Navin C. Khanna

Phosphorylation of lipocortins in vitro by protein kinase C.

Protein kinase C catalyzes the incorporation of about 1.1, 0.7 and 0.4 mole of phosphate per mole of Lipocortin-I (P35), Lipocortin-II (P36) and Lipocortin-85 (P36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While Lipocortin-I is phosphorylated on threonine residues, Lipocortin-II and Lipocortin-85 are phosphorylated on serine residues. The substoichiometric phosphorylation of Lipocortin-85 appears to preclude the potential regulation of this protein by protein kinase C. The phosphorylation of Lipocortin-I on threonine residues and Lipocortin-II on serine residues suggests these proteins may be regulated by distinct phosphorylation-dephosphorylation reactions.

Purification of three forms of lipocortin from bovine lung.

Experimental conditions are described for simultaneous purification of three forms of lipocortin (lipocortin I, lipocortin II and lipocortin-85) from bovine lung. The procedure yields milligram quantities of all three lipocortins. Using antisera against lipocortin I and lipocortin II, purified proteins show no cross contaminations. All forms of lipocortin exhibit equal potency as in vitro bovine pancreatic phospholipase A2 inhibitors. Protein kinase C catalyzes the in vivo incorporation of about 1.0, 0.7 and 0.4 mole of phosphate per mole of lipocortin I (p35), lipocortin II (p36) and lipocortin-85 (p36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While lipocortin I is phosphorylated on threonine residues, lipocortin II and lipocortin-85 are phosphorylated on serine residues.

Inhibition of Phospholipase-A2 by Protein-i

The 36 kDa substrate of several tyrosine protein kinases has been shown to exist in monomeric and oligomeric (362102) forms. Partial sequence data has suggested that the oligomer, referred to as protein I, is homologous to a group of phospholipase A2 inhibitory proteins, collectively called lipocortins. In the present communication we demonstrate that protein I inhibits bovine pancreas phospholipase A2 with similar potency to that of lipocortin. Approximately 44 pmol protein I was required to produce 50% inhibition of 7.2 pmol of phospholipase A2. Inhibition of phospholipase A2 activity by calmodulin, S-100, calregulin, parvalbumin, troponin-C, or CAB-48 was not observed. These results indicate that protein I is a potent and specific inhibitor of phospholipase A2 activity, and thus shares functional homology with the lipocortin proteins. We therefore propose that this protein be named lipocortin-85.

[6] Purification of novel calcium-binding proteins from bovine brain

Publisher Summary This chapter describes the purification of two novel bovine brain Ca 2+ - binding proteins: CAB-27 and CAB-48. In addition, the strengths and weaknesses of the Chelex assay as a probe for the measurement of Ca 2+ -binding activity in tissue homogenates are discussed. Purified CAB-27 inhibits phospholipase A 2 activity in a dose-dependent manner. Brain Ca 2+ -binding proteins, such as CAB-48, calregulin, calmodulin, S-100, and parvalbumin, have little effect on phospholipase A 2 activity. The Chelex-100 competitive Ca 2+ -binding assay ensures maximum linearity and sensitivity, and this assay is also used as a probe for the detection of Ca 2+ -binding proteins. The studies demonstrated that the majority of the Ca 2+ -binding activity, detected by the Chelex assay, is not associated with calmodulin activity and is based on a comparison of the apparent M r of the Ca2 + -binding activity peaks with those of characterized Ca 2+ -binding proteins. The Chelex-100 is a resin of styrene divinylbenzene with acetate as its functional group. The resin binds a variety of divalent cations including calcium and has been used to determine the stability constants of several cation-binding substances. The amount of Chelex to be used in the assay system should be carefully selected to maximize the sensitivity of this assay in the detection of unknown calcium-binding substances in tissue extracts.

Identification and Characterization of the Tyrosine Protein-Kinases of Rat Spleen

High levels of tyrosine protein kinase have been recently detected in the membranes of rat spleen. In the present report the tyrosine protein kinase activity of the 30,000 x g pellet of rat spleen has been solubilized and partially purified by ion exchange and gel permeation chromatography. Two peaks of tyrosine protein kinase of Mr 35,000 (TPK-I) and Mr 40,000 (TPK-II) have been resolved. These kinases were free of the EGF receptor and insulin receptor tyrosine protein kinases. Although TPK-I and TPK-II phosphorylated angiotensin II, casein, histone, tubulin, phosphorylase b, and p36 they differed from each other in preference for the substrates. Both tyrosine protein kinases did not phosphorylate anti-pp60v-src IgG.

Calregulin - Purification, Cellular-Localization, and Tissue Distribution

Publisher Summary This chapter describes a simple procedure for purifying calregulin from bovine liver and then documents a convenient, one-step method for radioimmunoassay for calregulin. Finally, a method for immunocytochemical localization of calregulin in bovine fibroblasts is described. This protein is purified from bovine liver by a procedure involving three chromatographic steps. DEAE cellulose separates calregulin from calmodulin and other calcium-binding proteins, a Con A-agarose column resolves calregulin from the bulk of contaminants, and finally, gel permeation chromatography removes the remaining contaminants and yields highly purified calregulin. Purified calregulin is radioiodinated. Calregulin is added to the preloaded iodobeads and the reaction mixture is incubated. The products are transferred directly to a Sephadex G-50 column. The major peak of radioactivity in the void volume is collected and used as tracer in the RIA. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gels show that the iodinated protein comigrates with unlabeled bovine liver calregulin, and is free from any other contaminants. For the immunocytochemical localization of calregulin and for immunoprecipitation of calregulin, primary cultures of bovine fibroblasts are prepared. The cells are subsequently cultured and subcultured in the same growth medium.

Identification of a major bovine heart Ca2+ binding protein.

The 100,000 x g supernatant of bovine heart has been chromatographed on DEAE-cellulose and the resultant fractions have been analyzed for both calcium binding activity and calmodulin activity. Of the four peaks of calcium binding activity detected by this procedure only a single peak (peak IV) was identified as calmodulin. The calcium binding activity of the largest peak (peak III) has been subjected to further purification and a single calcium binding protein of Mr 63,000 isolated. Biochemical and immunological results documented that the 63 kDa protein is identical to calregulin. The results of this study identify calregulin as a major bovine heart calcium binding protein.

Identification of bovine brain calcium binding proteins.

Three peaks of calcium binding activity have been identified by the Chelex-100 calcium binding assay of the fractions from DEAE cellulose chromatography of 100,000 X g supernatant of bovine brain. These calcium binding activity peaks have been subjected to extensive purification and three novel calcium binding proteins (Mr 27,000, Mr 48,000 and Mr 63,000) and two previously characterized proteins (calcineurin and calmodulin) have been identified as components of calcium binding activity peaks. Analysis of the calcium binding properties of the novel proteins by equilibrium dialysis suggests these proteins may be intracellular calcium receptors.

Chapter 3 The role of calcium binding proteins in signal transduction

Publisher Summary This chapter discusses the role of calcium (Ca)-binding proteins in signal transduction. Measurement of the free intracellular calcium [Ca 2+ ] within cells suggests that it is very low, of the order of 10–100 nM. This means that the vast majority of intracellular Ca is bound. Considering that the extracellular Ca concentration is about 1 mM (compared to 10 nM [Ca 2+ ] i ) and that the interior of the cell is negatively charged there is a large electrochemical gradient driving Ca ions into the cell, however the relative impermeability of the plasma membrane to Ca ions prevents large movements of Ca ions across the plasma membrane. The Ca transient is controlled by the uptake and release of Ca across the three major membrane systems which border the cytoplasm—the plasma membrane, the inner mitochondria1 membrane, and the endoplasmic reticulum. Each membrane possesses distinctive transport mechanisms which act in concert to regulate and modulate intracellular Ca. Cellular signal transduction can be divided into two separate components: (1) the generation of an intracellular signal from an external event and (2) transduction of the intracellular signal into the physiological response of the cell.

PHOSPHOLIPASE A2 INHIBITORY PROTEINS OF BOVINE LUNG

Publisher Summary This chapter examines the phospholipase A2 inhibitory proteins of bovine lung. There is an evidence suggesting that some of the substrates of protein-tyrosine kinases are the inhibitors of phospholipids turnover. Two phospholipase A2 inhibitors have been purified from human placenta. Amino acid sequence analysis of the first placental inhibitor (lipocortin I) has identified this protein as the EGF receptor protein–tyrosine kinase substrate p35. The sequence of the second placental inhibitor (lipocortin II) reveals it to be the major retroviral protein-tyrosine kinase substrate p36. In the study presented in this chapter, lipocortin I, lipocortin II and 1ipocortin-85 were purified from bovine lung. Phosphorylation by protein kinase C was carried out. Samples were tested for phospholipase A2 inhibitory activity. A time course of the protein kinase C-catalyzed phosphorylation of lipocortin I, lipocortin II and lipocortin-85 is presented. The results show that the protein kinase C incorporated about 1.0, 0.7 and 0.4 mole of phosphate per mole of lipocortin I, lipocortin II and lipocortin-85, respectively. No phosphorylation was observed when either Ca2+ or phospholipids were omitted from the reaction mixture.