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R B Trimble

Publications and source records attributed to R B Trimble.

At least 55 records · Page 3Linked to original sources
PubMed1986

An avian serum alpha 1-glycoprotein, hemopexin, differing significantly in both amino acid and carbohydrate composition from mammalian (beta-glycoprotein) counterparts.

We report here on physicochemical characteristics of chicken hemopexin, which can be isolated by heme-agarose affinity chromatography [Tsutsui, K., & Mueller, G. C. (1982) J. Biol. Chem. 257, 3925-3931], in comparison with representative mammalian hemopexins of rat, rabbit, and human. The avian polypeptide chain appears to be slightly longer (52 kDa) than the human, rat, or rabbit forms (49 kDa), and also the glycoprotein differs from the mammalian hemopexins in being an alpha 1-glycoprotein instead of a beta 1-glycoprotein. This distinct electrophoretic mobility probably arises from significant differences in the amino acid composition of the chicken form, which, although lower in serine and particularly in lysine, has a much higher glutamine/glutamate and arginine content, and also a higher proline, glycine, and histidine content, than the mammalian hemopexins. Compositional analyses and 125I concanavalin A and 125I wheat germ agglutinin binding suggest that chicken hemopexin has a mixture of three fucose-free N-linked bi- and triantennary oligosaccharides. In contrast, human hemopexin has five N-linked oligosaccharides and an additional O-linked glycan blocking the N-terminal threonine residue [Takahashi, N., Takahashi, Y., & Putnam, F. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2021-2025], while the rabbit form has four N-linked oligosaccharides [Morgan, W. T., & Smith, A. (1984) J. Biol. Chem. 259, 12001-12006]. In keeping with the finding of a simpler carbohydrate structure, the avian hemopexin exhibits only a single band on polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions, whereas the hemopexins of the three mammalian species tested show several bands.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1986

Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage.

N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.

Acetylglucosamine↗
PubMed1986

Structure of yeast external invertase Man8-14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy.

A series of high mannose oligosaccharides with the size range Man8-14GlcNAc was purified from Saccharomyces cerevisiae invertase, and the composition of each was determined by chemical analysis. Purity and composition were verified by 1H NMR spectroscopy at 500 MHz, and structures were assigned on the basis of chemical shifts in C1-H and C2-H protons of similarly substituted compounds of known structure. Such analyses showed that these invertase oligosaccharides were a homologous series of homogeneous compounds, each related to the next member by addition of 1 mol of mannose in a specific alpha-linked configuration. Man8GlcNAc purified from the total glycoprotein fraction of disrupted yeast was the smallest species found and had the same homogeneous structure as that previously reported for the Man8GlcNAc from invertase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Digestion of Man8-13GlcNAc species from invertase with Aspergillus satoi alpha 1,2-mannosidase provided products that were consistent with the structures assigned by 1H NMR as did fast atom bombardment-mass spectroscopy fragmentation analysis of the Man9,10GlcNAc oligosaccharides. These results lead to the proposal that Man8GlcNAc is the only trimming intermediate in Saccharomyces sp., and the remaining Man9-14GlcNAc oligosaccharides are biosynthetic intermediates which define the principal pathway of single-step mannose addition in the formation of the inner core of yeast mannan.

Aspergillus↗
PubMed1986

Characterization of cellular oligosaccharides from normal and cystic fibrotic fibroblasts using sequential endoglycosidase digestions.

A method was developed for obtaining detailed oligosaccharide profiles from [2-3H]mannose- or [6-3H]fucose-labeled cellular glycoproteins. The oligosaccharides were segregated first according to class, using endo-beta-N-acetylglucosaminidase H (Endo H) to release the high mannose species, and then with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F), which provided a complete array of complex oligosaccharide chains. The high mannose and complex oligosaccharides were fractionated subsequently according to net negative charge on QAE-Sephadex. High resolution gel filtration on TSK HW-40(S) resolved the neutral high mannose population into species of the type Man9-5 N-acetylglucosamine. Desialylation of the complex chains with neuraminidase allowed resolution of these oligosaccharides into their corresponding asialo bi-, tri-, and tetraantennary species. Fibroblasts from normal and cystic fibrosis cells were analyzed for differences in their glycosylation patterns using these techniques. Over 95% of the [2-3H]mannose-labeled glycoproteins were susceptible to the combined glycosidase digestions, but no difference in either the high mannose or complex oligosaccharides were observed. Nonetheless, the methodology developed in this study provides an important new approach for investigating oligosaccharides of different cell types and variants of the same type. Metabolic changes induced in cellular glycoproteins, as illustrated by use of the processing inhibitor swainsonine, demonstrated the versatility of this procedure for investigating questions relating to glycoprotein structure and enzyme specificity. Thus, by employing a variation of this method, it was possible to confirm the location of fucose in the core of PNGase F-released hybrid oligosaccharides by the subsequent release with Endo H of the disaccharide, fucosyl-N-acetylglucosamine.

Acetylglucosaminidase↗
PubMed1985

Comparative properties of amplified external and internal invertase from the yeast SUC2 gene.

Saccharomyces cerevisiae external and internal invertases have been amplified by introducing the normal and modified SUC2 genes into yeast multicopy plasmids, which were then used to transform a yeast strain resistant to repression by glucose. Amino acid compositional analysis of these enzymes, in addition to end group sequencing, confirmed the DNA sequence data of Taussig and Carlson (Taussig, R., and Carlson, M. (1983) Nucleic Acids Res. 11, 1943-1954), indicating that both enzymes were encoded in the same gene. Comparison of the properties of carbohydrate-containing external invertase and its nonglycosylated internal form revealed that although the carbohydrate did not appear to influence the conformation of the peptide backbone, as determined by circular dichroism analyses, its presence considerably enhanced the ability of guanidine HCl-denatured external invertase to be renatured relative to internal invertase. The Mr of the internal enzymes was found to be greatly dependent on pH with the enzyme being a monomer at pH 9.4, a dimer at pH 8.3, and an apparent octamer at pH 4.9.

Amino Acid Sequence↗
PubMed1985

Amplified expression of streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product.

The endo-beta-N-acetylglucosaminidase H (Endo H) gene from Streptomyces plicatus has been cloned into the Escherichia coli plasmid pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature 272, 128-132), thus placing expression of this gene under control of the strong lambda promoter pL. The construction, pKCE3, which includes a properly positioned E. coli ribosome binding site from the lac operon (Robbins, P.W., Trimble, R. B., Wirth, D.F., Hering, C., Maley, F. Maley, G. F., Das, R., Gibson, B.W., and Biemann, K. (1984) J. Biol. Chem. 259, 7577-7583), was used to transform an E. coli strain lysogenic for a lambda prophage containing a temperature-sensitive repressor. By shifting cultures of pKCE3 lysogens to 42 degrees C, the production of Endo H commenced and was linear for about 1 h. Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S. plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure. Although most of the cloned Endo H was secreted into the periplasmic space by E. coli, its 4 kDa leader sequence peptide (Robbins et al. (1984] was only partially removed during processing. As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S. plicatus. Despite the residual approximately 2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E. coli or from S. plicatus. Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants.

Acetylglucosaminidase↗
PubMed1985

Rheumatoid arthritis with bloody and cholesterol pleural effusion.

We describe a case of rheumatoid arthritis complicated by unilateral adhesive pleurisy and associated with bloody and cholesterol effusion in a 47-year-old man. Our case emphasizes that the diagnostician needs to consider rheumatoid disease in a differential diagnosis of bloody pleural effusion in patients with associated joint disease. Adequate histologic confirmation of the underlying pathology is required for definite diagnosis.

Arthritis, Rheumatoid↗
PubMed1984

Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H.

We report the DNA and primary amino acid sequences of the Streptomyces plicatus enzyme endo-beta-N-acetylglucosaminidase H. Peptide sequence information was derived from enzyme isolated from Streptomyces culture medium using a combination of mass spectrometric methods and conventional techniques, including Edman degradation and carboxypeptidase Y digestion. The DNA sequence was determined by analysis of the Endo-beta-N-acetylglucosaminidase H gene cloned into the Escherichia coli plasmid pBR322 (Robbins, P. W., Wirth , D. F., and Hering , C. (1981) J. Biol. Chem. 256, 10640-10644). The enzyme from Streptomyces medium is 271 (or 269) amino acids in length and has a ragged NH2-terminal sequence beginning primarily with Ala-Pro-Val or Ala-Pro-Ala-Pro-Val. DNA resection experiments as well as the DNA sequence itself suggest that a proenzyme or, more probably, " prepro " enzyme may be the primary product of translation. The long 42 (or 44) residue leader sequence of the preproenzyme shows striking similarities to leader sequences found on proteins secreted by Bacillus species. The leader sequence is partially removed by E. coli and, as reported previously, endo-beta-N-acetylglucosaminidase H made in E. coli appears in both the periplasmic space and in the cell.

Acetylglucosaminidase↗
PubMed1984

Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H.

The ability of endo-beta-acetylglucosaminidase H (Endo H) from Streptomyces plicatus to hydrolyze high-mannose oligosaccharides from glycoproteins is influenced by numerous factors, including the tertiary structure of the substrate glycoproteins, the amount of Endo H used, the time of incubation, and the presence or absence of reagents that affect protein configuration. Endo H levels below 10 to 20 milliunits/ml may incompletely hydrolyze oligosaccharides, regardless of the incubation time, because even though the enzyme remains active, it becomes trapped or sequestered and is unavailable. Endo H activity can be potentiated by first denaturing substrate glycoproteins in a 1.2-fold weight excess of sodium dodecyl sulfate prior to hydrolysis. However, low levels of Endo H are sensitive to inactivation by sodium dodecyl sulfate, with considerable activity being lost over 4 h when the unbound detergent concentration exceeds protein by 0.02% (0.2 mg/ml). Other denaturants such as the Tritons, the zwittergents, the Brij series, or octylglucoside do not enhance or inhibit Endo H removal of oligosaccharides, but the chaotropic salt sodium thiocyanate at 0.5 M enhances Endo H action on some glycoproteins, particularly bovine thyroglobulin. Under denaturing conditions, proteolytic contaminants are a potential problem. Addition of 1 mM phenylmethylsulfonyl fluoride to Endo H incubations completely inhibits the residual Endo H-associated protease(s). Furthermore, Endo H is unaffected by a wide range of proteolytic inhibitors that may be used to protect substrate glycoproteins.

Acetylglucosaminidase↗
PubMed1982

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing.

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).

Carbohydrate Conformation↗
PubMed1981

Revision of the structure for an endo-beta-N-acetylglucosaminidase H substrate using a novel modification of the Smith degradation.

(Man)5(GlcNAc)2Asn was shown in a previous study (Trimnble, R. B., Tarentino, A. L., Plummer, T. H., Jr., and Maley, F. (1978) J. Biol. Chem. 253, 4508-4511) to be hydrolyzed by alpha-mannosidase to Man alpha 1 leads to 6Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 2 leads to 4GlcNAc-Asn. The latter is the most effective substrate for endo-beta-N-acetylglucosaminidase H tested to date. By employing a new and highly sensitive modification of the Smith degradation, it is shown that this compound is in reality Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc beta 1eads to 4GlcNAc-Asn. The method entails the conversion of a glycosyl asparagine derivative to its corresponding dimethylaminonaphthyl sulfonyl analogue, which after periodate oxidation is treated directly with Dowex 50-H+ to eliminate the modified carbohydrate residues. The dansylated products, which are eluted from the resin with ammonium hydroxide, can be identified rapidly by thin layer chromatography.

Acetylglucosaminidase↗
PubMed1981

Ageing studies in rat liver. II. Patterns of cellular and cell-free protein synthesis from one to ten months of age.

Potential changes in liver protein biosynthesis were investigated in 1 to 10-month-old male Fischer F344 rats by isolation and translation of polysome-derived poly(A)-containing RNA and by incubation of isolated hepatocytes in Swim's S77 medium supplemented with [3H]leucine. Labeled protein products were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis followed by fluorography. The protein profiles revealed that the translation products of poly(A)-containing RNA derived from the livers of 1 -months-old animals lacked a major 22000 dalton band which was present in comparable profiles from livers of older animals. In parallel experiments, gel profiles of products from cultured hepatocytes isolated from 1-month-old animals were missing an 18000 dalton band which was seen in preparations from older animals. With livers from rats between 2 and 10 months of age, however, no age-related changes in the pattern of protein biosynthesis were detected either in the products of poly(A)-containing RNA translated in vitro or in the cellular and secreted proteins of intact hepatocytes. Furthermore, the polysome content (A1(260)cm/g tissue) was constant between 1 and 10 months of age, providing a constant amount of tissue was processed. A procedure for maximizing the yield of polysomes is described. These results suggest that the decline in protein synthesis observed in rat liver and in cultured hepatocytes during the first year of life is not due to changes in mRNA content or the variety of specific products synthesized, but results primarily from an overall decrease in protein biosynthesis, probably at the level of peptide chain elongation.

Animals↗
PubMed1980

Effect of glucosylation of lipid intermediates on oligosaccharide transfer in solubilized microsomes from Saccharomyces cerevisiae.

Glc3Man9GlcNAc2-P-P-dolichol and Man9GlcNAc2-P-P-dolichol isolated from Saccharomyces cerevisiae are substrates for the N-glycosylation of endogenous proteins in Triton X-100-solubilized yeast microsomes. The solubilized oligosaccharide transferase requires Mn2+ for activity; neither Mg2+ nor Ca2+ is an effective substitute. The pH optimum of the transfer reaction is between 6.5 and 7.5. Unlike animal systems, which utilize glucosylated oligosaccharide-lipid to a much greater extent than the unglucosylated species as a donor in the transferase rection, yeast extracts transfer more than 70% of Glc3Man9GlcNAc2 and Man9GlcNAc2 from their respective oligosaccharide-lipids to proteins. However, the rate of N-glycosylation in vitro is approximately 25-fold faster with Glc3Man9GlcNAc2-P-P-dolichol than with Man9GlcNAc2-P-P-dolichol. The apparent Km value for the glucosylated species is 75 nM, while that for the unglucosylated glycolipid is 55 nM.

Cations, Divalent↗
PubMed1980

Characterization of large oligosaccharide-lipids synthesized in vitro by microsomes from Saccharomyces cerevisiae.

Conditions are described for optimizing the synthesis of large oligosaccharide-lipids in microsomal preparations from Saccharomyces cerevisiae. On incubating microsomes, with GDP-[14C]Man, the major product obtained was Man9GlcNAc2-P-P-dolichol, but when both GDP-[14C]Man and UDP-[3H]Glc were present in the incubation mixture about half of the Man9GlcNAc2 was elongated to Glc3Man9GlcNAc2-P-P-dolichol. Unlike particulate fractions from mammalian systems, little glucosylation of the yeast microsomal oligosaccharide-lipid was obtained when the concentration of UDP-Glc was less than 10 microM, but the synthesis of this product could be maximized by raising the concentration of UDP-Glc to 50 microM. Analysis of the yeast Man9GlcNAc2 species confirmed that 8 of the 9 mannose residues could be released with alpha-mannosidase, while the remaining mannosyl residue was in the core trisaccharide, Manbeta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc. Treatment of Glc3Man9GlcNAc2 with alpha-mannosidase released 5 of 9 mannose residues and yielded Glc3Man4GlcNAc2. This product appeared to be identical with that obtained in parallel experiments with double labeled oligosaccharide-lipid synthesized in oviduct microsomes. Streptomyces plicatus endo-beta-N-acetylglucosaminidase H (Endo-H) treatment of yeast microsomal glycoproteins that were labeled with sugar nucleotides established that 15% of the label was associated with N-linked oligosaccharides. The remaining labeled sugars were released with alkali, indicating that they were linked to serine or threonine. Based on the size and distribution of [3H]glucose and [14C]mannose in the Endo-H-released oligosaccharides, it was concluded that Glc3Man9GlcNAc2 was the primary species transferred to proteins in the yeast system.

Glycolipids↗