Unspecific Arginine Kinase of Molecular Weight 150000. Amino Acid Composition, Subunit Structure and Number of Substrate Binding Sites

Abstract

The amino acid composition of unspecific arginine kinase of molecular weight 150000 of Sabella pavonina muscle has been determined. It was found to be very similar to that of the phosphagen kinases previously studied.The subunit structure of the enzyme has been investigated by physical and chemical means.The data obtained from ultracentrifugation studies in 6 M guanidine hydrochloride and from molecular sieving and disc electrophoresis in 8 M urea, as well as the tryptic peptide mapping, suggest that Sabella muscle arginine kinase is composed of four non-covalently linked polypeptide chains, with similar molecular weights. The number of binding sites for the nucleotide substrate ADP-MgZ' has been estimated, using differential spectrophotometry and gel filtration on Sephadex columns. By both methods it was demonstrated that the enzyme contains two catalytic sites per protein molecule of molecular weight 150 000.Thus, arginine kinase from Sabella muscle, of molecular weight 150000, consists of four similar polypeptide chains, but possesses only two substrate binding sites per tetrameric molecule.The purification and some properties of a new molecular form of arginine kinase, isolated from the body-wall muscle of the marine annelid, Sabella pavanina, have been reported earlier [ l ] . The most interesting features of the enzyme were its molecular weight around 150000 [1,2], which is two or four times that of the invertebrate arginine kinases studied previously [3-101, and its comparatively wide specificity towards the guanidine substrates, especially towards the L and D forms of arginine [1,2, 111, in contrast with the strict specificity of most arginine kinases for L-arginine [11 -131.The present work is concerned with the study of the amino acid composition of the new enzyme, with the investigation of its quaternary structure by physical and chemical methods, and with the determination of the number of binding sites for the nucleotide substrate ADP-Mg2+. The results show that, in spite of significant differences in the molecular size and in the ratio of number of catalytic sites to number of subunits per enzyme molecule, several structural similarities can be pointed out between Sabella muscle arginine kinase and other muscle phosphagen kinases. EXPERIMENTAL PROCEDURE MaterialsSabella muscle arginine kinase was prepared as described previously [l], using ultrafiltration in a Diaflo cell (Amicon) fitted with a PM-30 membrane instead of Lyphogel, for the concentration of the protein solutions. The enzyme is labile and was utilized within 24 h following the last purification step, the final precipitation with ammonium sulfate being omitted and the enzyme being brought to a protein concentration of 5 to 10 mg/ml by ultrafiltration. Unspecific Arginine Kinase of Molecular Weight 150000 grade) and egg albumin (B grade) were from Calbiochem, trypsin (treated with N,N-dicyclohexyl carbodiimide) from Miles. Urea (Merck) was purified according to Poulik [16]. Guanidine hydrochloride RP from Carlo Erba was used without fur...

Comparison and Characterization of Arginine Kinases Purified from the PrawnPenaeus japonicus(Kurumaebi) and the Swimming CrabPortunus trituberculatus(Gazami)

“…the lobster Homarus americanus,2) the crayfish Panulirus longipes,3) the tobacco hornworm Manduca sexta,4) and the sea urchins Hemicentrotus pulcherrimus, A nth 0 cidaris crassispina, and Pseudocentrotus depressus. S ) It has been purified and characterized from various species such as the sea crayfish Jasus verreauxi,6,7) the marine polychaetous annelid Sabella pavonina, 8,9) the honeybee Apis mellijera,!O) and the squid Symplectoteuthis oualaniensis.ll) After extensive studies, it was found that argmme kinase is important in muscle contraction. Watts 12 ) has compared certain characteristics of this widespread invertebrate arginine kinase with vertebrate creatine kinase and concluded that they had a common genetic origin.…”