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Lysosomal phospholipases A1 and A2 of bovine adrenal medulla.

ABSTRACT: 1. [(32)P]Lecithin and [(32)P]phosphatidylethanolamine were prepared by incubating rat liver mince with [(32)P]phosphate. With these (32)P-labelled phospholipids conditions for the quantitative assay of phospholipase A activity were established. 2. The distribution of phospholipase A activity between subcellular fractions of the bovine adrenal medulla was determined. Phospholipases A(1) and A(2), with pH optima at 4.2 and 6.5 respectively, were found in the large-granule fraction. By means of sucrose-density-gradient centrifugation it was shown that both these phospholipases were localized in lysosomes. 3. Lysosomal phospholipase A(1) catalysed the hydrolysis of [(32)P]lecithin and [(32)P]phosphatidylethanolamine at the same rate. The enzymic activity was inhibited by 70% in the presence of 10mm-calcium chloride. 4. Lysosomal phospholipase A(2) catalysed the hydrolysis of [(32)P]phosphatidylethanolamine more rapidly than it hydrolysed [(32)P]lecithin. The hydrolysis of [(32)P]phosphatidylethanolamine, but not that of [(32)P]lecithin, by phospholipase A(2) was activated by 0.8mm-calcium chloride. However, the hydrolysis of both substrates was inhibited by 8mm-calcium chloride. 5. The significance of the presence of phospholipase activity in lysosomes is discussed in relation to the functions of lysosomes in general and in the adrenal medulla.

SUBMITTER: Smith AD

PROVIDER: S-EPMC1198894 | biostudies-other | 1968 Aug

REPOSITORIES: biostudies-other

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Project description:Chromogranin A is produced in many endocrine cell types, and is widely used as a marker in endocrine-cell pathology and secretory-cell biology. There is some evidence that it may be proteolytically processed to yield the putative pancreatic regulatory peptide, pancreastatin, and, in order to characterize the relevant pathways in gastrointestinal and pancreatic endocrine cells, we have used, in radioimmunoassay, site-directed antibodies to pancreastatin itself (L331) and to a sequence of chromogranin A immediately C-terminal to pancreastatin (L300). The latter antibody revealed three major forms of immunoreactivity of 8 kDa and five peptides of approx. 3 kDa in bovine pancreas and gut extracts. The 8 kDa peptides were characterized as chromogranin A-(248-313)-peptides, i.e. C-terminally extended forms of pancreastatin; two of the 8 kDa variants differed in two positions, confirming a polymorphism predicted from cDNA sequencing. One of the 3 kDa peptides was characterized as chromogranin A-(297-313)-peptide, i.e. the C-terminal heptadecapeptide of the 8 kDa peptide that would be liberated after cleavage to yield pancreastatin. On the basis of chromatographic studies, immunohistochemistry and the stoichiometry of different immunoreactive peptides, three different pathways of chromogranin A processing were identified: in adrenal chromaffin cells chromogranin A existed mainly as the unmodified intact protein, in pancreatic islet and gastric antral endocrine cells pancreastatin and the 3 kDa peptides were major products, but in small intestine and gastric corpus endocrine cells there was little nor no pancreastatin and the 8 kDa cleavage product predominated. There are therefore important differences in the distribution of chromogranin A-derived peptides between quite closely related populations of endocrine cells that are attributable not only to variable post-translational cleavage but also to the expression of different primary sequences. It seems possible that in different cell types chromogranin A-derived peptides might subserve a variety of different functions.

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Lysosomal phospholipases A1 and A2 of bovine adrenal medulla.

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