[53] β-Galactosidase from bovine testes

Distler, Jack J.; Jourdian, George W.

doi:10.1016/0076-6879(78)50055-4

Cited by 61 publications

(33 citation statements)

References 4 publications

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“…Digestions with jack bean ␤-hexosaminidase, jack bean ␤-galactosidase, and coffee bean ␣-galactosidase were all negative. However, bovine testes ␤-galoactosidase successfully digested all of structure B to structure C. Resistance to jack bean ␤-galactosidase and sensitivity to bovine testes ␤-galoactosidase is consistent with the presence of a non-reducing terminal ␤Gal residue linked 1-3 to a subterminal residue (Li et al, 1975;Distler and Jourdain, 1978). Thus neutral glycan structure B appears to differ from structure C by the presence of a non-reducing terminal 1-3-linked ␤Gal residue.…”

Section: Resultsmentioning

confidence: 99%

Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase

Brewis

Ferguson

Mehlert

et al. 1995

Journal of Biological Chemistry

123

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The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal membrane dipeptidase (EC 3.4.13.19) were determined following deamination and reduction by a combination of liquid chromatography, exoglycosidase digestions, and methylation analysis. The glycan core was found to exhibit microheterogeneity with three structures observed for the porcine GPI anchor: Man␣1-2Man␣1-6Man␣1-4GlcN (29% of the total population), Man␣1-2Man␣1-6(GalNAc␤1-4)Man␣1-4GlcN (33%), and Man␣1-2Man␣1-6(Gal␤1-3GalNAc␤1-4)Man␣1-4GlcN (38%). The same glycan core structures were also found in the human anchor but in slightly different proportions (25, 52, and 17%, respectively). Additionally, a small amount (6%) of the second structure with an extra mannose ␣(1-2)-linked to the non-reducing terminal mannose was also observed in the human membrane dipeptidase GPI anchor. A small proportion (maximally 9%) of the porcine GPI anchor structures was found to contain sialic acid, probably linked to the GalNAc residue. The porcine GPI anchor was found to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion electrospray-mass spectrometry revealed the presence of exclusively diacyl-phosphatidylinositol (predominantly distearoyl-phosphatidylinositol with a minor amount of stearoyl-palmitoyl-phosphatidylinositol) in the porcine membrane dipeptidase anchor. Porcine membrane dipeptidase was digested with trypsin and the C-terminal peptide attached to the GPI anchor isolated by removal of the other tryptic peptides on anhydrotrypsin-Sepharose. The sequence of this peptide was determined as ThrAsn-Tyr-Gly-Tyr-Ser, thereby identifying the site of attachment of the GPI anchor as Ser 368. This work represents a comprehensive study of the GPI anchor structure of porcine membrane dipeptidase and the first interspecies comparison of mammalian GPI anchor structures on the same protein. Glycosyl-phosphatidylinositol (GPI)1 membrane anchors are present in organisms at most stages of eukaryotic evolution, including protozoa, yeast, slime molds, invertebrates, and vertebrates, and are found on a diverse range of proteins. They are primarily responsible for the anchoring of cell-surface proteins in the plasma membrane and may be considered as an alternative to the hydrophobic transmembrane polypeptide anchor of integral membrane proteins. Many other functions have been proposed for GPI anchors, including roles in intracellular sorting, transmembrane signaling, and the novel endocytic process of potocytosis. A GPI anchor might also allow the protein to associate in membrane microdomains or be selectively released from the cell-surface by phospholipases. These functions, as well as the structure, biosynthesis, and distribution of GPI anchors have been extensively reviewed (Ferguson and

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Section: Resultsmentioning

confidence: 99%

Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase

Brewis

Ferguson

Mehlert

et al. 1995

Journal of Biological Chemistry

123

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“…Aggregation of Man6P-containing monomers could produce a multivalent ligand. Although the molecular weight of the purified bovine enzyme corresponds to that expected for a monomer (27), changes in the aggregation state of mammalian 3-galactosidases have been amply documented (28)(29)(30)(31). Of particular importance was the report that high molecular weight aggregates were enriched in fractions eluting at higher salt concentrations from DEAEcellulose (28).…”

Section: Discussionmentioning

confidence: 99%

Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts.

Natowicz¹,

Maggie²,

Lowry³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

208

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Human fB-glucuronidase (fl-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycopr tein lysosomal hydrolases, is subject to receptor-mediated endocytosis by fibroblasts. Prior work demonstrated charge heterogeneity in P-glucuronidase and showed that high-uptake forms are more acidic than slowly internalized forms. Considerable indirect evidence implicated mannose 6-phosphate as an essential part of the recognition marker on high-uptake enzyme forms. Here we report the purification of fl-glucuronidase from human spleen and demonstrate enzymatically that mannose 6-phosphate is released on acid hydrolysis of pure enzyme. Furthermore, the mannose 6-phosphate content of the enzyme varies directly with its susceptibility to pinocytosis by fi roblasts. Enzyme forms resolved by CM-Sephadex chromatography differed over an 18-fold range in uptake rate and in mannose 6-phosphate content. The most acidic forms had 4.4 mol of mannose 6-phosphate per mol of enzyme. The mannose 6-phosphate was released from the enz me b treatment with endoglycosidase H with concomitant loss of susceptibility to adsorptive endocytosis. Thus, these studies provide direct evidence that mannose 6-phosphate is present on high-uptake enzyme forms,.that it is present in the recognition marker for uptake, and that it is present on oligosaccharide that is released by endoglycosidase H.

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“…Generalized gangliosidosis skin fibroblasts, strain KD, were obtained from the Repository for Mutant Human Cell Strains (Montreal Children's Hospital). Bovine testicular f3-galactosidase was prepared and assayed as described (17). Assimilation and inhibition of assimilation of f3-galactosidase were tested by the procedure of Hieber et al (2).…”

Section: Methodsmentioning

confidence: 99%

Identification of mannose 6-phosphate in glycoproteins that inhibit the assimilation of β-galactosidase by fibroblasts

Distler

Hieber

Sahagian

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

104

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Observations that extracellular lysosomal enzymes are assimilated by fibroblasts and serve to correct genetic lysosomal enzyme deficiencies has stimulated interest in the mechanisms by which the enzyme assimilation occurs. It has been proposed that the assimilation (absorptive endocytosis or uptake) of lysosomal enzymes by fibroblasts is initiated by cellular "recognition" of a carbohydrate-containing "marker" bound to the lysosomal enzymes (1). Studies in this laboratory implicated enzyme-bound mannosyl residues in the assimilation of bovine testicular f-galactosidase (f-D-galactoside galactohydrolase, EC 3.2.1.23) (2). Recently, Kaplan et al. (3) suggested that phosphate, probably as mannose 6-phosphate, participated in the assimilation of platelet 3-glucuronidase by fibroblasts. This conclusion was based on the strong inhibition of the assimilation of (-glucuronidase by free mannose 6-phosphate and the lack of assimilation of 3-glucuronidase when the enzyme was pretreated with alkaline phosphatase. Subsequently, similar experiments implicated mannose phosphate in the assimilation of other lysosomal enzymes by fibroblasts. These enzymes included highly purified a-L-iduronidase (4) and 3-galactosidase (5) as well as several other partially purified lysosomal enzymes from platelets (6) and urine (7). These observations strongly suggested that mannose 6-phosphate was a normal constituent of many lysosomal enzymes. However, direct evidence of the occurrence of mannose 6-phosphate in lysosomal enzymes has not been presented. Furthermore, the type of carbohydrate unit of these glycoproteins that may contain mannose 6-phosphate and constitute the "recognition marker" was not apparent.Elucidation of the structural features that characterize the recognition marker has been restricted by the limited amounts of highly purified enzymes. Fortunately, glycoproteins prepared from urine (4), platelets (6), and bovine testes (2, 5) furnish abundant materials which, based on inhibition studies, bear recognition markers similar to those of lysosomal enzymes. * These glycoprotein inhibitors were found to contain several lysosomal enzymes that may compete for the same cell receptors (4, 5). MATERIALS AND METHODSDiphenylcarbamyl chloride-treated (DCC) trypsin, soybean trypsin inhibitor, jack bean ca-mannosidase, and mannose 6-phosphatet were obtained from Sigma. Potato acid phosphatase, glycoaldehyde phosphate diethylacetal, and DL-glyceraldehyde-3-phosphate diethylacetal were obtained from Calbiochem; acetals were hydrolyzed as recommended by the supplier. Concanavalin A (Con A)-Sepharose was obtained from Pharmacia. Sodium borol3H]hydride [specific activity, 422 mCi/mmol (1 Ci = 3.7 X 1010 becquerels)] was obtained from Amersham. Radioactivity was measured by liquid scintillation spectrometry on squares of Whatman 3 MM paper immersed in a toluene liquid scintillation cocktail. Methyl 3-mannopyranoside was prepared from methyl tetra-O-acetyl-3-mannopyranoside. The latter compound and synthetic a-mannopyranosyl-(1l-2)-m...

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[53] β-Galactosidase from bovine testes

Cited by 61 publications

References 4 publications

Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase

Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase

Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts.

Identification of mannose 6-phosphate in glycoproteins that inhibit the assimilation of β-galactosidase by fibroblasts

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