SEARCH · PubMed Health

Results for “GLUCOSYLTRANSFERASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed1991

Flavonol ring B-specific O-glucosyltransferases: purification, production of polyclonal antibodies, and immunolocalization.

UDP-glucose: flavonol 2'- and 5'-O-glucosyltransferases (E.C.2.4.1.-) from leaves of Chrysosplenium americanum were copurified to apparent homogeneity by successive chromatography on Sephacryl S-200, UDP-glucuronic acid-agarose, Mono P, Superose 12, and Mono Q columns. Both enzymes have similar properties except for their substrate specificity and stability (J. Chromatogr. 388, 235, 1987). The purified protein was used as the source of antigen to produce polyclonal antibodies in rabbits. In situ localization of the O-glucosyltransferases was studied by applying a postembedding immunogold labeling technique on ultrathin sections of Lowicryl K4M- and LR White-embedded tissues. Postfixation with osmium tetroxide followed by embedding in LR White resulted in good preservation of membrane ultrastructure, although protein antigenicity was greatly reduced. Leaf sections embedded in Lowicryl K4M had an extracted appearance; however, they retained a high degree of protein antigenicity revealing the deposition of gold particles in the periplasmic region of cells. Considering the compromise chosen in this study to retain antigenicity over preservation of membrane ultrastructure, the results suggest that the "easily solubilized" O-glucosyltransferases of C. americanum may actually be associated with vesicle-like structures and cytoplasmic membranes.

Antibody Formation↗

PubMed1985

UDPglucose-ceramide glucosyltransferase from porcine submaxillary glands is associated with the Golgi apparatus.

Subcellular distribution of pig submaxillary gland UDPglucose-ceramide glucosyltransferase (EC 2.4.1.80), the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The results presented strongly suggest that in pig submaxillary gland, the transfer of glucose on endogenous or exogenous ceramides takes place in the Golgi apparatus: the specific activity of UDPglucose-ceramide glucosyltransferase increased in parallel with the activity of a known marker of the Golgi apparatus, UDPgalactose-ovomucoid galactosyltransferase. The specific activity of the glucosyltransferase was 18-times higher in the purified Golgi membranes than in the postnuclear supernatant and the yield was over 30%. An apparent Km of 22 microM for UDPglucose and 54 microM for ceramides was determined. Maximal glucosylation of endogenous ceramides was achieved at pH 6.5 in the presence of NADH (1 mM) as inhibitor of pyrophosphatases and with Mn2+ (5 mM). It was found that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) is an efficient activator for the glucosylation of exogenous ceramides.

Animals↗

PubMed1980

The distribution of collagen:glucosyltransferase in human blood cells and plasma.

Collagen:glucosyltransferase (UDP-glucose:5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) present in platelets, plasma, granulocytes and lymphocytes has been compared in order to determine whether the platelet enzyme has unique properties or distribution which would support a possible role in platelet-collagen interaction. The enzyme was purified 5400-fold from human plasma and 4400 from human platelets. The two enzymes were similar in terms of Km values for reacting with galactosylhydroxylysine (2.75 mM) and UDPglucose (7.4 microM), optimal Mn2+ concentration (10--15 mM) and pH optimum (7.0). The enzyme was not detectable in red cells. As in platelets, the enzyme was detected in membrane-bound and soluble forms in lymphocytes and granulocytes. Identical mobilities were obtained after elution following polyacrylamide gel electrophoresis of the enzymes from plasma, platelets, granulocytes and lymphocytes. These studies do not support a unique role for the collagen:glucosyltransferase of platelets in platelet-collagen interaction.

Blood Platelets↗

PubMed1983

Mechanism of synthesis of D-glucans by D-glucosyltransferases from Streptococcus mutans 6715.

Two glucosyltransferases from Streptococcus mutans 6715 were purified and separated. One of the glucosyltransferases synthesized an insoluble glucan, and the other, a soluble glucan. The enzymes were immobilized on Bio-Gel P-2 beads, and the mechanism of glucan synthesis was studied by pulse and chase techniques with 14C-sucrose. Label was associated with the immobilized enzymes. The label could be quantitatively released by heating at pH 2. Analysis of the labeled products from the pulse experiment showed labeled glucose and labeled glucan; the chase experiment showed labeled glucan and a significant decrease in labeled glucose. The glucans from the pulse and the chase experiments were separated from glucose by chromatography on Bio-Gel P-6. They were reduced with sodium borohydride, and the products hydrolyzed with acid. Analysis of the labeled products from the reduced and hydrolyzed, pulsed glucans showed labeled glucose and labeled glucitol; label in the glucitol was greatly decreased in the chase experiment. These experiments showed that glucose and glucan were covalently attached to the active site of the enzymes during synthesis, and that the glucose was being transferred to the reducing end of the glucan chain. A mechanism for the synthesis of the glucans is proposed in which there are two catalytic groups on each enzyme that holds glucosyl and glucanosyl units. During synthesis, the glucosyl and glucanosyl units alternate between the two sites, giving elongation of the glucans from the reducing end. The addition of increasing amounts of B-512F dextran to the insoluble-glucan-forming glucosyltransferase produced a decrease in the proportion of insoluble glucan formed and a concomitant increase in a soluble glucan. The total amount of glucan synthesized (soluble plus insoluble) was increased 1.6 times over the amount of insoluble glucan formed when no exogenous dextran was added. It is shown that the addition of B-512F dextran affects the solubility of the synthesized alpha-(1 to 3)-glucan by accepting alpha-(1-3)-glucan chains at various positions along the dextran chain, to give a soluble, graft polymer.

Enzymes, Immobilized↗

PubMed1977

Age-related changes in human skin collagen galactosyltransferase and collagen glucosyltransferase activities.

Collagen galactosyltransferase and collagen glucosyltransferase activities were assayed in human skin specimens of about 100 mg wet weight. The assay of the glucosyltransferase activity was found to be highly specific. The assay of the galactosyltransferase activity was somewhat less specific, but there was no difference in specificity between the foetal and adult human skin samples. The activities of the two collagen glycosyltransferases in human skin extract were found to vary with age, being highest in foetal skin, and higher in the skin of young children that in that of adults. The galactosyltransferase and glucosyltransferase activities in foetal skin were respectively about 4 times and 6 times those in adult skin. The magnitudes of the changes with age in the two collagen glycosyltransferase activities were smaller than those occurring in the activities of the two other intracellular enzymes of collagen biosynthesis namely prolyl and lysyl hydroxylase. This difference suggests that the four intracellular enzyme activities of collagen biosynthesis are not regulated in an identical manner.

Adolescent↗

PubMed1985

Elevated serum galactosylhydroxylysyl glucosyltransferase, a collagen synthesis marker, in fibrosing lung diseases.

The activity of galactosylhydroxylysyl glucosyltransferase, an enzyme catalyzing collagen biosynthesis, was measured in the sera of 101 patients with various pulmonary diseases to study whether detectable enzyme amounts are liberated into the serum from the lung tissue, and whether this is associated with the development of lung fibrosis. Increased serum galactosylhydroxylysyl glucosyltransferase activity was found in all the patients with progressive pulmonary fibrosis and in about half of the patients with acute stages of farmer's lung and infectious pneumonia. In one third of the patients with stage I sarcoidosis the serum enzyme activity was slightly increased, whereas in bronchial asthma and chronic bronchitis the values were mostly within the normal range. In conclusion, elevated serum enzyme activity was demonstrated in connection with those respiratory diseases in which pulmonary fibrosis was already verifiable or relatively often develops later. Measurements of serum galactosylhydroxylysyl glucosyltransferase may, thus, be useful in evaluating actual lung collagen synthesis in human pulmonary diseases.

Adolescent↗

PubMed2004

Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity.

Mutans streptococci are oral bacteria with a key role in the initiation of dental caries, because their glucosyltransferases synthesize polysaccharides from sucrose that allow them to colonize the tooth surface. Among the strategies to prevent dental caries that are being investigated are (1) the inhibition of bacterial growth of mutans streptococci or (2) the inhibition of glucosyltransferases involved in polysaccharide formation. Pure fatty acid esters of sucrose, maltose and maltotriose were synthesized by an enzyme-catalyzed process and tested as inhibitors of two glucosyltransferases of great homology, those from Streptococcus sobrinus and Leuconostoc mesenteroides NRRL B-512F. In spite of having their nonreducing end glucose blocked at 6-OH, they did not inhibit dextran synthesis. However, their effect on the growth of S. sobrinus in the solid and liquid phase was notable. 6-O-Lauroylsucrose, 6'-O-lauroylmaltose and 6"-O-lauroylmaltotriose at 100 microg/mL showed complete inhibition of S. sobrinus in agar plates. Consequently, these nontoxic derivatives are very promising for inclusion in oral-hygiene products aimed at disrupting plaque formation and preventing caries.

Agar↗

PubMed2006

Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris.

Two full-length cDNAs encoding flavonoid-specific glucosyltransferases, UGT73A4 and UGT71F1, were isolated from a cDNA library of Beta vulgaris (Amaranthaceae) cell suspension cultures. They displayed high identity to position-specific betanidin and flavonoid glucosyltransferases from Dorotheanthus bellidiformis (Aizoaceae) and to enzymes with similar substrate specificities from various plant families. The open reading frame of the sequences encode proteins of 476 (UGT73A4) and 492 (UGT71F1) amino acids with calculated molecular masses of 54.07kDa and 54.39kDa, and isoelectric points of 5.8 and 5.6, respectively. Both enzymes were functionally expressed in Escherichia coli as His- and GST-tagged proteins, respectively. They exhibited a broad substrate specificity, but a distinct regioselectivity, glucosylating a variety of flavonols, flavones, flavanones, and coumarins. UGT73A4 showed a preference for the 4'- and 7-OH position in the flavonoids, whereas UGT71F1 preferentially glucosylated the 3- or the 7-OH position. Glucosylation of betanidin, the aglycone of the major betacyanin, betanin, in B. vulgaris was also observed to a low extent by both enzymes. Several O-glycosylated vitexin derivatives isolated from leaves of young B. vulgaris plants and rutin obtained from B. vulgaris tissue culture are discussed as potential endogenous products of UGT73A4 and UGT71F1. The results are analyzed with regard to evolution and specificity of plant natural product glucosyltransferases.

Base Sequence↗

PubMed1977

Comparison of the action of glucoamylase and glucosyltransferase on D-glucose, maltose, and malto-oligosaccharides.

The action patterns of glucoamylase (amyloglucosidase) and glucosyltransferase (transglucosylase) on D-[1-14C]glucose, [1-14C]maltose, and [1-14C]malto-oligosaccharides (labeled at position 1 of the D-glucose group at the reducing end) have been investigated by paper-chromatographic and oligosaccharide-mapping techniques. Under the conditions of the experiments, the extent of conversion of D-glucose and of maltose into new oligosaccharides was 2.2 and 1.9% with glucoamylase, and 5.7 and 33% with glucosyltransferase. The major oligosaccharides produced by both enzymes were isomaltose (6-O-alpha-D-glucopyranosyl-alpha-D-glucose), panose (O-alpha-D-glucopyranosyl (1 leads to 6)-O-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-glucose), and nigerose (3-O-alpha-D-glucopyranosyl-alpha-D-glucose). The glucosyltransferase also synthesized oligosaccharides from malto-oligosaccharides of higher molecular weight to yield compounds having alpha-(1 leads to 6)-linked D-glucosyl groups at the non-reducing ends. Glucoamylase exhibited little, if any, such activity on malto-oligosaccharides.

Glucan 1,4-alpha-Glucosidase↗

PubMed1980

Specific inhibition of glucosyltransferase of Streptococcus mutans.

Clinical dextran, partially oxidized with sodium periodate, acts as a potent inhibitor of the extracellular glucosyltransferases of several cariogenic strains of oral Streptococcus mutans. Preincubation with oxidized dextran resulted in a rapid loss of up to 80% of the ability of the enzyme preparation to synthesize polysaccharide from sucrose, but there was no loss of enzyme activity when the oxidized dextrans were reduced with sodium borohydride before preincubation with enzyme. The presence of unoxidized clinical dextran during the preincubation period afforded the enzymes protection against inhibition by partially-oxidized dextran, but clinical dextran did not readily restore activity when it was added after incubation of the enzyme with oxidized polysaccharide. Fructosyltransferase, and glycogen and starch phosphorylase, activities were not inhibited by oxidized dextran, and the bacterial glucosyltransferases were not inhibited by partially oxidized glycogen and amylose. It is proposed that the potent and specific inhibition of glucosyltransferase by oxidized dextran results from the interaction of dialdehyde groups with reactive functional groups close to the dextran-binding site of the enzyme.

Animals↗

PubMed1998

Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid.

Two tobacco genes (TOGT) with homology to glucosyltransferase genes known to be induced by salicylic acid (SA) also responded rapidly to a fungal elicitor or to an avirulent pathogen. SA, although an efficient inducer, was shown not to be essential in the signal transduction pathway regulating TOGT gene expression during the resistance response. Recombinant TOGT proteins produced in Escherichia coli exhibited low, but significant, glucosyltransferase activity towards SA, but very high activity towards hydroxycoumarins and hydroxycinnamic acids, with glucose esters being the predominant products. These results point to a possible important function in defense of these glucosyltransferases in conjugating aromatic metabolites prior to their transport and cross-linking to the cell wall.

Benzoates↗

PubMed1992

Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.

It was found, in cell-free assays, that the Man8GlcNAc2 and Man7GlcNAc2 isomers having the mannose unit to which the glucose is added were glucosylated by the rat liver glucosyltransferase at 50 and 15%, respectively, of the rate of Man9GlcNAc2 glucosylation. This indicates that processing by endoplasmic reticulum mannosidases decreases the extent of glycoprotein glucosylation. All five different glycoproteins tested (bovine and porcine thyroglobulins, phytohemagglutinin, soybean agglutinin, and bovine pancreas ribonuclease B) were found to be poorly glucosylated or not glucosylated unless they were subjected to treatments that modified their native conformations. The effect of denaturation was not to expose the oligosaccharides but to make protein determinants, required for enzymatic activity, accessible to the glucosyltransferase because (a) cleavage of denatured glycoproteins by unspecific (Pronase) or specific (trypsin) proteases abolished their glucose acceptor capacities almost completely except when the tryptic peptides were held together by disulfide bonds and (b) high mannose oligosaccharides in native glycoproteins, although poorly glucosylated or not glucosylated, were accessible to macromolecular probes as concanavalin A-Sepharose, endo-beta-N-acetylglucosaminidase H, and jack bean alpha-mannosidase. In addition, denatured, endo-beta-N-acetylglucosaminidase H deglycosylated glycoproteins were found to be potent inhibitors of the glucosylation of denatured glycoproteins. It is suggested that in vivo only unfolded, partially folded, and malfolded glycoproteins are glucosylated and that glucosylation stops upon adoption of the correct conformation, a process that hides the protein determinants (possibly hydrophobic amino acids) from the glucosyltransferase.

Animals↗

PubMed1997

UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli.

Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067-1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.

Amino Acid Sequence↗

PubMed2002

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmentally regulated expression.

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-0-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.

Amino Acid Sequence↗

PubMed2004

Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance.

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.

Coumarins↗

PubMed2002

Effects of apigenin and tt-farnesol on glucosyltransferase activity, biofilm viability and caries development in rats.

Propolis, a resinous hive product secreted by Apis mellifera bees, has been shown to reduce the incidence of dental caries in rats. Several compounds, mainly polyphenolics, have been identified in propolis. Apigenin and tt-farnesol demonstrated biological activity against mutans streptococci. We determined here their effects, alone or in combination, on glucosyltransferase activity, biofilm viability, and development of caries in rats. Sprague-Dawley rats were infected with Streptococcus sobrinus 6715 and treated topically twice daily as follows: (1) tt-farnesol, (2) apigenin, (3) vehicle control, (4) fluoride, (5) apigenin +tt-farnesol, and (6) chlorhexidine. Apigenin (1.33 mM) inhibited the activity of glucosyltransferases in solution (90-95%) and on the surface of saliva-coated hydroxyapatite beads (35-58%); it was devoid of antibacterial activity. tt-Farnesol (1.33 mM) showed modest antibacterial activity against biofilms and its effects on glucosyltransferases were minimal. The incidence of smooth-surface caries was significantly reduced by apigenin +tt-farnesol (60%), fluoride (70%), and chlorhexidine (72%) treatments compared to control (P < 0.05).

Analysis of Variance↗

PubMed2003

Remote glucosyltransferase-microparticle vaccine delivery induces protective immunity in the oral cavity.

Intranasally administered dental caries vaccines show significant promise for human application. Alternate mucosal routes may be required, however, to induce caries-protective salivary IgA antibody in children with respiratory diseases. Since rectal mucosa contains inductive lymphoid tissue, we hypothesized that the rectal route could be used to induce salivary immunity to mutans streptococcal glucosyltransferase (GTF), resulting in protective immunity to experimental dental caries. We first explored the ability of glucosyltransferase, incorporated into polylactide-co-glycolide (PLGA) microparticles (MP), and administered rectally together with mucosal adjuvant, to induce a salivary IgA antibody response. Groups of Sprague-Dawley rats (6/group) were immunized rectally on days 0, 7, 14 and 21 with a) GTF-MP alone, b) GTF-MP with cholera toxin, c) GTF-MP with detoxified mutant Escherichia coli toxin (dLT), or d) sham immunized with PLGA and cholera toxin. An additional group was immunized intranasally with GTF-MP alone. Saliva and nasal washes of all intranasally immunized rats contained IgA antibody to glucosyltransferase on day 28. Salivary IgA antibody was also detected in 7/12 rats rectally immunized with GTF-MP and cholera toxin or dLT, although responses were lower than those obtained by intranasal immunization. Most fecal extracts from rectally delivered GTF-MP plus cholera toxin or dLT rats contained IgA antibody to GTF-MP. Low levels of fecal IgA antibody were detected in 3/6 intranasally immunized rats and 2/6 rats rectally immunized with GTF-MP alone. We then examined the extent to which salivary IgA antibody induced by the rectal route could be protective. At 25, 31 and 38 days of age, two groups of female Sprague-Dawley rats (13/group) were rectally immunized with GTF-MP and cholera toxin or with empty microparticles and cholera toxin (sham group). A third group was intranasally immunized with GTF-MP alone. After demonstrating salivary IgA responses to GTF in most GTF-immunized rats, all animals were infected with streptomycin-resistant Streptococcus sobrinus and placed on diet 2000. After 79 days of infection, total caries on molar surfaces were lower in both rectally (7.9 +/- 1.0) and intranasally (7.1 +/- 0.9; P < 0.0.03) immunized groups compared with the sham-immunized group (11.9 +/- 1.6). Smooth surface caries were significantly lower (P < 0.05) in both rectally and intranasally immunized groups. These results support the interconnectedness of the mucosal immune system and indicate that rectal immunization with GTF-MP, together with adjuvant, or intranasal immunization with GTF-MP alone, can induce protective levels of salivary antibody in rats.

Administration, Rectal↗

PubMed1975

Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells.

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.

Animals↗