A hypothetical model of the influence of inorganic phosphate on the kinetics of pyruvate kinase.

This paper presents a simple solution to the problem of approximating the calculated curve of reaction progress to the measured curve which is usually disturbed by initial oscillation of auxiliary lactate dehydrogenase (LDH) reaction. The experiments leading to the determination of the apparent Km for phosphoenolpyruvate (PEP) and Vm were performed. For precise estimation of kinetic parameters (Km and Vm) of the M1 isozyme of pyruvate kinase (PK), measured by coupling it to LDH reaction, the sequence of Michaelis-Menten for pyruvate kinase and second-order kinetics for lactate dehydrogenase reaction as well as a non-zero initial concentration of lactate was assumed. The functions of apparent Km and Vm of pyruvate kinase with respect to phosphate concentration, computed by an analysis of the total reaction progress curves, indicate that the reaction mixture contains an uncompetitive inhibitor of pyruvate kinase, and that the phosphate binds this inhibitor. The proposed simple mathematical model of pyruvate kinase Km and Vm increase by inorganic phosphate assumes that the pyridine nucleotides (NAD-derivatives) are kinase inhibitors. An approximate dissociation constant for pyridine nucleotides-phosphate complex and true Km of pyruvate kinase for PEP were estimated. The proposed model fits exactly the entire measured reaction process.

Hydrophobic labelling of spectrin in erythrocytes using arylisothiocyanates.

Labelling of spectrin with the hydrophobic probes 2-naphthylisothiocyanate and 4-[125I]-iodophenylisothiocyanate dispersed in lipid vesicles was studied. Although spectrin is a peripheral protein of the erythrocyte membrane, it was found to be labelled as a result of incubation of washed red blood cells with hydrophobic arylisothiocyanates.

Labelling of erythrocyte spectrin in situ with phenylisothiocyanate.

The labelling of erythrocyte spectrin in situ with the hydrophobic reagent phenylisothiocyanate (Sigrist, H. and Zahler, P. (1978) FEBS Lett. 95, 116-120) is studied. Spectrin isolated from erythrocytes which have been incubated with phenylisothiocyanate is covalently modified by the probe. The modification in the spectrin molecule is stable under an excess of nucleophile in alkaline conditions. The labelling is very little or not affected by preincubation of erythrocytes of membranes with the polar, structural analogue of phenylisothiocyanate, p-sulfophenylisothiocyanate. When erythrocyte ghosts are subjected to labelling, a substantial increase in the degree of spectrin modification is observed. Subunits of labelled spectrin separated electrophoretically show similar amounts of attached label.

Specificity of acid protease from Fusarium moniliforme.

Specificity of acid protease from Fusarium moniliforme was investigated using beta chain of oxidized insulin. The enzyme is highly specific for the bonds involving aromatic and hydrophobic amino acid residues. It shows high affinity for the following bonds: Leu(15)-Tyr(16), Tyr(16)-Leu(17), Phe(24)-Phe(25), Phe(25)-Tyr(26), and somewhat lower for other three bonds: His(10)-Leu(11), Leu(11)-Val(12) and Tyr(26)-Thr(27) in oxidized beta chain of insulin.

Amino acid sequence of the basic trypsin inhibitor from bovine splenic capsule. A new fluorescent reagent for protein sequence analysis.

The amino acid sequences of tryptic peptides from the performic acid-oxidized trypsin inhibitor were determined. The degradation was performed with a new reagent, 3-isothiocyanato-4-methoxy-4'-nitrostilbene, and compared with the dansyl-Edman technique. The amino acid sequence of the trypsin inhibitor from bovine splenic capsule was found to be the same as that of the basic trypsin inhibitor from bovine pancreas, established by Kress & Laskowski (J. Biol. Chem. 1967, 242, 4925-4928).

Amino acid sequence of human muscle glyceraldehyde-3-phosphate dehydrogenase. Isolation and amino acid sequences of tryptic peptides.

1. The amino acid compostion, N- and C-terminal amino acid sequences, and the subunit molecular weight of glyceraldehyde phosphate dehydrogenase from human muscle, were determined. The obtained results and the maps of tryptic peptides suggest that the enzyme is composed of four identical or very similar polypeptide chains. 2. From the tryptic digest of performic acid-oxidized enzyme, 32 peptides were isolated. The amino acid sequence analysis showed a high degree of homology with the corresponding tryptic peptides of the dehydrogenase from pig muscle, with 9 replacements and probably two additional amino acids in the examined sequences of the human muscle enzyme.