[49] α-d-Mannosidase from rat liver lysosomes

Opheim, Dennis J.; Touster, Oscar

doi:10.1016/0076-6879(78)50051-7

Cited by 31 publications

(41 citation statements)

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“…Enzyme and Protein Assays-The human lysosomal ␣-mannosidase was assayed using p-nitrophenyl-␣-D-mannopyranoside (pNP-Man) as substrate as described previously (6). Enzyme assays of crude culture medium or of purified enzyme preparations were performed in a 50-l total reaction volume containing 0.1 M sodium acetate (pH 4.5), 4 mM pNP-Man, and 25 l of enzyme solution at 37°C for 2 h or as specified.…”

Section: Methodsmentioning

confidence: 99%

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Liao

Lal

Moremen

1996

Journal of Biological Chemistry

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We have cloned and expressed two cDNAs encoding the human lysosomal ␣-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells. The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from ␣-mannosidosis fibroblasts, and p-nitrophenyl-␣-D-mannopyranoside substrates. In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, K m , and V max characteristic of the enzyme purified previously from human liver. The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing. No ␣-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme. Antiserum raised to the recombinant product of the 3-kb ␣-mannosidase cDNA immunoprecipitated lysosomal ␣-mannosidase activity from human fibroblast extracts. Northern blots identified a 3-kb RNA transcript in all human tissues tested, including ␣-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues. Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19. Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal ␣-mannosidase (Nebes, V. L., and Schmidt, M. C. (1994) Biochem. Biophys. Res. Commun. 200, 239 -245) and several differences were found relative to the functional lysosomal ␣-mannosidase encoded by the 3-kb spleen cDNA.The lysosomal catabolism of N-glycans on mammalian glycoproteins occurs through the sequential exoglycosidase digestion of oligosaccharides from the non-reducing terminus down to the carbohydrate-peptide core region (1). Included among the hydrolytic activities responsible for this oligosaccharide degradation is a broad specificity exo-␣-mannosidase activity (EC 3.2.1.24) that catalyzes the hydrolysis of ␣1,2-, ␣1,3-, and ␣1,6-mannoside linkages present in complex, hybrid, and high mannose Asn-linked glycans (2). This enzyme is distinguished from other ␣-mannosidase activities by a combination of a low pH optimum (pH 4.5), a broad natural substrate specificity, activity toward the synthetic substrate p-nitrophenyl-␣-mannoside, and sensitivity to inhibition by swainsonine (3, 4). An enzyme activity with these characteristics has been identified and purified from several species sources including Dictyostelium discoideum (5), and a variety of mammalian tissues (6 -11). Metabolic radiolabeling studies have indicated that the human enzyme is initially synthesized as a polypeptide of ϳ110 kDa that is sub...

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Section: Methodsmentioning

confidence: 99%

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Liao

Lal

Moremen

1996

Journal of Biological Chemistry

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“…It is now evident that almost all lysosomal enzymes are glycoproteins. Rat liver lysosomal 8-glucuronidase [25] and cc-L-fucosidase [26] were shown to contain five sugars; mannose, glucosamine, glucose, galactose and fucose. Therefore, the carbohydrate composition of cathepsin D is of special interest, and in order to clarify the role of carbohydrate moieties in the translocation of lysosomal enzymes, cathepsin D purified from rat spleen was also subjected to chemical analysis.…”

mentioning

confidence: 99%

“…We also determined the carbohydrate composition of cathepsin D-I and compared the carbohydrate moiety with those of other lysosomal enzymes [25,26].…”

mentioning

confidence: 99%

Cathepsin D of Rat Spleen Affinity Purification and Properties of Two Types of Cathepsin D

Yamamoto

Katsuda

Himeno

et al. 1979

European Journal of Biochemistry

102

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Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin-Sepharose 4B and concanava h -A -Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85 % of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15 %), termed cathepsin D-11, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pl: 4.2, 4.9, 6.1 and 6.5) and three (pl: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corrcsponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6 % carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8 : 2 : 1 : 1 : 5 with a trace of sialic acid. The properties of purified enzymes were also compared.Cathepsin D is a major proteolytic enzyme of lysosomes [l, 21 and widely distributed in animal tissues. It is presumed that cathepsin D plays an important role in physiological protein degradation and pathological processes [2 -51. However, the precise role of cathepsin D is still unclear, largely because the mechanism of protein degradation is presumably a complex and multistep process. Consequently it is clear that significant progress in understanding cathepsin D function can be made if a reasonably large quantity of pure enzyme can be obtained. For this purpose we used a rapid and convenient method for the purification of cathepsin D, which involved affinity chromatography with pepstatin -Sepharose 4B and concanavalin-A -Sepharose 4B. Although a number of lysosoma1 cathepsin D have been isolated from various tissues, including spleen [6-91, liver [lo-121, uterus [13,14], thyroid [15,16], small intestine [17], lung [18,19] and erythrocytes [20], we have undertaken a study of cathepsin D from rat spleen because cathepsin D from rat spleen has not been extensively studied and a specific activity of the enzyme in this organ is about 5 times higher than that of the liver. Recently several workers have isolated cathepsin D by affinity chromatography using suitable ligands such as substrate [8] and inhibitors [12,21,22]. In the previous work we have shown that rat spleen possesses two types of acid proteinases strongly inhibited by pepstatin [23,24]. One of these was isolated in a pure form by the use of affinity chromatography on pepstatinSepharose 4B and concanavalin-A -Sepharose 4B, and identified as a cathepsin-E-like enzyme that differs from cathepsin D [24]. In the study described here on the isolati...

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“…This fraction contained predominantly Kupffer cells. The cells were recovered by centrifuging the eluates at 600 g for 7 (27). Cathepsin B1 (EC 3.4.4.-) was estimated using a-N-benzyloxycarbonyl-L-lysine-p-nitrophenyl ester-HCI (28 30 s. The cell pellet in the KOH layer was removed by slicing through the silicone oil layer and radioactivity in the pellet measured using a Nuclear Chicago gamma counter (Nuclear Chicago Corp., Des Plaines, IL).…”

mentioning

confidence: 99%

Modulation of a Glycoprotein Recognition System on Rat Hepatic Endothelial Cells by Glucose and Diabetes Mellitus

Summerfield¹,

Vergalla²,

Jones³

1982

J. Clin. Invest.

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A B S T R A C T The cellular location and carbohydrate specificities of a glycoprotein recognition system on rat hepatic sinusoidal cells have been determined. Purified preparations of endothelial, Kupffer, and parenchymal cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. '251-labeled agalactoorosomucoid, an N-acetylglucosamine-terminated glycoprotein, was selectively taken up in vitro by endothelial cells. Uptake was shown to be protein dependent, calcium ion dependent, and saturable, and could be described by Michaelis-Menten kinetics (apparent Km 0.29 ,M; apparent maximum velocity 4.8 pmol/h per 5 X 106 cells). Uptake was inhibited not only by Nacetylglucosamine, mannose, and mannan but also by glucose, fructose, and a glucose-albumin conjugate. Inhibition by glucose was competitive over a wide range of concentrations and was almost 100% at a glucose concentration of 56 mM. Fasting and the induction of diabetes mellitus prior to isolation of cells was associated with 60% reductions in the recovery of endothelial cells. Uptake by cells isolated from fasted rats was enhanced (apparent maximum velocity 14.3 pmol/ h per 5 X 10 cells without change in the apparent K.).These observations suggest that fasting is associated with a marked increase in the mean number of glycoprotein receptors per endothelial cell isolated from normal rats. This effect of fasting could be due to upregulation of glycoprotein receptors on endothelial cells or to the selective isolation of a subpopulation of endothelial cells from fasted animals that bears more glycoprotein receptors per cell than does another sub-

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[49] α-d-Mannosidase from rat liver lysosomes

Cited by 31 publications

References 9 publications

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Cathepsin D of Rat Spleen Affinity Purification and Properties of Two Types of Cathepsin D

Modulation of a Glycoprotein Recognition System on Rat Hepatic Endothelial Cells by Glucose and Diabetes Mellitus

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