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K Docherty

Publications and source records attributed to K Docherty.

120 records · Page 7Linked to original sources
PubMed1983

Carboxypeptidase activity in the insulin secretory granule.

Carboxypeptidase activity was studied in subcellular fractions from a transplantable rat insulinoma and found to be localised principally in the insulin secretory granule. The activity, which was specific for peptide substrates with C-terminal basic amino acids, appeared to be a single enzyme with Mr 54000. This enzyme differed with respect to size and pH optimum from other basic amino acid-specific carboxypeptidases, such as carboxypeptidases B and N, and may be a secretory granule-specific enzyme involved in propolypeptide processing.

Adenoma, Islet Cell↗
PubMed1983

Sugar transport in rat liver lysosomes. Direct demonstration by using labelled sugars.

Purified rat liver lysosomes ('tritosomes') were prepared from rats injected with Triton WR-1339. 2. The water space of tritosomes, measured by using [3H]water and [14C]sucrose, was 2.15 +/- 0.72 microliter/mg of protein (mean +/- S.E.M., n = 12). 3. Tritosomes, when compared with a crude preparation of normal lysosomes by an indirect method of study, showed sugar specificity but decreased stereospecificity of sugar uptake. 4. At 125 mM the relative rates of net uptake of D-[14C]ribose, D-[14C]- or D-[3H]glucose and 2-deoxy-D-[3H]glucose were the same as that inferred from the indirect study. 5. The entry of D-[3H]glucose into tritosomes showed concentration-dependence suggestive of saturation, with a Km of 48 +/- 18 mM (4). 6. D- and L-glucose, D-ribose, 2-deoxy-D-glucose and D-mannose competed with D-[14C]glucose or D-[14C]ribose for uptake. 7. Cytochalasin B inhibited D-[3H]glucose uptake. 8. Uptake of 1 mM-L-[14C]glucose was slower than for 1 mM-D-[14C]glucose. 9. It is concluded that a facilitated-diffusion transport system is present in purified rat liver lysosomes.

Acetylglucosaminidase↗
PubMed1983

Identification of a 31,500 molecular weight islet cell protease as cathepsin B.

A method for the preparation of a radioisotopically labeled active-site directed reagent for proteases (125I-Tyr-Ala-Lys-ArgCH2Cl) is described, and an example of its use as a sensitive method for identifying trypsin-like proteases is provided. This high specific activity reagent was then used in an attempt to identify proteases in rat islets of Langerhans involved in the conversion of proinsulin to insulin. Previous studies have indicated that the endoprotease involved in proinsulin conversion is a cysteine proteinase and that 125I-Tyr-Ala-Lys-ArgCH2Cl affinity labels an islet crude granule fraction protein having a molecular weight of 31,500. Here we demonstrate, using a probe of higher specific activity, that the major affinity-labeled proteins of the islet crude granule fraction, when displayed by sodium dodecyl sulfate gel electrophoresis, have molecular weights of approximately 39,000 (5%), 31,500 (53%), and 5,000-6,000 (37%), with several other minor proteins (less than 5%) also labeled. The two predominant labeled proteins were mainly soluble rather than membrane bound, and they exhibited patterns of competition with various inhibitors that were similar to the pattern shown by the conversion of proinsulin to insulin in vitro. A rabbit antibody to rat liver cathepsin B immunoprecipitated both affinity-labeled 31,500 and 5,000-6,000 molecular weight proteins, and on the basis of this and structural considerations the 31,500 molecular weight cysteine protease is identified as cathepsin B. The 5,000-6,000 molecular weight peptide is an NH2-terminal, active site cysteine-containing, proteolytic fragment of the 31,500 molecular weight protein. Because cathepsin B is not per se a candidate for the proinsulin convertase because of its excessively broad substrate specificity, these studies suggest that a similar enzyme or a modified form of this enzyme is active within the secretory progranules, whereas the more typical cathepsin B may be largely confined to lysosomal contaminants in our granule preparations.

Animals↗
PubMed1982

Conversion of proinsulin to insulin: involvement of a 31,500 molecular weight thiol protease.

A lysed crude secretory granule fraction from rat islets of Langerhans was shown to process endogenous proinsulin to insulin with a pH optimum of 5.0--6.0. The converting activity in the lysed fraction was not inhibited by serine protease inhibitors (diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin) or metalloprotease inhibitors (EDTA and o-phenanthroline) but was inhibited by some thiol protease reagents (p-chloromercuribenzenesulfonic acid, antipain, and leupeptin) but not by others (N-ethylmaleimide and iodoacetamide). N alpha-p-Tosyl-L-lysyl chloromethyl ketone only mildly inhibited at higher concentrations, whereas L-alanyl-L-lysyl-L-arginyl chloromethyl ketone was a powerful inhibitor. L-Alanyl-L-lysyl-L-arginyl chloromethyl ketone was [125I]iodotyrosylated and used as an affinity labeling agent for the converting activity. Because the crude granule preparation contained contaminating lysosomes the affinity labeling of the granule preparation proteins was compared with that in liver lysosomes purified from rats injected with Triton WR1339. In the crude granule fraction the affinity label bound in a cysteine-enhanced manner to a single 31,500 molecular weight protein, but in purified liver lysosomes the major affinity-labeled protein had a molecular weight of 25,000 and minor 31,500 and 35,000 molecular weight proteins were also labeled. Evidence suggests that these proteins are thiol proteases and that in islets the 31,500 molecular weight thiol protease is involved in the conversion of proinsulin to insulin.

Affinity Labels↗
PubMed1979

The permeability of rat liver lysosomes to sugars. Evidence for carrier-mediated facilitated diffusion.

1. By the osmotic-protection method, the penetration of sugars through the rat liver lysosomal membranes was studied with a view of determining whether sugar uptake was by facilitated diffusion. 2. The following criteria for this type of transport were established: sugar specificity, the order of uptake being 2-deoxy-D-glucose less than D-glucose less than D-mannose less than D-galactose less than D-ribose less than 2-deoxy-D-ribose; stereospecificity, the uptake of L-glucose and L-ribose being 50% slower than their D-stereoisomers; inhibition by 1 MM-phlorrhizin and 1 M-cytochalastin B; competition between sugars for uptake, and a Q10 (rate difference over a 10 degrees C temperature range) for uptake of approx. 2.8. 3. It is proposed that sugar uptake into lysosomes from rat liver is by facilitated diffusion.

4-Nitrophenylphosphatase↗