Inactivation of phosphoglycerate mutase and creatine kinase isoenzymes in human serum

Abstract

Aims/background: Total phosphoglycerate mutase (PGM) activity in serum has been shown to be increased in acute myocardial infarction with the same time course as creatine kinase (CK) activity. However, the increase in the muscle (MM) and in the cardiac (MB) PGM isoenzymes was not as high as expected. The present study was undertaken to characterise PGM inactivation by serum and to compare it with serum CK inactivation.

Methods: The PGM and the CK activities of extracts of human heart, skeletal muscle, and brain were determined spectrophotometrically after incubation with different media, namely: plasma, whole serum, dialysed serum, heated serum, serum ultrafiltrate, urate solution, and buffer solution.

Results: Type MM PGM was inactivated by plasma, whole serum, heated serum, dialysed serum, and serum ultrafiltrate. Inactivation in dialysed serum was reduced by EDTA and largely reversed by thiol agents. Inactivation in serum ultrafiltrate was not prevented by EDTA and only partially reversed by dithiothreitol. The muscle and type BB CK isoenzymes were inactivated in all the tested media. The incubation of human and rabbit skeletal muscle PGM and CK in urate solution showed that urate does not affect mutase activity under conditions that inactivate CK.

Conclusions: These results confirm the mechanisms of CK inactivation proposed by others and show that the type M PGM subunit is inactivated by two different mechanisms, which appear to involve the thiol groups of the enzyme. One mechanism is caused by either a protein component or a protein bound serum component and involves calcium ions and/or another chelatable metal ion. The other mechanism is caused by a lower molecular weight serum component and is metal ion independent.

Figure 1

Electrophoretogram of phosphoglycerate mutase (PGM) isoenzymes in extracts of human heart incubated in plasma and serum. (A) Plasma at 0°C; (B) plasma at 37°C: lane 1, 0 minutes; lane 2, 12 hours; lane 3, 24 hours; lane 4, 48 hours. (C) Serum at 37°C; (D) preheated serum (60°C, 20 minutes) at 37°C: lane 1, 0 minutes; lane 2, four hours; lane 3, eight hours; lane 4, 24 hours.

Figure 2

Inactivation of phosphoglycerate mutase (PGM) in human muscle extracts incubated at 37°C in 20mM Tris/HCl buffer pH 7.5 (A,B), in whole serum (C,D), in dialysed serum (E,F), and in serum ultrafiltrate (G,H). Extracts were incubated in the absence (A,C,E,G) and in the presence (B,D,F,H) of 5mM EDTA. The activity of PGM was measured directly (continuous lines) and after 30 minutes of incubation at 30°C with 10mM dithiothreitol (discontinuous lines).

Figure 3

Inactivation of creatine kinase (CK) activity of human muscle extracts incubated at 37°C in 20mM Tris/HCl buffer, pH 7.5 (A,B), in whole serum (C,D), in dialysed serum (E,F), and in serum ultrafiltrate (G,H). Extracts were incubated in the absence (A,C,E,G) and in the presence (B,D,F,H) of 5mM EDTA. The activity of CK was measured directly (continuous lines) and after 30 minutes of incubation at 30°C with 10mM dithiothreitol (discontinuous lines).

Figure 4

Inactivation of phosphoglycerate mutase (PGM) in human brain extracts incubated at 37°C in 20mM Tris HCl buffer pH 7.5 (A,B) and in whole serum (C,D). Extracts were incubated in the absence (A,C) and in the presence (B,D) of 5mM EDTA. The activity of PGM was measured directly (continuous lines) and after 30 minutes of incubation at 30°C with 10mM dithiothreitol (discontinuous lines).

Figure 5

Inactivation of creatine kinase (CK) in human brain extracts incubated at 37°C in 20mM Tris HCl buffer pH 7.5 (A,B) and in whole serum (C,D). Extracts were incubated in the absence (A,C) and in the presence (B,D) of 5mM EDTA. The activity of PGM was measured directly (continuous lines) and after 30 minutes of incubation at 30°C with 10mM dithiothreitol (discontinuous lines).