ABSTRACT
Heparinase was released from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock procedure. The procedure involves resuspending exponentially growing cells consecutively into (1) 40% sucrose, (2) 10 mM sodium phosphate, 2 mM magnesium chloride, pH 7, and (3) 10 mM sodium phosphate, 300 mM sodium chloride, 2 mM magnesium chloride, pH 7. Typically, 50-75% of the total heparinase activity is recovered by this procedure with an observed 7-15-fold increase in purity. The majority of heparinase activity is released in the final step of the procedure allowing for resolution from cytoplasmic and nonspecific periplasmic material. F. heparinum cells can be stored in 40% sucrose at 4 degrees C for up to one week without significant losses in recovery yields.
Subject(s)
Flavobacterium/enzymology , Polysaccharide-Lyases/metabolism , Biotechnology , Cell Membrane/enzymology , Edetic Acid , Flavobacterium/growth & development , Heparin Lyase , Hydrogen-Ion Concentration , Magnesium , Osmolar Concentration , Osmotic Pressure , SucroseABSTRACT
The localization of the protein tyrosine kinase pp60c-src to the plasma membrane and to the membrane of secretory vesicles in neurally derived bovine chromaffin cells has suggested that tyrosine phosphorylations may be associated with the process of secretion. In the present study we have identified two cytosolic proteins of approximately 42 and 45 kD that become phosphorylated on tyrosine in response to secretagogue treatment. Phosphorylation of these proteins reached a maximum (3 min after stimulation) before maximum catecholamine release was observed (5-10 min after stimulation). Both secretion and tyrosine phosphorylation of p42 and p45 required extracellular Ca2+. Tyrosine-phosphorylated proteins of similar Mr have previously been identified in 3T3-L1 adipocytes stimulated with insulin (MAP kinase; Ray, L. B., and T. W. Sturgill. 1987. Proc. Natl. Acad. Sci. USA. 84:1502-1506) and in avian and rodent fibroblasts stimulated with a variety of mitogenic agents (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273; Nakamura, K. D., R. Martinez, and M. J. Weber. 1983. Mol. Cell. Biol. 3:380-390). Comparisons of the secretion-associated 42-kD protein of chromaffin cells with the 42-kD protein of Swiss 3T3 fibroblasts and 3T3-L1 adipocytes provide evidence that these three proteins are highly related. This evidence includes comigration during one-dimensional SDS-PAGE, cochromatography using ion exchange and hydrophobic matrices, similar isoelectric points, identical cyanogen-bromide peptide maps, and cochromatography of MAP kinase activity with the tyrosine-phosphorylated form of pp42. This protein(s), which appears to be activated in a variety of cell types, may serve a common function, perhaps in signal transduction involving a cascade of kinases.
Subject(s)
Adrenal Medulla/metabolism , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Adrenal Gland Neoplasms , Animals , Cattle , Cell Line , Cells, Cultured , Cyanogen Bromide , Molecular Weight , Norepinephrine/metabolism , Peptide Mapping , Pheochromocytoma , Phosphoproteins/isolation & purification , Phosphorylation , Phosphotyrosine , Substrate Specificity , Tyrosine/analogs & derivatives , Tyrosine/analysisABSTRACT
High levels of the proto-oncogene product, pp60c-src, have been found in developing and adult neural tissues as well as in certain fully mature cells of the hematopoietic lineage, e.g., platelets and myelomonocytes. Adrenal medullary chromaffin cells exhibit characteristics of both types of cells, i.e., they are derived from the neural crest and carry out exocytosis in response to specific stimuli. Earlier studies have shown that pp60c-src localizes not only to the plasma membrane of chromaffin cells but also to the membranes of chromaffin granules, the secretory vesicles of these cells that store catecholamines and other secretory products. To investigate the possible involvement of pp60c-src in exocytosis, cultured bovine chromaffin cells were analyzed for changes in c-src tyrosine kinase activity in response to stimulation by several secretagogues. Results of in-vitro immune complex kinase assays showed that pp60c-src, derived from cells that had been stimulated for various lengths of time, exhibited decreased auto- and transphosphorylating activities as compared to pp60c-src immunoprecipitated from control cells. The greatest reduction in activity was observed 10 min post-stimulation, while normal levels were regained 2-6 hr after secretagogue treatment. Western immunoblot analysis of the immunoprecipitated pp60c-src revealed that approximately 50% less c-src protein was present in immune complexes prepared 10 min after stimulation as compared to those prepared from mock-stimulated controls, resulting in a specific autophosphorylating activity that was 42-47% of control and little or no reduction in the transphosphorylating specific activity. In experiments in which the rate of secretion of [3H]-norepinephrine from cells preloaded with this compound was compared to the rate of modulation of pp60c-src activity, 50% of the maximal reduction in pp60c-src activity occurred within 2-4 min while 50% maximal release of [3H]-norepinephrine occurred within 1-3 min. Taken together, these results suggest that pp60c-src may play some role (direct or indirect) in the exocytotic process.
Subject(s)
Adrenal Medulla/enzymology , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins/physiology , Adrenal Medulla/cytology , Adrenal Medulla/drug effects , Animals , Antigen-Antibody Complex/metabolism , Blotting, Western , Carbachol/pharmacology , Cattle , Down-Regulation/physiology , Exocytosis/physiology , In Vitro Techniques , Kinetics , Microscopy, Fluorescence , Nicotine/pharmacology , Norepinephrine/metabolism , Phosphorylation , Proto-Oncogene Proteins pp60(c-src)ABSTRACT
At least 23 soluble proteins (chromobindins) bind to chromaffin granule membranes in the presence of Ca2+. In order to further the identification of the chromobindins and to determine the roles they may play in exocytosis or other aspects of chromaffin cell biology, several of these proteins were compared to other known membrane-binding proteins. Chromobindin 4 was identified as a 32-kDa protein called calelectrin or endonexin. Immunologically related proteins were detected in bovine brain and human platelets. Chromobindin 20 was identified as a 67-kDa variant of calelectrin and was found to have the activities of the synexin inhibitory protein, synhibin. Chromobindin 8 was identified as p36, a substrate for the tyrosine-specific kinase, pp60v-src. Chromobindin 8 was also demonstrated to undergo phosphorylation predominantly on alkali-sensitive sites during stimulation of the chromaffin cell with 20 microM nicotine. Chromobindin 6 was identified as p35, a substrate for the tyrosine kinase activity associated with the epidermal growth factor receptor. Chromobindin 9, which is known to be a substrate for protein kinase C (Ca2+/phospholipid-dependent enzyme), was found to be immunologically related to p35 and may be a precursor of chromobindin 6. The identification of these proteins from the chromaffin system may be useful in the characterization of similar, complex groups of membrane-binding proteins that have been observed in other systems.
Subject(s)
Calcium-Binding Proteins/analysis , Carrier Proteins/analysis , Chromaffin Granules/analysis , Chromaffin System/analysis , Membrane Proteins/analysis , Protein-Tyrosine Kinases/metabolism , Amino Acids/analysis , Animals , Annexins , Cattle , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Phosphoproteins/metabolismABSTRACT
R-plasmid transfer in Escherichia coli was investigated in nutrient broth, sterile soil, and sterile stream water. Plasmid transfer occurred in broth cultures at 30 and 22 degrees C, but not at 15 degrees C. R-plasmid transfer was not observed at 30, 22, and 15 degrees C in nonamended sterile soil and stream water. The addition of nutrients to sterile stream water and soil allowed plasmid transfer to occur at temperatures ranging from 15 to 30 degrees C. R-plasmid transfer was also observed in soil adjusted from 20 to 100% of its water-holding capacity.