Brain adenosine 5′-triphosphate–creatine phosphotransferase. Purification, thiol group reactivity and the amino acid sequence around the reactive thiol groups

Abstract

1. The purification of creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) from ox brain by a method that is quicker, simpler and gives much higher yields than other published procedures is described. 2. Stoicheiometric inhibition studies with iodoacetate showed that the enzyme, like that from muscle, has two reactive thiol groups that are essential for enzyme activity. 3. The amino acid sequence around the essential thiol groups was determined and found to be virtually identical with that in creatine kinases from rabbit and ox muscle, and very similar to that found in arginine kinase; the evolutionary significance of this is discussed. 4. The identification of DNS-amino acids on thin layers of silica gel was found to have, in many cases, distinct advantages over that on polyamide layers.

Complete nucleotide sequence of Torpedo marmorata mRNA coding for the 43,000-dalton nu 2 protein: muscle-specific creatine kinase.

“…Furthermore, the deduced amino acid sequence includes amino acid 275 to amino acid 291, the sequence previously established by chemical degradation of a peptide containing the active site cysteine residue from rabbit muscle CK (30). Also, amino acids 379 and 380 of the Torpedo se- quence are identical to the known COOH-terminal residues of rabbit muscle CK (31).…”

Comparative Structural Studies of the Active Site of ATP: Guanidine Phosphotransferases

“…The remaining radioactivity is distributed between peptides TI (25. 5 and TIII (6.6*lo). Therefore, it is suggested that peptide TII must represent the essential cysteine tryptic peptide of lombricine kinase.…”

“…Amino acid sequences around the reactive cysteine residues of creatine kinase, arginine kinase and lmbricine kinase. The sequence for creatine kinase from rabbit muscle was determined by Thomson et al [3] and Mahowald [28], and that from OX brain by Atherton et al [ 5 ] ; the sequence for arginine kinase was determined by Der Terrossian et al [6] of creatine kinase tryptic peptide whereas its C-terminal sequence can be superimposed on the same portion of arginine kinase tryptic peptide. The deletion of one amino acid (glycine or threonine) next to the proline in creatine kinase peptides is compensated by the addition of one glycine in the C-terminal region.…”

“…Smyth et al [14] pointed out the formation of a ninhydrin-negative thiazine derivative ; this intramolecular rearrangement could be produced when compound I was exposed to mild alkaline conditions. We must not forget that phenyl isothiocyanate reacts with the a-NH, group at pH 9, this pH value being suitable for the formation of the compound IV : If we make a comparison between the tryptic peptide of lombricine kinase containing the essential thiol group of the enzyme, and the corresponding tryptic peptides of muscle [3,4] and brain creatine kinase [5] and of lobster arginine b a s e [6] we notice several points of considerable similarity (Fig.5). I n these peptides, the unusual presence of proline next to the reactive sulfhydryl is of particular interest.…”

“…The amino acid sequences around the two essential cysteinyl residues of creatine kinases from rabbit [3] and ox muscle [4] and bovine brain [5] (mol. wt = 80000) have been determined previously.…”