Tetrathionate-blocked creatine kinase as a substrate for human plasma ‘creatine kinase conversion factor’

Purification and determination of the modifying protein responsible for the post-synthetic modification of creatine kinase (EC 2.7.3.2) and enolase(EC 4.2.1.11)

“…Removal of more than 95% of the serum proteins, which can possibly inhibit the modifying activity, and of other eventual disturbing factors may be an explanation for this > 100% recovery. The same phenomenon was recently described by Edwards and Watts [9]. Addition of albumin to the modification test medium resulted in substantial inhibition of modifying activity (data not shown) which supports our supposition.…”

Human ‘creatine kinase conversion factor’ identified as a carboxypeptidase

“…Incubation mixtures contained 50mM-Tris/HCl buffer, pH 8.0, 2Oil (1 .3 units) of partially purified CK conversion factor (preparation described by Edwards & Watts, 1983), tetrathionate-treated human or rabbit CK, as previously described by Edwards & Watts (1983), to a final activity of 500 units/I (1 unit consumes 1 pmol of phosphocreatine/min at 30°C in the assay mixture; Szasz et al, 1976) and any other additions in a total volume of 100 ul. All incubations were carried out at 37°C.…”

“…This 'converts' the single band of a skeletal-muscle extract, resolved by agarose-gel electrophoresis, into a triple-banded pattern. The slowest band corresponds to the original MM isoenzyme, and the intermediate and fastest forms are thought to represent modification of one and both subunits respectively (Wevers et al, 1978;Yasmineh et al, 1981;Falter et al, 1982;Edwards & Watts, 1983). Some enzyme activity is lost during conversion of the native MM isoenzyme in vitro; thiols stabilize the enzyme, but also inhibit CK-conversion-factor activity.…”

“…Some enzyme activity is lost during conversion of the native MM isoenzyme in vitro; thiols stabilize the enzyme, but also inhibit CK-conversion-factor activity. Reversibly blocking the reactive thiol group with tetrathionate, however, protects against activity loss, but does not inhibit conversion (Edwards & Watts, 1983). The same workers excluded the possibility of CK conversion factor acting as an endopeptidase.…”

Captopril-induced creatine kinase elevations: a possible role of the sulfhydryl group.

“…9 ' l0 Sulfhydryl reagents including penicillamine reportedly inhibit CK conversion factor. 9 ' 10 In addition, one of the sulfhydryl reagents, 2-mercaptoethanol, inhibits carboxypeptidase N (kininase I)," and captopril is a potent inhibitor of angiotensin converting enzyme (kininase II). Although the effect of carboxypeptidase N or angiotensin converting enzyme on CK is not known, some carboxypeptidases, especially A or B, and proteinase K, have been reported to influence the sulfhydryl group of CK and change the molecular configuration, resulting in a loss of activity.…”