2-L-fucosyltransferase. Immunochemistry and immunohistochemistry showed alpha-2-L-fucosylated structures in normal caecum from secretors and in tumour tissue from both secretors and non-secretors. We conclude that the alpha-2-L-fucosyltransferases control the expression of ABH, and Lewis(b) structures in normal and malignant colon.

Medulloblastoma biopsies are heterogenous and might contain normal brain tissue, which limits the usefulness of such tumor material for biochemical analyses. We have, therefore, examined the gangliosides and their metabolism using the medulloblastoma cell lines. Daoy and D341 Med, cultured both in vitro and as xenografts in nude mice. The ganglioside patterns in the Daoy showed a switch from a high GM2, 70% (mol% of total ganglioside sialic acid) and low lactoseries gangliosides (2%) content in monolayer cultures, to a high proportion of lactoseries gangliosides (50%) and virtually no GM2 (1%) in xenografts, but an increased proportion of other a-series gangliosides. The D341 Med showed a similar change regarding the lacto-series gangliosides from 1% in suspension culture to 10% in xenografts. The activity of five glycosyltransferases, GM3, GD3, GM2, GM1 and LA2 synthases, did not parallel the ganglioside patterns and could not account for the noted variations therein. In the Daoy cell line the LA2 synthase as well as the GM2 synthase activity was relatively high in both culture systems, despite the marked difference in the expression of GM2 and the lactoseries gangliosides. These results suggest that environmental factors play a crucial role for the in vivo activity of the glycosyltransferases.

The oral Streptococcus salivarius strain S3 possessed extracellular glucosyltransferase (GTF) and cell-associated fructosyltransferase activities. Chromatofocusing column chromatography of crude GTF yielded multiple peak fractions, which were categorized into two types which catalysed (1) water-insoluble glucan synthesis and (2) water-soluble glucan formation. The GTF isozyme eluted in fraction 29 synthesized mutanase- and dextranase-sensitive insoluble glucan from sucrose, while fraction 81 GTF synthesized soluble glucan which was insensitive to both glucanases. The ability to agglutinate Streptococcus sobrinus cells was high for insoluble glucan but negligible for soluble glucan. The insoluble glucan-synthesizing activity of fraction 29 GTF was inhibited by the presence of dextran T10. Isoelectric focusing yielded a single activity band but SDS-polyacrylamide gel electrophoresis gave multiple bands for both GTF fractions 29 and 81. The extracellular crude GTF activity was inactivated by 0.1% SDS or 1% Tween 80, but the activity was completely restored by the addition of 1% Triton X-100.

The synthesis of glucosylceramide from ceramide and UDP-[3H]glucose by mouse kidney homogenates is very sensitive to the concentration of tissue. This was shown to be due to the presence of a UDP-glc pyrophosphatase, which could be blocked by adding NAD to the medium. A new solvent partitioning system is described, containing t-butyl methyl ether, isopropyl alcohol, and aqueous sodium sulfate, which separates the original substrate (UDP-[3H]glc) from the enzyme product, [3H]cerebroside. A particular advantage of the solvent system is that only a single partitioning step is needed, without backwashes, and the enzyme product appears in the upper phase, making transfer to a counting vial more reliable. A novel incubation device, a thermostatically controlled ultrasonic bath, is used to produce highly uniform enzyme reaction rates. Ca2+, as well as Mg2+ and Mn2+, was found to be a good stimulator of the glucosyltransferase. The enzyme activity in kidney of 22-day old mice, approximately 240 pmol/h/mg tissue, is significantly greater than previously demonstrated. The enzyme was stable in intact kidneys stored at -70 degrees C but unstable at 4 degrees C. The enzyme, when acting on endogenous ceramides, showed no demonstrable glucosylation of the C24 family of ceramides although this family is the predominant one in kidney.

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.

Diabetes acutely impairs the ability of the liver to synthesize glycogen. However, the effect of chronic diabetes on the glycogenic function of the liver is not known. We measured hepatic glycogen contents in streptozotocin (STZ)-diabetic rats 3 weeks or 9 months after the induction of diabetes, in the fed state and following a 24-hour fast. In the fed state, liver glycogen levels were markedly decreased in short-term diabetic animals (5.8 +/- 2.0 v 33.9 +/- 2.3 mg/g, P less than .001), but not in long-term diabetic rats (18.3 +/- 4.4 v 20.7 +/- 1.3 mg/g, P = NS) as compared with age-matched nondiabetic animals, despite comparable hyperglycemia (portal plasma glucose levels of 424 +/- 21 and 449 +/- 24 mg/100 mL, short- and long-term diabetics, respectively). In the fasted state, on the other hand, liver glycogen was depleted in acute diabetes (4.5 +/- 2.2 mg/g v 1.9 +/- 0.5 of control rats), but significantly increased in chronic diabetes (10.1 +/- 3.1 v 0.2 +/- 0.03 mg/g, P less than .001). The latter finding was confirmed by electron-microscopical examination of liver cells. Furthermore, the percentage of hepatic glycogen synthase in the active form (synthase a) was lower than normal in short-term diabetic rats and in old nondiabetic rats. In long-term diabetic animals, on the other hand, synthase a was significantly higher than in old controls (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this work was to use preparations from germinating seeds of Pisum sativum to determine the apparent equilibrium constant of the reaction catalysed by sucrose-phosphate synthase (EC 2.4.1.14) and to compare this with the mass-action ratio of the reaction in the seeds. The apparent equilibrium constant ranged from 5.3 at 0.25 mM-MgCl2, pH 7.0, to 62 at 10 mM-MgCl2, pH 7.5. The sucrose phosphate content of the seeds, 23 nmol/g fresh wt., was determined by separating sucrose phosphate from sucrose by ion-exchange chromatography and then measuring the sucrose released by alkaline phosphatase. Comparison of equilibrium constants and mass-action ratios in the cotyledons of 38 h-germinated seeds showed that the reactions catalysed by glucose-6-phosphate isomerase, phosphoglucomutase and UDP-glucose pyrophosphorylase are close to equilibrium, and those catalysed by sucrose-phosphate synthase and sucrose phosphatase are considerably displaced from equilibrium in vivo.

A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.

The development of an enzyme-linked immunosorbent assay (ELISA) for uridine 5'-diphospho-N-acetyl-glucosamine: alpha mannoside beta 1----6 N-acetylglucosaminyltransferase (GnT-V) is reported. The assay quantitates the enzymatic conversion of the specific synthetic GnT-V acceptor GlcNAc beta 1----2Man alpha 1----6Man beta-R (5) to the product GlcNAc beta 1----2[GlcNAc-beta 1----6]Man alpha 1----6Man beta-R (6) when these oligosaccharide structures were covalently attached to bovine serum albumin which was then coated on microtiter wells. Conversion of 5 to 6 was detected using a polyclonal antiserum raised against the product 6 and from which antibodies cross-reacting with acceptor 5 had been removed by affinity adsorption. GnT-V activity detected by ELISA was linearly proportional to both enzyme concentration and time under appropriate experimental conditions where 50-300 fmol of product was formed per microtiter well. GnT-V activity could be measured by ELISA in Triton X-100 extracts of hamster kidney acetone powder and in human serum. The twofold increase in GnT-V activity which is known to accompany Rous sarcoma virus transformation of baby hamster kidney cells could also be quantitated using the ELISA.

Certain strains of Streptococcus sanguis adhere (Adh+) selectively to human platelets and, in plasma, induce them to aggregate (Agg+) into in vitro thrombi. In this study, we examined 18 recent endocarditis and dental plaque isolates of microorganisms that were biotyped as S. sanguis for coexpression of platelet interactivity phenotypes with another possible virulence factor in bacterial endocarditis, dextran synthesis. Detectable production of extracellular glucosyltransferase ranged from 0.2 to 66 mU/mg of culture fluid for 10 representative strains tested. Production of extracellular or cell-associated glucosyltransferase, fructosyltransferase, and soluble or insoluble dextrans was not necessarily coexpressed with platelet interactivity phenotypes, since the levels of production of soluble and insoluble dextrans varied among representative Adh+ Agg+ and Adh- Agg- strains. Analysis of a second panel of 38 fresh dental plaque isolates showed that S. sanguis distributes in a reproducible manner into the possible phenotype groups. Strains with different platelet interactivity phenotypes were distinguished with a panel of four murine monoclonal antibodies (MAbs) raised against Adh+ Agg+ strain 133-79 and screened to rule out artifactual reactions with antigenic components in culture media. The MAbs reacted selectively with Adh+ Agg+ strains in a direct-binding, whole-cell, enzyme-linked immunosorbent assay and also inhibited their interactions with platelets. Analysis of minimal tryptic digests of many strains, including variants that failed to bind the MAbs, suggested that some noninteractivity phenotypes possess cryptic surface determinants. Since the ability to adhere to platelets and induce them to aggregate is relatively stable, these traits may be useful in a phenotyping scheme for these Lancefield nontypeable streptococci.

Trehalose-6-phosphate synthase activity was determined by colorimetric, spectrophotometric and trehalose specific assays. All methods gave comparable results thus confirming our previous findings (1) and those reported by Elander (2). Different strains and mutants of Saccharomyces were carefully re-investigated in relation with the recent claim made by Vandercammen et al. (3) that our spectrophotometric assay over-estimated the enzyme activity and that no differences exist between wild type and mutant strains. In this paper we also confirm the de-activation of the trehalose synthase complex in response to a "glucose signal", and present trehalose-6-phosphate synthase and trehalase activities in different strains measured during all phases of growth on glucose.

1. Collagens are the most important components of the connective tissue. 2. Collagen synthesis involves greater than 12 different enzymes whereas three enzymatic systems are involved in the ordered degradation. 3. Some enzymes are found in the rough endoplasmic reticulum (RER). The subcellular localization of disulfur isomerase, alpha D-glucosidase, proteases, galactosyltransferases and glucosyltransferases specific to collagen is unknown. 4. After having determined the best subcellular fractionation conditions for the chick embryo liver, we demonstrate that the galactosylhydroxylysyl glucosyltransferase specific to collagen is located in the RER and in the Golgi apparatus.

The interaction between Streptococcus mutans dextransucrase (EC 2.4.1.5) and high-molecular-weight dextran was studied in both the presence and absence of substrate sucrose. Equivalent weight-percent solutions of primer dextrans that differed 200-fold in molecular weight were found to be equally efficient in priming new dextran synthesis. Sodium borohydride reduction of dextran had no effect on its priming ability. These results suggest that dextran synthesis proceeds by addition of glucosyl residues to nonreducing termini of primer dextrans and that several enzyme molecules simultaneously bind to single high-molecular-weight dextran molecules. Kinetic data suggested that dextransucrase contains only one dextran binding site per enzyme molecule. The nature of the commonly observed highly aggregated state of dextransucrase was also studied. Two types of enzyme aggregates were distinguished: (i) oligomeric enzyme aggregates that formed in the absence of dextran and were dissociated by 1 M KCl; and (ii) dextran-induced enzyme aggregates that were stable to 3 M salt. Oligomeric enzyme aggregates were obtained from supernatants of fructose-grown cultures, whereas dextran-induced enzyme aggregates appeared to be present in glucose-grown cultures. The molecular weight of the smallest species of dextran-free detransucrase observed in solutions of 1 M KCl was estimated to be 40,000 by gel column chromatography. Addition of dextran to primer-dependent dextransucrase resulted in formation of complexes that were stable in CsCl density gradients and exhibited a buoyant density of 1.382 g/cm3 as compared with a buoyant density of 1.302 g/cm3 exhibited by dextransucrase. The enzyme-dextran complexes observed in CsCl density gradients contained about 25% dextran. This corresponded to 150 enzyme molecules (molecular weight, 40,000) per dextran molecule (molecular weight, 2 X 10(6)). The implication of these results to the mechanism of sucrose- and dextran-induced aggregation of S. mutans is discussed.

A mutant of Streptococcus mutans GS-5 has been isolated as a smooth colonial variant on mitis salivarius agar. This mutant, designated SNG-1, adheres to glass surfaces as well as the parental organism when grown in the presence of sucrose. However, in contrast to the parental organism, glucose-grown cultures of the mutant did not adhere to smooth surfaces when incubated with sucrose under nongrowing conditions. The inability of the mutant organism to adhere to glass surfaces under the latter condition was a result to markedly reduced levels of mutant cell-associated glucosyltransferase activity. In addition, the extracellular activity of the mutant was also severely depressed relative to the parental activity. The reduced levels of mutant enzyme activity appear to be a result of a mutation in a structural gene coding for glucosyltransferase activity since (i) mutant glucosyltransferase activity is much less resistant to heat inactivation compared to the parental enzymes and (ii) the migration patterns of the mutant and parental enzymes differ on polyacrylamide gels and after isoelectric focusing on polyacrylamide gels. However, the kinetic properties of the mutant enzymes are similar to those of the comparable parental activities in terms of pH and temperature optima and Km values for sucrose. The mutant enzyme responsible for soluble glucan synthesis has been purified approximately 300-fold. These results are discussed in terms of the mechanism of glucan synthesis by S. mutans.

The nucleotide sequence of the glg C gene of Escherichia coli K12, coding for ADP-glucose synthetase, has been determined. The structural gene consists of 1293 base pairs, which specify a protein of 431 amino acids. The amino acid sequence deduced from the DNA sequence is consistent with the known NH2-terminal amino acid sequence and the amino acid composition of ADP-glucose synthetase. The translation start of the structural gene of glycogen synthase, glg A, starts immediately after termination of the glg C gene.

IgA, IgG, albumin, lysozyme, alpha-amylase and glucosyltransferase were identified in the saliva coat which forms on hydroxyapatite exposed to whole saliva. It is suggested that these proteins may be involved in binding microorganisms to saliva coated hydroxyapatite in model studies.

Antibodies directed against Streptococcus mutans GS-5 intracellular invertase and glucosyltransferase fractions capable of synthesizing primarily water-soluble or insoluble glucans were used to ultrastructurally localize the enzymes by means of the unlabeled antibody peroxidase-antiperoxidase method. This immunocytochemical procedure revealed that the intracellular invertase was associated primarily with the cytoplasmic membrane of the cariogenic organism. The glucosyltransferase complex responsible for insoluble glucan synthesis was localized as aggregates attached to the cell surface or extracellular polysaccharides of strain GS-5. In contrast, the glucosyltransferase activity synthesizing primarily water-soluble glucans was distributed uniformly over the cell surface or in association with extracellular polysaccharides. These results are discussed relative to the sucrose-metabolizing ability of Streptococcus mutans.

A novel glucosyltransferase that synthesizes low molecular weight water-soluble glucan was partially purified from Streptococcus mutans AHT cell-free culture supernatant. This enzyme was compared with another water-soluble glucan synthase which synthesized high molecular weight glucan.

A glucosyltransferase which synthesized insoluble glucan in polyacrylamide gel was isolated from the culture supernatant of Streptococcus mutans Ingbritt (serotype c) by ultrafiltration, ethanol fractionation, isoelectric focusing, and preparative gel electrophoresis. The enzyme preparation was electrophoretically homogeneous and immunologically distinct from the highly branched 1,6-alpha-D-glucan synthase and fructosyltransferase from the same strain and glucosyltransferases from serotypes a and g. The molecular weight was 99,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was 8.5. The enzyme had the optimum pH of 6.0 and Km value for sucrose of 9.4 mM. Besides the insoluble glucan with 96% 1,3-alpha linkage, this enzyme synthesized a considerable amount of diffusible glucan with 84% 1,6-alpha linkage, separately. This enzyme may be the one released from the enzyme aggregates by extracellular proteases, because the addition of extraneous trypsin to the crude enzyme preparation increased the amount of the enzyme species.

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [(14)C]lysine were assayed for hydroxy[(14)C]lysine and hydroxy[(14)C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[(14)C]lysine and glucosylgalactosylhydroxy[(14)C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [(14)C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. (14)C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5'-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.

Two glucan synthetase preparations from Candida albicans were obtained by lysis of regenerating protoplasts (enzyme A) or mechanical breakage of yeast cells (enzyme B). Enzyme A was insensitive to EDTA or GTP but it was stimulated by a combination of both agents. Enzyme B was inhibited by EDTA, this inhibition being released by increasing the concentration of the chelating agent or by addition of GTP to the assay mixtures. Enzyme A was further activated by glycerol and sodium fluoride. The reaction products were characterized as linear beta-1,3-linked glucans on the basis of their resistance to periodate and susceptibility to beta-glucanases. In both cases the "in vitro" synthesized radioactive chains were added to the non-reducing end of cold, performed glucan or to and acceptor other than glucan. At least, part of the preformerd glucan chains of enzyme A, but no those of enzyme B, showed a free reducing terminal. On the basis of the origin of both enzyme preparations it is suggested that glucan molecules are synthesized while bound to an acceptor of a different nature which is subsequently excised.

A case report of a child with glycogen storage disease type IV is presented. The diagnosis was confirmed by enzyme assay on cultured fibroblasts. Some unusual features of this disorder are discussed and the possibility of antenatal diagnosis is emphasized.

The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The K(m) for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a K(i) of 27 mM.

The glucosyltransferases of S mutans 6715 were resolved into two major fractions. One fraction synthesized water-soluble glucans and the other made water-insoluble glucans. Each fraction was found by polyacrylamide gel electrophoresis to be composed of several catalytically active species, apparently glycoprotein in nature. Treatment of the glucosyltransferases with dextranase in the absence of sucrose caused an interconversion of enzyme forms concomitant with a time-dependent loss of enzyme activity, but did not appear to remove significant amounts of the carbohydrate associated with the enzymes. Comparison of enzyme activity patterns on polyacrylamide gels of the five different S mutans serotypes further emphasizes the complexity of the glucosyltransferase system from this group of microorganisms.

Cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans has been purified, crystallized and analyzed by X-ray diffraction. The enzyme is monomeric. SDS/polyacrylamide gel electrophoresis gave an Mr of 73,600(+/- 1000), corresponding to 670(+/- 10) amino acid residues. The structure of the crystalline enzyme has been elucidated at a resolution of 3.4 A, using multiple isomorphous replacement and solvent flattening for phase determination. The resulting electron density map allowed tracing of the polypeptide chain; 664 residue positions have been assigned. The chain fold has been subdivided into five domains. The N-terminal domain forms a (beta alpha)8-barrel, which contains the second domain of about 55 residues as an insert after the third beta-strand. The three remaining domains form almost exclusively beta-pleated sheet structures and consist of about 90, 80 and 95 residues. The chain fold of the three N-terminal domains of 492 residues resembles closely the two known structures of alpha-amylases. This geometric similarity corresponds to the observed amino acid sequence homology. On the basis of the sequence homology with alpha-amylases, the active center can be located. The fourth domain has an immunoglobulin fold and is far away from the active center, while the fifth domain participates in the formation of the broad depression at the active center. Accordingly, the cyclodextrin glycosyltransferase chain fold can be considered as an alpha-amylase chain fold with two additional domains.

Methods previously described for glycogen or amylopectin branching enzymatic activity are insufficiently sensitive and not quantitative. A new, more sensitive, specific, and quantitative one was developed. It is based upon the quantitation of the glucose residues joined by alpha 1,6 bonds introduced by varying amounts of branching enzyme. The procedure involved the synthesis of a polysaccharide from Glc-1-P and phosphorylase in the presence of the sample to be tested. The branched polysaccharide was then purified and the glucoses involved in the branching points were quantitated after degradation with phosphorylase and debranching enzymes. This method appeared to be useful, not only in enzymatic activity determinations but also in the study of the structure of alpha-D-glucans when combined with those of total polysaccharide quantitation, such as iodine and phenol-sulfuric acid.

Deficiency of amylo-1,6-glucosidase activity was expressed in parallel in liver and skin fibroblasts from a patient with type III glycogenosis. In crude extracts of control liver and muscle, amylo-1,6-glucosidase (M.W. 164000) was identified by immunoprecipitation; no cross-reacting material was found in the patient's liver. Assay of amylo-1,6-glucosidase activity in cultured skin fibroblasts from the affected family revealed less than 10 per cent of control value in mutant homozygous cells whereas in cells from the parents, activity was reduced to 40-60 per cent of the control value. Activity in cultured amniotic fluid cells was similar to that of control fibroblasts. In cultured amniotic fluid cells obtained during the mother's subsequent pregnancy, the normal amylo-1,6-glucosidase activity measured, predicted correctly the outcome of this pregnancy prior to the 20th week of gestation.

Extraction of lipids with CHCl3/CH3OH/1 M HCl from fresh and frozen cells of Candida bogoriensis showed the presence of a steryl glucoside. The product was purified and identified as a beta-cholesteryl glucoside. It hydrolyzes in methanolic HCl and therefore it is a steryl glucoside. The quantitation of the extracted cholesteryl glucoside was pursued with high-pressure liquid chromatography. The chemical ionization mass spectra of the isolated and the synthesized beta-cholesteryl glucosides were compared and found identical. The cell wall of Candida bogoriensis was weakened with enzyme (glusulase) and after homogenisation and sucrose density gradient centrifugation, five layers separated. Only the pellets of one layer showed the presence of cholesteryl glucoside in thin-layer chromatography after extraction with solvents. In the same layer, the activity of sterol glucosyl transferase was found.