Dataset Information

The distributions of some granule-associated enzymes in guinea-pig polymorphonuclear leucocytes.

ABSTRACT: 1. Homogenates of guinea-pig polymorphonuclear leucocytes were separated by differential centrifugation into six particulate fractions and a soluble fraction. 2. The distributions in these fractions of protein, DNA, succinate dehydrogenase, beta-glucuronidase, peroxidase, alkaline phosphatase, acid phosphatase (against p-nitrophenyl phosphate and beta-glycerophosphate), cathepsin, and catalase were compared. 3. Almost all of the DNA sedimented in the first two pellets, indicating that the nuclei were relatively intact. 4. The four hydrolases and peroxidase showed different distribution patterns, although these activities were previously reported to be localized mainly in the single ;granule' fraction isolated from leucocytes. 5. The particles containing peroxidase, acid phosphatase and alkaline phosphatase all exhibited latency. Maximum activity for each enzyme was obtained at roughly similar concentrations of Triton X-100. 6. The acid phosphatase of these cells was distributed between two populations of particles that differed in both sedimentation characteristics and density. The acid phosphatase(s) of the two populations showed slightly different substrate specificities. This bimodal distribution was not an artifact of the procedure used to elicit the cells. 7. Catalase was recovered almost entirely in the soluble fraction and showed no latency in freshly prepared homogenates. No urate oxidase was detected. 8. We conclude that the ;granule' fraction of the polymorphonuclear leucocyte, as isolated by previous workers, contains at least three, probably more, populations of particles with different enzyme contents, and that these cells probably do not contain peroxisomes.

SUBMITTER: Michell RH

PROVIDER: S-EPMC1185347 | biostudies-other | 1970 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:1. Two cyclic AMP-independent casein/glycogen synthase kinases were purified from pig polymorphonuclear leucocytes by chromatography on phosphocellulose followed by affinity chromatography on casein-Sepharose 4B or gel filtration on Bio-Gel A-1.5m. When the affinity step was used, the specific activities were 86 and 43units/mg of protein for casein kinase 1 and 2, respectively, whereas these values were 94 and 90units/mg of protein when the gel-filtration step was used. 2. These kinases differ as follows: (a) the molecular weight of casein kinase 1 (38000) is very much lower than that of casein kinase 2 (185000); (b) the K(m) for casein (0.46mg/ml) and K(a) for Mg(2+) (0.3mm) of casein kinase 1 are lower than those of casein kinase 2 (0.90mg/ml and 1.7mm respectively); (c) KCl stimulates the phosphorylation of casein by casein kinase 1, whereas it inhibits phosvitin phosphorylation by this enzyme; on the contrary, the effect of KCl on casein kinase 2 is very similar with either casein or phosvitin as substrate; (d) although both kinases phosphorylate rabbit muscle glycogen synthase I, the ratio of glycogen synthase to casein phosphorylation by casein kinase 1 is about 4-fold greater than that by casein kinase 2. Furthermore, (32)P incorporation into glycogen synthase promoted by casein kinase 1 (3.6mol of (32)P/mol of 85000-dalton subunit) is twice that observed with casein kinase 2 (1.8mol of (32)P/mol of 85000-dalton subunit). Such a phosphorylation results in a decrease in the glucose 6-phosphate-independence ratio of glycogen synthase to 10-15 with casein kinase 1 and to 35-45 with casein kinase 2. 3. The activity of both kinases is neither stimulated by cyclic AMP, Ca(2+) and calmodulin nor inhibited by cyclic AMP-dependent protein kinase inhibitor protein. 4. No phosphorylation kinase activity was observed with casein kinase 1 and 2 at either pH6.8 or 8.2 in the presence of Ca(2+). 5. Activities of both kinases on casein and glycogen synthase decreased in parallel when incubated at 50 degrees C.

Project description:Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.

Dataset Information

The distributions of some granule-associated enzymes in guinea-pig polymorphonuclear leucocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets