Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.     

Purification and characterization of a NAD+-dependent secondary alcohol dehydrogenase from propane-grown Rhodococcus rhodochrous PNKb1

NAD⁺-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD⁺-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C₂–C₈) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K _m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC high performance liquid chromatography - DEAE diethyl amino ethyl - IEF isoelectrofocusing - NTG nitrosoguanidine - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - pI isoelectric point     

Cellular localization of cytochrome c 553 in the N2-fixing cyanobacterium Anabaena variabilis

The in situ location of the electron carrier protein cytochrome C ₅₅₃ (cyt c ₅₅₃) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c ₅₅₃ specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c ₅₅₃ during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c ₅₅₃ is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c ₅₅₃ cytochrome c ₅₅₃ - PBS phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4) - PMSF phenylmethylsulfonyl fluoride Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz.     

Protein phosphorylation in Streptomyces albus

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.     

Development of mutants of the mosquitocidal bacterium Bacillus thuringiensis subspecies morrisoni (PG-14) toxic to lepidopterous or dipterous insects

The parasporal body of the mosquitocidal isolate (PG-14) of Bacillus thuringiensis subsp. morrisoni (BTM) contains five major proteins with molecular masses of, respectively, 27.3, 65, 128, 135, and 144 kDa. Proteins corresponding in mass to the first four of these also occur in the mosquitocidal strain, B. thuringiensis subsp. israelensis (BTI), and it is thought therefore that the mosquitocidal activity of both strains is due to these four proteins. In other studies it has been shown that each of these proteins exhibits from moderate to high toxicity to mosquitoes, though the specific toxicity of the 144 kDa protein in PG-14 to mosquitoes remains unknown. In the present study, two parasporal body mutants (M146 and M242) of PG-14 were developed growing the wild-type strain at 42 degrees C. The parasporal body of M146 contained less of the 65-kDa protein and was less toxic (LC50 = 108 ng/ml) to mosquitoes than the wild-type strain (LC50 = 8.3 ng/ml). The parasporal body of M242 consisted of a bipyramidal crystal composed of a 144-kDa protein that was not toxic to the mosquito, Aedes aegypti, but exhibited substantial toxicity (LC50 = 2.5 micrograms/ml) to the lepidopteran. Trichoplusia ni. Because the parasporal bodies of BTI and BTM PG-14 are similar in mosquitocidal toxicity on a weight basis, the latter results suggest the 144-kDa protein, though not mosquitocidal alone, can contribute to mosquitocidal, activity when in the presence of other mosquitocidal proteins.     

中国昆明小鼠亚群蛋白质多态性的研究

本文报道采用电泳技术对北京、上海、长春3市的4个中国昆明小鼠(简称KM)实验群体中24个蛋白质标志研究的结果,与我国1981年从美国引进的NIH小鼠进行比较,显示出:(1)KM小鼠亚群间等位基因组成无明显差异,它们间遗传距离为0.008—0.027,与群体封闭时间成正相关;(2)4个KM小鼠群体间聚类分析发现S:KM群体为特殊一类,该结果与KM亚群间下颌骨分析结果相符;(3)KM与Swiss来源的NIH小鼠群体间在E(?)-3、Es-10、Got-2、Glo-1、Gpt-1、和Mpi-1座位上的等位基因组成存在显著差异,它们之间的遗传距离平均值为0.131±0.011,证实中国KM小鼠为非Swiss来源的一个亚种。     

Anisotropy decay of fluorescence as an experimental approach to protein dynamics

This minireview makes an initial assessment of the progress made using anisotropy decay measurements for investigating the conformational changes and molecular dynamics in soluble systems. A critical analysis of available data is presented. The anisotropy decays of the tryptophan fluorescence of staphylococcal nuclease, adrenocorticotropin, melittin and of labeled transfer RNA were studied for investigating the functional conformational changes of these systems. The emissions of variously labeled immunoglobulins have been used to elucidate the conformations of these proteins before and after the binding of specific antibodies. Labeled myosin and its fragments have given information on the functional motions of the protein domains. The anisotropy decays of labeled and natural hemoglobin systems have been utilized for exploring the allosteric behavior of these molecules. The data suggest a wide applicability of this technique to the study of protein dynamics and conformational changes of macromolecules.     

An improved purification procedure and properties of casein kinase II from brain

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that theK _m for ATP is 12.5 M and 25.1 M when using phosvitin and casein respectively as phosphoryl acceptors. TheK _m for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while theV _max is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 g/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.     

Regulation fo protein kinase C activity by various lipids

Protein kinase C has recently attracted considerable attention because of its importance in the control of cell division, cell differentiation, and signal transduction across the cell membrane. The activity of this enzyme is altered by several lipids such as diacylglycerol, free fatty acids, lipoxins, gangliosides, and sulfatides. These lipids may interact with protein kinase C either directly or through calcium ions and produce their regulatory effect (activation or inhibition) on the activities of the enzymes phosphorylated by this kinase. These processes widen our perspective of the regulation of intercellular and intracelluular communication.Abbreviations used (PK-C) Protein kinase C - (cAMP-PK) cAMP dependent protein kinase - (DAG) diacylglycerol - (PtdSer) phosphatidylserine - (InsP ₃) inositol 1,4,5-trisphosphate - (PtdIns 4,5-P₂) inositol 4,5 bisphosphate - (FFA) free fatty acid - (MBP) myelin basic protein - (ATP) adenosine triphosphate - (GTP) guanine triphosphate - (TPA) 12-tetradecanoylphorbol-13-acetate - (EGF) epidermal growth factor - (PDGF) platelet derived growth factor - (NeuNAc) and N-acetylneuraminic acid