Non-requirement of calcium on protamine phosphorylation by calcium-activated, phospholipid-dependent protein kinase

Phosphorylation of clupeine sulfate by purified rat brain calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was studied. In the absence of Ca2+, phosphatidylserine and diolein markedly stimulated its phosphorylation. However Ca2+ did not stimulate but inhibit this phosphorylation about 30% in the presence of phospholipids. Random polymer (Arg, Ser) 3:1 and (Lys, Ser) 3:1 could be phosphorylated by protein kinase C. In the presence of phospholipids Ca2+ is not needed for the phosphorylation of polymer (Arg, Ser) 3:1, while Ca2+ is necessary for polymer (Lys, Ser) 3:1. Non-requirement of Ca2+ on clupeine phosphorylation by protein kinase C is briefly discussed.     

Accumulation of CK-MM is impaired in innervated and contracting cultured muscle fibers of Duchenne muscular dystrophy patients

No specific abnormalities have been reproducibly manifested in aneurally cultured muscle of Duchenne muscular dystrophy (DMD) patients. We now report that the accumulation of the muscle-"specific" isozyme of creatine kinase (CK-MM) was significantly and preferentially impaired in long-term innervated contracting muscle fibers cultured from 4 DMD patients (DMD-InnCMFs) compared to: i) their noninnervated sister-cultured muscle fibers, and ii) innervated contracting control cultured human muscle fibers (Control-InnCHMFs). Accumulation of other muscle-"specific" isozymes (MSIs), viz. glycogen phosphorylase, phosphoglycerate mutase, and lactic dehydrogenase, was not significantly impaired. We have not observed preferentially-impaired CK-MM accumulation in any Control-InnCHMFs from 22 patients (children and adults) with a variety of neuromuscular diseases. There was no apparent difference between DMD-InnCMFs and Control InnCHMFs regarding: acceptance of innervation; neuronally-driven, virtually continuous muscle-fiber contractions; characteristic myofiber organization by phase-contrast microscopy, and increased longevity of the innervated fibers.     

Effects of Exogenous Amino Acids on Iron Uptake in Relation to their Effects on Photoperiodic Flowering in Lemna paucicostata 6746

The flowering of Lemna paucicostata 6746 grown on 14-h photoperiodwas enhanced by the addition of high concentrations of ironto the medium, which also increased the endogenous iron concentration.The addition of asparagine, aspartate, glutamate, -alanine,glycine or serine to the medium also increased the endogenousiron level, resulting in the promotion of flowering. In contrast,the addition of cysteine, cystine, glutamine, arginine, threonineor phenylalanine lowered the endogenous iron level, resultingin the inhibition of flowering. Glycine and asparagine added to the medium during an inductive96-h dark period did not promote iron uptake and had no effecton flowering, but when added during the subsequent 120-h lightperiod, they promoted both iron uptake and flowering response.The increase in the endogenous iron level seems to favor floraldevelopment rather than induction of photoperiodic floweringof Lemna paucicostata 6746. (Received September 8, 1986; Accepted March 31, 1987)     

Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

Acyl-peptide hydrolase from rat liver. Characterization of enzyme reaction

Acyl-peptide hydrolase, which catalyzes the hydrolysis of an N-terminally acetylated peptide to release an N-acetylamino acid, was isolated from rat liver and found to be N-terminally blocked. The kinetics of the hydrolysis of acetyl (Ac)-Ala-Ala, Ac-Ala-Ala-Ala, acetylalanine p-nitroanilide, and acetylalanine beta-naphthylamide were investigated. The Km values were between 1 and 9 mM, and the Vmax values were between 100 and 500 nmol/min/micrograms of enzyme. The enzyme activity toward acetylalanine p-nitroanilide and acetylalanine beta-naphthylamide was activated by the presence of Cl- and SCN- at concentrations between 0.1 and 0.5 M. By contrast, the activity toward Ac-Ala-Ala and Ac-Ala-Ala-Ala was inhibited by these anions. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by the addition of diisopropyl fluorophosphate, diethyl pyrocarbonate. Woodward's Reagent K, and glycine methyl ester/carbodiimide. Titration by diisopropyl fluorophosphate showed 0.7 mol of active serine/mol of enzyme subunit, which was confirmed by the incorporation of [3H]diisopropyl fluorophosphate into the enzyme. Acetylalanine chloromethyl ketone inactivated the enzyme following pseudo-first order kinetics; and Ac-Ala, a competitive inhibitor, protected the enzyme from this inactivation. Acyl-peptide hydrolase appears to be a serine protease utilizing a charge relay system involving serine, histidine, and, probably, a carboxyl group(s). Two series of acetyl dipeptides, acetylamino acid p-nitroanilides and acetylamino acid beta-naphthylamides, were prepared in order to determine enzyme specificity. The enzyme preferentially removed Ac-Ala, Ac-Met, and Ac-Ser, the most common acetylated N-terminal residues (Persson, B., Flinta, C., von Heijne, G., and J?rnvall, H. (1985) Eur. J. Biochem. 152, 523-527). The enzyme was shown to be useful for deblocking peptides (e.g. alpha-melanocyte-stimulating hormone and acetyl-renin substrate), and the crude enzyme/substrate mixtures were amenable to direct protein sequence analysis.     

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.