‘Fingerprinting’ of 1-dimethylaminonaphthalene-5-sulphonyl-labelled protein digests

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

“…Atherton, Laws, Miles & Thomson (1970a) gaveapreliminaryaccount of the sequence around the essential thiol group of ox BB isoenzyme, and this was largely identical with the MM isoenzyme. These studies confirmed the conclusion reached by Eppenberger et al (1967), Thomson et al (1968a) and Atherton & Thomson (1969) that creatine kinases from the same tissue of different species are more alike than those from different tissues ofthe same species. Der Terrossian, Pradel, Kassab & Thoai (1969) found that the amino acid sequence around the essential thiol group of lobster arginine kinase is very similar to the sequences mentioned above, showing that this part of the sequence of guanidinophosphotransferases has been substantially conserved throughout their evolution.…”

“…Thus the two major radioactive peptides T2 and T5 accounted for 16.8 and 52.1% respectively of the radioactivity in the tryptic digest, and subsequent purification showed that they contained no other contaminating radioactive peptides; purified peptides T2 and T5 contained respectively 11.6 and 27.5% of the radioactivity in the tryptic digest; they had identical specific radioactivities, and peptide T2 was shown to be a hydrolysis product of peptide T5. This evidence, together with the results of peptide 'mapping' (Thomson et al 1968a;Atherton & Thomson, 1969), therefore strongly suggests that the two essential thiol groups are identical and that ox brain creatine kinase itself consists of two identical subunits each with a single catalytic site and one reactive and essential thiol group. Arginine kinase of invertebrate muscle fulfils the same role as creatine kinase in vertebrates and is generally regarded as its evolutionary precursor, a view supported in general terms by its amino acid composition (Virden & Watts, 1966;Der Terrossian, Kassab, Pradel & Thoai, 1966;Thomson et al 1968a) and by mechanistic studies (Smith & Morrison, 1969).…”

Synthesis of O‐phosphonotyrosyl peptides

“…Dimethyl, diethyl, and dibenzyl phosphorochloridates were prepared by reaction of the corresponding hydrogen phosphonates (Fluka) as described (25) and used immediately. Dimethyl, diethyl, and dibenzyl phosphorochloridates were prepared by reaction of the corresponding hydrogen phosphonates (Fluka) as described (25) and used immediately.…”

Bovine brain creatine phosphotransferase: amino acid sequence around the essential thiol groups

“…The guanidino phosphotransferases form a closely related group of enzymes of similar properties and function. When creatine phosphotransferases from muscle and brain tissues of the same species are compared significant differences in their Km values, stability, response to alkylation, amino acid composition and peptide 'maps' are observed Thomson, Eveleigh, Laws & Miles, 1968;Atherton & Thomson, 1969). However, there is good evidence that in the region of their essential thiol groups the amino acid sequences are similar (Thomson et al 1968).…”