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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.

We describe an improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations. These procedures were used to examine the subcellular distribution of chitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in homogenates from exponentially growing walled cells of a wild-type strain of yeast. Chitin synthetase (Chs1) activity was mainly found in two distinct vesicle populations of nearly equal abundance but with markedly different buoyant densities and particle diameters. One population contained 45-65% of the total chitin synthetase and was identified as chitosomes because of microvesicular size (median diameter = 61 nm) and characteristic low buoyant density (1.15 g/ml); it also lacked 1,3-beta-glucan synthetase activity. The second population (35-55%) was identified as plasma membrane because of its high buoyant density (1.22 g/ml), large vesicle size (median diameter = 252 nm), and presence of vanadate-sensitive ATPase. This fraction cosedimented with the main peak of 1,3-beta-glucan synthetase. A third, minor population of chitin synthetase particles was also detected. Essentially all of the chitin synthetase in the two vesicle populations was zymogenic; therefore, we regard these vesicles as precursors of the final active form of chitin synthetase whose location in the cell has yet to be unequivocally determined.

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.

We have studied through the late foetal period of the rat, the evolution of two enzymes involved in glycogen metabolism of the liver : UDPG glycogen synthetase (a and b forms) and branching enzyme. The activities were measured during the development of normal foetuses and of foetuses experimentally deprived of corticosteroids. In normal foetus the activity of glycogen synthetase and the branching enzyme increased progressively between days 18 and 21 ; but the percentage of glycogen synthetase a increased promptly between days 18 and 19. This variation coincides with the beginning of glycogen accumulation in the liver. In foetuses submitted to corticosteroid shortage, the activity of each enzyme, and the amount of glycogen in the liver, were reduced at term. Cortisol given to the decapited foetus restores subnormal glycogen storage and normal activity of branching enzyme. The activity of synthetase a was slightly increased but remained very low ; only synthetase b was restored to normal. It seems that there is no relationship between the synthesis of glycogen, in the foetal rat liver, and the activity of synthetase a.

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.

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.

An N-acetylglucosaminyltransferase III which catalyzes the addition of N-acetylglucosamine through a beta 1-4 linkage (bisecting N-acetylglucosamine) to the beta-linked mannose of the trimannosyl core structure of N-linked oligosaccharides of glycoproteins was measured in human serum, and liver and hepatoma tissues. The enzyme activity in serum was significantly elevated in patients with hepatomas and liver cirrhosis, and the activity markedly decreased on the transcatheter arterial embolization treatment. High activities were also found in the hepatoma and cirrhotic liver tissues, indicating that the serum activity reflected the activity in the tissues. The assaying of the enzyme activity in serum appears to be useful for the detection and monitoring of primary hepatomas.

Porcine thyroid cells were cultured in serum-free medium and thyrotropin was or was not added at day 4 and [3H]glucosamine at day 6 for 24 h. The major glycoprotein secreted outside the follicles proved to be thyroglobulin by immunoprecipitation, polyacrylamide gel electrophoresis, and amino acid composition. Thyroglobulin glycopeptides were analyzed by sequential affinity chromatography on immobilized lectins and compared to chemically labeled carbohydrate chains released from thyroid-derived thyroglobulin by hydrazinolysis. 82% and 85% of the glucosamine-labeled oligosaccharides of thyroglobulin from control and stimulated cells, respectively, were unretained on concanavalin A (ConA)-Sepharose compared to 46% only for in vivo thyroglobulin. 35-42% and 33-35% of the ConA-unbound glycopeptides were retarded on erythrophytohemagglutinin and leukophytohemagglutinin under basal or stimulatory conditions, respectively, while none of the triantennary structures of in vivo thyroglobulin was. Moreover, binding to Bandieraea-agarose showed that 20% of these complex structures contained alpha-linked galactose in thyroglobulin secreted by control cells, but only 10% in the molecules derived from thyroid. When analyzed on ricin-agarose after neuraminidase treatment, the ConA-unbound glycopeptides were retained to an extent of 65% for those from control cells and 98% for those from stimulated cells. Furthermore, 15% of desialylated ConA-unbound glycopeptides from cellular origin were also found to bind to wheat germ agglutinin. Carbohydrate composition, gel chromatography, and exoglycosidase treatment further demonstrated that thyroglobulin carbohydrate chains synthesized under serum-free cell culture were essentially composed of heterogeneous multiantennary structures instead of usual biantennary and high mannose type species. Under thyrotropin stimulation, 85% of the carbohydrate chains of thyroglobulin was shown to be sialylated by high performance liquid chromatography analysis instead of 65% under basal conditions, suggesting that thyrotropin may shift terminal glycosylation of thyroglobulin from alpha-galactose to sialic acid.

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.

Optimal assay conditions have been determined in human liver preparations for the catalytic transfer of mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine, respectively, to dolichyl phosphate. Both enzymatic reactions have an absolute requirement for divalent cation (5 mmol/l Mn2+ optimal), detergent (Triton X-100 or Nonidet P-40) and dolichyl phosphate (as acceptor substrate) and both reactions have optimal activity at a pH value of 7.8. Preliminary characterization of the glycolipid products for both enzymatic reactions indicates that phosphorylated dolichol is the major acceptor substrate for radiolabeled mannose and N-acetylglucosamine. The activity levels and specific activities of dolichyl phosphate-mannosyltransferase are comparable in liver homogenates from normal controls and patients with cystic fibrosis and diabetes mellitus. The activity levels and specific activities of dolichyl phosphate-N-acetylglucosaminyltransferase are comparable in liver homogenates from normal controls and patients with cystic fibrosis and diabetes mellitus but considerably lower than the activity levels of dolichyl phosphate-mannosyltransferase. It appears that two of the initial steps of the lipid-mediated glycosylation pathway are normal in livers from patients with cystic fibrosis and diabetes mellitus.

Three muscle biopsies were performed in 53 overt type II diabetics over a period of approximately 2 years. At baseline, 21 (40%) had an abnormally increased capillary basement membrane width in muscle. Thirty-five subjects received glipizide and 18, placebo. At baseline, no statistically significant difference was found in the muscle capillary basement membrane width between the two groups (P = NS). In the subjects receiving placebo, the mean width of the muscle capillary basement membrane increased (P = NS), but in those receiving glipizide, the mean decreased from 193 +/- 13 nm (SEM) to 161 +/- 10 nm (P = .02). Fasting plasma glucose and glycosylated hemoglobin A1 significantly decreased (P less than .001) after two years in those receiving glipizide. In 15 subjects, mean glycosylated hemoglobin A1 reached the normal range, and mean muscle capillary basement membrane width decreased to a level close to that found in subjects without diabetes (P = NS). Determinations of enzyme activities involved in the synthesis and degradation of glycoproteins revealed a 2-year significant decrease of muscle glucosyltransferase (synthesis) activity (P less than .01) in the glipizide-treated subjects as opposed to a significant increase (P less than .001) in those receiving placebo. Muscle N-acetyl-beta-glucosaminidase activity (degradation) was statistically increased (P less than .001) in those subjects taking glipizide, but decreased in those taking placebo (P less than .001).

The sequences of glucosyltransferase genes from Streptococcus sobrinus (gtfI) and Streptococcus mutans (gftB) were compared and show a high degree of homology. There is a 57.7% homology of nucleotides in the genes and a 56.7% homology of amino acids in the deduced protein sequences. The G + C content for the protein-coding region is 43.6% for S. sobrinus and 41.2% for S. mutans. Internal repeating sequences present in both proteins exhibit some difference in sequence pattern.

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 extracellular glucosyltransferase (GTF) enzymes of Streptococcus mutans GS-5 and avirulent mutant GS-511 were fractionated using agarose and DEAE-cellulose columns. GTF of GS-5 produced both water-soluble and -insoluble glucans, while those of GS-511 made soluble products almost exclusively. Nearly all (98%) of the small amount of insoluble GTF enzyme made by GS-511 was bound to the cell wall.

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.

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.

The localization of chitin synthase in the cells of Mucor rouxii was studied by a method which combined permeabilization of the cells with toluene/ethanol and incubation with the radioactive substrate UDP-[3H]GlcNAc followed by high resolution autoradiography. By this technique it was demonstrated that most of the chitin synthesized by these cells was located within the cytoplasm, and only a small amount of the enzyme product appeared at the cell surface. It was concluded that most of the chitin synthase of M. rouxii is located in the cytoplasm of the cells.

Multiple forms of purified dextransucrase have been observed in the presence of low detergent concentrations ( Luzio , G.A., Grahame , D. A. and Mayer, R.M. (1982) Arch. Biochem. Biophys. 216, 751-757). We now show these forms to arise partly as a result of proteolysis, and partly due to incomplete dissociation of the enzyme. Upon 25 degrees C incubation of the crude enzyme, several new bands appeared with little or no change in total activity. The electrophoretic pattern of aged, crude enzyme was similar to that of partially purified enzyme. Specific detection of dextransucrase on SDS gels revealed a single polypeptide of 174 kDa, which is converted to a 156 kDa protein during the aging process. The observation indicates the occurrence of proteolysis. The polypeptide composition of several of the enzyme forms was determined by two-dimensional electrophoresis. Forms Ia and IIa are composed exclusively of 174 kDa polypeptides. Forms III and IVa consist of 156 kDa units, as does the newly observed form Ic. It is likely that form Ib contains both 174 and 156 kDa polypeptides. The results indicate that incomplete dissociation of aggregates of the 174 kDa unit accounts for all of the bands observed on native gels run on fresh culture extracts. Additional enzyme forms result from aggregation of the 156 kDa proteolysis product alone, and from aggregation with unproteolyzed units to form hybrid aggregates.

Debranching enzyme was purified 150-fold from rabbit skeletal muscle by a three-step procedure which utilised ammonium sulphate precipitation, ion-exchange chromatography on DEAE-cellulose and "hydrophobic" chromatography on Sepharose-NH(CH2)4NH2. The preparation was completed within three days, and 200 mg enzyme was isolated from 1000 g muscle, which represented an overall yield of 60%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation coefficient, s20,w, was 8.1 S. The amino acid composition was determined, and the absorption coefficient, A280 1%, measured refractometrically was 17.5. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 166000 and this value was supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride (1550oo). The molecular weight of the native enzyme measured by high-speed sedimentation equilibrium was 164000, showing that the debranching enzyme is a monomeric protein at the concentrations which exist in muscle (0.7 mg/ml). The results indicate that the two different enzyme activities which are associated with debranching enzyme, 1,4-glucan-4-glycosyltransferase and amylo-1,6-glucosidase, reside on the same polypeptide chain. Protein-glycogen particles isolated from skeletal muscle showed seven major protein-staining components by polyacrylamide gel electrophoresis, one of which was identified as debranching enzyme. ,our of the other components were the alpha and beta subunits of phosphorylase kinase, glycogen phosphorylase and glycogen synthetase. A new titrimetric assay for debranching enzyme was developed; it was used to demonstrate that the maximum potential activity of debranching enzyme is only 5--10% that of phosphorylase at the concentrations of the two enzymes in skeletal muscle. Since the activity of debranching enzyme is unaffected by every mechanism which leads to the activation of glycogen phosphorylase and phosphorylase kinase, the evidence suggests that the hormonal control of muscle glycogenolysis by adrenalin might be confined to a stimulation of rate of degradation of the outermost branches of glycogen.

Implantation of cobalt powder in the cerebral cortex of rat determines an epileptogenic focus where two types of reactive astrocytes are observed. The first type is mostly represented in the subcortical white matter but it does exist in the cortex around the implant. Phosphorylase and branching enzyme are both very active in these cells which are filled with glycogen. The second type is limited to the cortex and phosphorylase activity leads to an unbranched polysaccharid. These cells correspond to the "activated astrocytes" described by the authors in a previous paper and observed round irritative lesions which, in the cerebral cortex, produce epileptogenic foci.

Dextran stimulation (priming) of the dextransucrase (EC 2.4.1.5) from Streptococcus mutans strain 6715 was studied. The dextransucrase activity in supernatant fluids from glucose-grown cultures was shown to be partially primer dependent. During extended storage at 4 C the enzyme retained its activity. However, the ability to make dextran became increasingly primer dependent. Hydroxylapatite-chromatographed enzyme preparations were completely dependent upon added dextran for rapid synthesis of methanol-insoluble glucan from sucrose. Half-maximal stimulation of new dextran synthesis occurred with dextran at a concentration of 2 to 3 muM and with a molecular weight of about 2,600. Neither glycogen, amylose, inulin, nor isomaltose functioned as primer. Studies with the dextransucrase activities detectable by in situ assay in polyacrylamide gels subjected to electrophoresis under nondenaturing conditions revealed that the major activity was detectable in the presence of sucrose alone and was stimulated by addition of primer dextran. The minor activity was only detected when primer dextran was present. Homogeneous preparations of both enzymes contained 30 to 40% carbohydrate.

The activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, and the concentration of hydroxyproline (HYP) were measured in the gastrocnemius, soleus, and tibialis anterior muscles of rats after sciatic nerve neurectomy combined with cast immobilization of the denervated limb for 1 and 3 wk. PH and GGT were also observed in Achilles and tibialis anterior tendons after cast immobilization without neurectomy. After neurectomy the specific PH activity in the denervated gastrocnemius muscle increased by 215% (P less than 0.001). The specific GGT activity increased by 92-110% (P less than 0.01) in the denervated gastrocnemius, soleus, and tibialis anterior muscles. Elevation of the muscular HYP concentration by 118-170% (P less than 0.001) in the denervated muscles was observed. The PH, GGT, and HYP responses of the denervated muscles immobilized at a lengthened or shortened position during denervation atrophy did not generally differ significantly from those of the unfixed denervated ones. The specific PH and GGT activities of the disused tendons decreased by 62 (P less than 0.01) and 25% (P less than 0.001), respectively, in tendons immobilized in a chronically shortened position. The results suggest that denervation atrophy of skeletal muscle is associated with both an increased level of muscular collagen biosynthesis and with an increased muscular collagen concentration. The PH and GGT responses of the cast-immobilized tendons suggest adaptive changes in collagen biosynthesis of the disused tendon.

Experiments were designed to define the ability of retinoic acid to block the estrogen-induced metaplasia in the mouse anterior prostate gland (coagulating gland), and to elucidate some of the biochemical correlates of the actions and interactions of these two compounds. In castrated mice, the estrogen-induced metaplasia of epithelial cells consisted of multi-layered, nonpolarized cells, which accumulated to fill the lumen of the acini. Retinoic acid had no discernable effect on epithelial morphology of castrates, but significantly reduced the estrogen-induced metaplasia. Likewise, the estrogen-induced increases in the prostatic wet weight, ratio of ribonucleic to deoxy ribonucleic acids (RNA/DNA) and glycosyltransferases, as well as the decreases in 21,000 and 28,000 Mr soluble proteins were prevented by retinoic acid; the retinoid had no effect on these various parameters when administered alone to castrates. In contrast, the cyclic 3',5'-adenosine monophosphate (cAMP)-dependent phosphorylations at 16,000, 18,000, and 25,000 Mr and the activities of the type II cAMP-dependent kinase were uniformly reduced by both estradiol and retinoic acid. Tests of the action of the anti-estrogen LY-156758 on estrogen and retinoid effects showed that for those parameters on which retinoic acid was anti-estrogenic, LY-156758 was also anti-estrogenic. However, the qualitatively similar effects of retinoic acid and estradiol, which were confined to selected aspects of protein phosphorylation, were not antagonized by LY-156758. It is concluded that in the mouse anterior prostate, retinoic acid has both anti-estrogenic and estrogenic actions, and the latter may occur independently of the estrogen receptor.

The isolation and characterization of cellulase-containing membranes of Achlya ambisexualis Raper was attempted by differential and density gradient centrifugations. Maximum cellulase activity was found at an isopycnic density of 1.19 g/cm3, although some activity was found at other densities. A similar distribution of activity was shown by IDPase, ATPase, UDPG transferase, and by sedimentable carbohydrate. The coequilibration and steady enrichment of these activities during purification suggests their presence in a single type of subcellular particle. It was not possible to identify clearly the particle(s) in question from isolated fractions by electron microscopy, but when compared with the cytochemical localization of carbohydrate and IDPase in intact hypha, cytoplasmic vesicles with 150-mm diameters seem to be likely candidates.

Human skin fibroblasts from patients with Type IV glycogen storage disease, in which there is a demonstrable deficiency of glycogen branching enzyme, were shown to be able to synthesize [14C]glycogen containing [14C]glucose at branch points when sonicates containing endogenous glycogen synthase a were incubated with UDP[14C]glucose. The branch point content of the glycogen synthesized by the Type IV cells was essentially the same as that formed by normal cells, but the total synthetic capacity of the Type IV cells was lower. A new assay for the branching enzyme using glycogen synthase as the indicator enzyme has been developed. Using this assay it has been shown that the residual branching enzyme of affected children and of their heterozygote parents is less easily inhibited by an IgG antibody raised in rabbits against the normal human liver enzyme than is the branching enzyme of normal fibroblasts.

A fluorescence assay method for UDP-GlcNAc:glycopeptide beta 1-4 N-acetylglucosaminyltransferase (Gn T-III) has been developed involving a pyridylaminated sugar as a substrate. A fluorescent sugar chain, in which the reducing end of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc has been aminated with 2-aminopyridine, is incubated with an enzyme sample, and then the fluorescent product with a bisecting N-acetylglucosamine is separated by reverse-phase high performance liquid chromatography and quantitated with a fluorescence detector. This assay method was found to be sensitive enough for the detection of 0.1 pmol of a reaction product. This assay is a reliable alternative to the use of a radiolabeled substrate and can be used for assaying N-acetylglucosaminyltransferase activity in crude extracts of various rat tissues. The kinetic experiments were carried out using crude enzyme extracts from the rat kidney. The enzyme has a pH optimum of 6.25 and requires Mn2+. The Km values for UDP-GlcNAc and a sugar acceptor substrate were found to be 3.1 mM and 190 microM, respectively. The enzyme activity in the rat kidney was higher than those in the other tissues examined.

Two methods were used to purify the bifunctional extracellular enzyme sucrose: (1-6)- and (1-3)-alpha-D-glucan-6-alpha-D-glucosyltransferase (EC 2.4.1.5; dextransucrase) from continuous cultures of a serotype c strain of Streptococcus mutans. The first method, based on a previously published report, involved Sepharose 6B gel filtration and DEAE cellulose anion exchange chromatography. This resulted in a dextransucrase preparation with an apparent molecular mass of 162 kDa and a specific activity of 125 mg of glucan formed from sucrose h-1 (mg of protein)-1, at 37 degrees C. It was almost homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ratio of carbohydrate to protein was 0.14 and the recovery was 14% relative to the total glucosyltransferase activity in the original culture fluid. In the subsequently preferred method, hydroxyapatite-Ultrogel was used to purify dextransucrase with a 24% yield. The specific activity, 197 mg of glucan formed h-1 (mg of protein)-1, was the highest yet reported and this preparation contained less than 0.5 glucose-equivalent per subunit of molecular mass 162 kDa. Dextransucrase is therefore not a glycoprotein. Exogenous dextran stimulated activity, but was not essential for activity. The purified protein slowly degraded to multiple lower molecular mass forms during storage at 4 degrees C and 87% of the activity was lost after 20 days. The molecular mass of the most prominent, active degradation product was 140 kDa, similar to that of one of the multiple forms of dextransucrase detected in other laboratories. Preparations in which either the 140-kDa or the 162-kDa species predominated catalyzed the synthesis of a water-soluble glucan with sucrose alone, but catalyzed that of an insoluble glucan with sucrose and a high concentration of either (NH4)2SO4 or polyethylene glycol. The water-insoluble glucan was shown to lack sequences of 1,3-alpha-linked glycosyl residues typical of the insoluble glucan, mutan, which has been implicated in dental caries. We conclude that mutan is synthesized by the concerted action of two independent glucosyltransferases rather than by interconvertible forms of a single enzyme, as was proposed previously.

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.

The complete nucleotide sequence was determined for the Streptococcus sobrinus MFe28 gtfI gene, which encodes a glucosyltransferase that produces an insoluble glucan product. A single open reading frame encodes a mature glucosyltransferase protein of 1,559 amino acids (Mr, 172,983) and a signal peptide of 38 amino acids. In the C-terminal one-third of the protein there are six repeating units containing 35 amino acids of partial homology and two repeating units containing 48 amino acids of complete homology. The functional role of these repeating units remains to be determined, although truncated forms of glucosyltransferase containing only the first two repeating units of partial homology maintained glucosyltransferase activity and the ability to bind glucan. Regions of homology with alpha-amylase and glycogen phosphorylase were identified in the glucosyltransferase protein and may represent regions involved in functionally similar domains.

Metabolic activities of S mutans were selectively affected by Procion Blue, known to cause covalent bonds and stable cross links in relation to carbohydrate and protein. In this study, despite the reduction in extra polysaccharide, the level of activity of glucosyltransferase was not significantly changed from the control level and colonial morphology was not transformed from smooth to rough. The results of the investigation imply at least interference with formation of extra cellular polysaccharide, possibly in the bacterial cell wall.

We measured UDP-GlcNAc:Gal (beta 1-4) Glc (or GlcNAc) (beta 1-3) N-acetylglucosaminyltransferase activities in bovine (Holstein and Jersey cow) and human colostrums, and found in human colostrums sufficient activity to study the enzyme properties while not in bovine colostrums. The properties (requirements, pH optimum, acceptor specificity and Km values for lactose and N-acetyllactosamine) of the enzyme from human colostrum were very similar to those from human serum and urine. The reaction product was hydrolyzed by beta-N-acetylhexosaminidase, indicating that the N-acetylglucosaminyl residue was beta-linked to lactose. Methylation and hydrolysis of the reaction product from lactose [3H] labeled at the terminal galactose yielded 2, 4, 6-tri-O-methyl [3H] galactose. Thus the structure of the product was demonstrated to be GlcNAc (beta 1-3) Gal (beta 1-4) Glc (lacto-N-triose II). On the other hand, bovine sera contained N-acetylglucosaminyltransferase catalyzing the transfer of N-acetylglucosamine from UDP-GlcNAc to lactose. The enzyme activities were approximately 1/6-1/4 of that contained in human serum. The presence of (beta 1-3) N-acetylglucosaminyltransferase in human colostrum and its absence in bovine colostrums, apparently corresponds with the presence and absence of oligosaccharides containing lacto-N-triose II structure in colostrum.

Transglucosylative activity was examined in the serum and liver of mice with transplantable leukaemias, using following synthetic substrates: p-nitrophenyl-N-maltoside, as well as p-nitrophenyl-N-glucoside and maltose. In the serum of mice with leukaemias L 1210/ara-C and L 1210/CH3G as well as lymphoma AKSL-4 and plasmocytoma ADJ PC-5 there was observed a statistically significant decrease in the transglucosylative activity compared with the control group. Examination of extracts from the liver of mice proved increased transglucosylative activity at leukaemias L 1210/ara-C and AKSL-4. No significant changes in the transglucosylative activity were found at leukaemias L 1210/CH3G and ADJ PC-5.