scholarly journals Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity

1987 ◽  
Vol 241 (1) ◽  
pp. 137-143 ◽  
Author(s):  
J Spaltro ◽  
J A Alhadeff
Keyword(s):  
Plasma Membrane ◽  
Substrate Specificity ◽  
Isoelectric Focusing ◽  
Human Liver ◽  
Cellular Localization ◽  
Human Liver Tissue ◽  
Neuraminidase Activity ◽  
Acetylneuraminic Acid ◽  
Acidic Form ◽  
Hydrolysis Of

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2′-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.

Does plasma membrane and total transporter abundance differ between suspended, plated, sandwich culture hepatocytes and human liver tissue?

2019 ◽  
Vol 34 (1) ◽  
pp. S17
Author(s):  
Vineet Kumar ◽  
Laurent Salphati ◽  
Cornelis E.C.A. Hop ◽  
Christopher Rowbottom ◽  
Guangqing Xiao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liver Tissue ◽  
Human Liver ◽  
Human Liver Tissue ◽  
Sandwich Culture

scholarly journals Purification and Cloning of a Broad Substrate Specificity Human Liver Carboxylesterase That Catalyzes the Hydrolysis of Cocaine and Heroin

1997 ◽  
Vol 272 (23) ◽  
pp. 14769-14775 ◽  
Author(s):  
Evgenia V. Pindel ◽  
Natalia Y. Kedishvili ◽  
Trent L. Abraham ◽  
Monica R. Brzezinski ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Human Liver ◽  
Broad Substrate Specificity ◽  
Hydrolysis Of

scholarly journals Alteration of the isoform composition of plasma-membrane-associated rat sperm α-l-fucosidase during late epididymal maturation: comparative characterization of the acidic and neutral isoforms

1998 ◽  
Vol 333 (1) ◽  
pp. 201-207 ◽  
Author(s):  
Irene ABASCAL ◽  
Sheri R. SKALABAN ◽  
Karen M. GRIMM ◽  
Manuel AVILÉS ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Isoelectric Focusing ◽  
Human Liver ◽  
Polyclonal Antibodies ◽  
Enzymic Activity ◽  
Ph Optimum ◽  
Neuraminidase Treatment ◽  
Proximal Half ◽  
Epididymal Maturation ◽  
Isoform B

In a previous study, evidence was provided for the presence of a novel plasma-membrane-associated neutral-pH-optimum α-l-fucosidase in rat sperm. In the present study, rat sperm α-l-fucosidase was characterized during epididymal maturation. The pH 7 activity optimum of α-l-fucosidase and its subunit composition (one or two closely spaced immunoreactive protein bands of about 53±2 kDa) did not appear to change during transit through the epididymis. Isoelectric focusing of α-l-fucosidase indicated the presence of a major isoform (B) with a pI near 7 in sperm from testis, caput, corpus and the proximal half of the cauda. α-l-Fucosidase from sperm from the distal half of the cauda, which contained a significant enrichment of sperm and α-l-fucosidase activity, contained isoform B and an additional minor isoform (A) with a pI near 5.2. Isoform B and small amounts of isoform A were present in sperm from the vas deferens. The two fucosidase isoforms present in sperm from the distal cauda were separated by isoelectric focusing and comparatively characterized. They had similar pH–activity curves (with optima near pH 7) and comparable apparent KM values (0.4±0.04 mM) for 4-methylumbelliferyl α-l-fucopyranoside. Preincubation of the isoforms at different temperatures indicated that isoform A is considerably more thermostable than isoform B. Immunoprecipitation studies using polyclonal antibodies against human liver α-l-fucosidase indicated that approx. 90% of the enzymic activity for both isoforms was immunoprecipitable under conditions that immunoprecipitated essentially all the human liver enzyme. Neuraminidase treatment of sperm α-l-fucosidase from distal cauda (when compared with the appropriate heat-treated control) led to disappearance of isoform A and a concomitant increase in isoform B. The overall results suggest that isoform A is derived by sialylation of isoform B near the end of epididymal maturation.

scholarly journals Isoelectric focusing of glutathione S-transferases from rat liver and kidney

1978 ◽  
Vol 175 (3) ◽  
pp. 937-943 ◽  
Author(s):  
Barbara F. Hales ◽  
Valerie Jaeger ◽  
Allen H. Neims
Keyword(s):  
Substrate Specificity ◽  
Isoelectric Focusing ◽  
Transferase Activity ◽  
Sprague Dawley ◽  
Substrate Specificities ◽  
Liver Cytosol ◽  
Sprague Dawley Rats ◽  
Isoelectric Points ◽  
Liver And Kidney ◽  
Glutathione S Transferases

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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