Characterization of MB creatine kinase isoform conversion in vitro and in vivo in dogs.

Abstract

Time-dependent removal of the COOH-terminal lysine residue from each subunit of tissue MM creatine kinase by plasma carboxypeptidase N produces two additional isoforms that are readily separated, thereby permitting sensitive, early detection of acute myocardial infarction. Only two isoforms of MB creatine kinase have been detected in plasma leading to speculation that the COOH-terminal lysine on the B subunit is resistant to hydrolysis. To define the biochemical changes resulting in MB creatine kinase isoform conversion, we incubated highly purified MB creatine kinase from canine myocardium with plasma carboxypeptidase N. Quantitative anion-exchange chromatography of incubation mixtures and serial plasma samples from dogs subjected to coronary occlusion revealed a second, more acidic form evolved with time that was separated from the tissue isoform. Cyanogen bromide digestion of the two isoforms followed by amino acid sequencing of COOH-terminal peptides showed that MB creatine kinase undergoes removal of the COOH-terminal lysine residue from both M and B subunits. An intermediate form lacking lysine on the M subunit was delineated during incubations by the combined use of anion-exchange chromatography and conventional electrophoretic techniques. Thus, sequential cleavage of lysine from subunits of MB creatine kinase produces an intermediate isoform that has not been detected previously because of difficulties separating it from the tissue and fully converted isoforms.

Carboxypeptidase N and Creatine Kinase-MB Isoforms in Acute Myocardial Infarction

“…Several studies have shown that carboxypeptidase N is the main factor accounting for isoform conversion in human plasma under physiological conditions (7)(8)(9)(10)(11) and after myocardial infarction, elucidating in particular the relation between carboxypeptidase N activity in plasma and observed rates of creatine kinase-MM isoform conversion (13).…”

“…(circulating des-lysine isoform) ratio is a sensiis available for their ™asurement, this assay is also an tive and specific marker for the early diagnosis of acute mterestm S too1 for the earl y diagnosis of acute Wocardial infarction in emergency (5,6). Previous studies (1)(2)(3)(4)(5)(6)(7)(8)(9) indicated that the tissue creatine kinase-MB 2 iso-1) Enzymes:…”

Regulation of expression of M, B, and mitochondrial creatine kinase mRNAs in the left ventricle after pressure overload in rats.

“…17 The column was equilibrated with 20 mM Tris-Cl, pH 8.4. A sample of supernatant containing 1 mg protein18 was applied to the column and eluted with a linear gradient of NaCl from 0 to 400 mM (10 mmol/ml) in 20 mM Tris-Cl, pH 8.4, containing 5 mM 2-mercaptoethanol.…”

Noninvasive assessment of reperfusion and reocclusion after thrombolysis in acute myocardial infarction

“…Especially, the existence of perhaps 3 CK-MB isoforms must be clarified. 42,43 Myoglobin: Myoglobin is a cytosolic hemoprotein in cardiac and skeletal muscles (M, 17,700) necessary for the last step in the oxygen transport to the mitochondria.71*72 Normal serum levels of myoglobin are reported to be in the range of 6-85 ng/mL and 4-60 ng/mL for men and women, respectively. 3o After acute myocardial infarction, the serum concentration of myoglobin becomes abnormal in about 2 hours, peaks in about 6-9 hours, and becomes normal in 24-36 hours.…”