83
Clinica Chimica Acta, 97 (1979) 83.-88 0 Elsevier/Norkh-Holland Biomedical Press
CCA 1113
SERUM CREATINE
KINASE BB LINKED TO IMMUNOGLOBULIN
G
JtiRGEN BOHNER, WOLFGANG STEIN *, ELISABETH KUHLMANN and MANFRED EGGSTEIN Medizinische Klinik Tiibingen, Abteilung fiir Znnere Medizin ZV, 7400 Tiibingen 1 (F.R.G.) (Received March 24th, 1979)
Summary
We describe an atypical form of creatine kinase in the serum of a woman after myocardial infarction. Electrophoresis on agarose gel showed a single fraction between CK-MM and CK-MB isoenzymes. After ion-exchange chromatography on DEAE-Sephadex A-50 we isolated the atypical CK and CK-BB as detected by electrophoresis of the eluted fractions. Results of our immunological investigations and exclusion chromatography clearly demonstrate that the atypical CK consists of complexes with high molecular masses formed by CK-BB isoenzyme and immunoglobulin G. The clinical significance, if any, of this Macro CK has yet to be determined.
Introduction Three main forms of ATP:creatine phosphotransferase (EC 2.7.3.2), CK, have been described: CK-MM from skeletal muscle, CK-MB of myocardial origin and CK-BB from brain cells, having a relative molecular mass of 80 000 each [l]. Hitherto, in a few cases some atypical ratios of CK-MM/CK-MB/ CK-BB [ 2-41 and atypical isoenzyme patterns after electrophoresis [ 3,5] have been described. Yuu et al. [6] and Henderson et al. [ 71 describe macromolecular CK, but either the Macro CK was extremely labile [ 71 or the authors failed to demonstrate the macromolecular part [6] of their Macro CK. Urdal and Landaas [8] reported a case of persistently above normal activity of CK. By exclusion chromatography and immunological experiments they were able to conclude that this CK is CK-BB bound to immunoglobulin G, but they did not calculate the molecular weight. Here we describe a further case of abnormal CK. In addition we are able to give data about the molecular weights, to show the nature of the bound CK isoenzyme after dissociation and to determine the macromolecular parts of the stable complexes. * To whom comespondence should be addressed.
84
Materials and methods If possible, investigations were done within 6 h after the blood was drawn. One part of the serum was frozen at -20°C with mercaptoethanol (20 mmol/l). Total CK activity was measured by a N-acetyl-cysteine activated method (Boehringer GmbH, Mannheim) according to the recommendations of the German Society of Clinical Chemistry [9]. Fractions from ion-exchange chromatography were assayed by the method for the ACA (Du Pont Ltd.), except that we mixed a l-ml aliquot of each fraction with 4 ml of water and manually injected this into the CPK test packs, bypassing the ACA filling station. Sephadex G-200 sf (50 X 0.9 cm) (Pharmacia, Uppsala) was used with an eluting buffer of 50 mmol/l tris(hydroxymethyl)-aminomethane (Tris) (pH 7.3 at 25°C) containing 10 mmol/l of N-acetyl-cysteine, 10 mmol/l of ethylenediaminetetraacetate, 20 mmol/l sodium chloride, and 200 mg/l of sodium azide. The flow rate was 0.8 ml/h. The molecular masses were calculated from the K,, value [lo] and the relationship between the elution volumes (u, - u,) of proteins and their molecular masses. The isoenzymes (from 1 ml serum) were separated by ion-exchange chromatography on DEAE-Sephadex A-50 (5 X 0.8 cm bed) with g-ml portions of 50 mmol/l Tris buffer containing 100, 200 or 500 mmol/l sodium chloride
[ill. CK isoenzymes were separated electrophoretically in 0.9% agarose gel for 40 min (4.5 V/cm) with kit reagents from Sigma Chemie GmbH, Miinchen. CK-B activity was determined by use of an inhibiting antibody against the CK monomer M (Merck-l-Test, CK-MB, E. Merck, Darmstadt) [12]. Precipitating antibodies from sheep against CK-nn and CK-BB (E. Merck, Darmstadt) were used according to the vendor’s instructions [ 121. For characterisation of the CK-linked immunoglobulins the double immunodiffusion method (Ouchterlony) was performed in agarose. The precipitin lines formed by specific antisera (anti IgG, anti IgA, anti IgM) were stained for CK activity as described above. Quantitative precipitation of the immunoglobulinCK complexes was performed by adding increasing volumes of antisera against human immunoglobulins (Behring Werke, Marburg) to the patient’s serum (incubation time: 1 h at 37°C plus overnight at 4°C). After centrifugation at 7500 X g, the CK activities were determined in the supernatants. Case report
The serum studied was obtained from patient Doe. (Pat. No. 12580/6/17), a woman aged 58 years with coronary heart disease who had an acute myocardial infarction in August 1978. Diagnosis was based upon characteristic clinical signs and ECG changes. Surprisingly, for 7 months, we observed a persistently elevated total CK (130 U/l) and CK-MB isoenzyme activities, the latter being determined by an immunoinhibition test. CK-MB activity was 86% of the total CK activity on the average. Results The persistently raised activity of total CK and the atypical and contradictory ratios of total CK versus CK-MB or CK-BB found in the routine tests [ 11,121 were investigated and are explained as makro CK.
85
n/lolecular size: Fig. 1 shows the profile of Sephadex G-200 exclusion chroma~~aphy. This elution pattern suggests the presence of two different complexes of CK with relative molecular masses of about 350 000 (fraction 39) and 650000 (fraction 34), whereas normal CK (fraction 54) has a relative molecular mass of 80 000 [ 11. Ion-exchange chromatography and electrophoresis: Fig. 2 shows the elution pattern from ion-exchange chromatography. CK activity is observed in all three fractions with a large tailing between fraction B and fraction C, Evidently no CK remained on the column for recovery was 104%. However, electrophoresis of the fractions A, B, C clearly showed (Fig. 2) an atypical CK isoenzyme which was eluted as fraction A (simulating normal CK-MM) and fraction B (simulating CK-MB). Only fraction C (CK-BB) migrated to the expected position on eleetrophoresis. The amount of this atypical CK eluted during ionexchange chromato~p~y was found to be ~m~ependent. Upon treatment of the serum with DEAE-Sephadex A-50 up to 24 h at room temperature, the ratio of atypical CK : CK-BB was shifted towards CK-BB. After 24 h only a small amount of atypical CK is found in fraction A and B and almost the total CK activity is eluted as fraction C (CK-BB). After electrophoresis of the serum an atypical isoenzyme with only little electrophoretic mobility was detected (Fig. 2) between CK-MB and CK-MM bands. All these bands were absent, when creatine phosphate was omitted from the staining reagents. Immu~oZogic~l ~nuest~gat~o~s: Reacting the serum with precipitating anti CK-nn serum -did not influence CK activity in the supernatant, whereas, after incubation with anti CK-BB serum, no activity remained in the supemaht ? +
1EW-T234 CK
CU/‘lI
+
78.0 03. 0
Fig. 1. Elution pattern of exclusion chromatography on Sephadex E2OOsf (50 X O.Qcm column). Elution buffer: 50 mmoln Tris, pH 7.3; flow rate: 0.8 ml/h; fractions: 0.3 ml. + * =* * * *+. Transmission of proCK activity patient Doe.
Clinica Chimica Acta, 97 (1979) 83.-88 0 Elsevier/Norkh-Holland Biomedical Press
CCA 1113
SERUM CREATINE
KINASE BB LINKED TO IMMUNOGLOBULIN
G
JtiRGEN BOHNER, WOLFGANG STEIN *, ELISABETH KUHLMANN and MANFRED EGGSTEIN Medizinische Klinik Tiibingen, Abteilung fiir Znnere Medizin ZV, 7400 Tiibingen 1 (F.R.G.) (Received March 24th, 1979)
Summary
We describe an atypical form of creatine kinase in the serum of a woman after myocardial infarction. Electrophoresis on agarose gel showed a single fraction between CK-MM and CK-MB isoenzymes. After ion-exchange chromatography on DEAE-Sephadex A-50 we isolated the atypical CK and CK-BB as detected by electrophoresis of the eluted fractions. Results of our immunological investigations and exclusion chromatography clearly demonstrate that the atypical CK consists of complexes with high molecular masses formed by CK-BB isoenzyme and immunoglobulin G. The clinical significance, if any, of this Macro CK has yet to be determined.
Introduction Three main forms of ATP:creatine phosphotransferase (EC 2.7.3.2), CK, have been described: CK-MM from skeletal muscle, CK-MB of myocardial origin and CK-BB from brain cells, having a relative molecular mass of 80 000 each [l]. Hitherto, in a few cases some atypical ratios of CK-MM/CK-MB/ CK-BB [ 2-41 and atypical isoenzyme patterns after electrophoresis [ 3,5] have been described. Yuu et al. [6] and Henderson et al. [ 71 describe macromolecular CK, but either the Macro CK was extremely labile [ 71 or the authors failed to demonstrate the macromolecular part [6] of their Macro CK. Urdal and Landaas [8] reported a case of persistently above normal activity of CK. By exclusion chromatography and immunological experiments they were able to conclude that this CK is CK-BB bound to immunoglobulin G, but they did not calculate the molecular weight. Here we describe a further case of abnormal CK. In addition we are able to give data about the molecular weights, to show the nature of the bound CK isoenzyme after dissociation and to determine the macromolecular parts of the stable complexes. * To whom comespondence should be addressed.
84
Materials and methods If possible, investigations were done within 6 h after the blood was drawn. One part of the serum was frozen at -20°C with mercaptoethanol (20 mmol/l). Total CK activity was measured by a N-acetyl-cysteine activated method (Boehringer GmbH, Mannheim) according to the recommendations of the German Society of Clinical Chemistry [9]. Fractions from ion-exchange chromatography were assayed by the method for the ACA (Du Pont Ltd.), except that we mixed a l-ml aliquot of each fraction with 4 ml of water and manually injected this into the CPK test packs, bypassing the ACA filling station. Sephadex G-200 sf (50 X 0.9 cm) (Pharmacia, Uppsala) was used with an eluting buffer of 50 mmol/l tris(hydroxymethyl)-aminomethane (Tris) (pH 7.3 at 25°C) containing 10 mmol/l of N-acetyl-cysteine, 10 mmol/l of ethylenediaminetetraacetate, 20 mmol/l sodium chloride, and 200 mg/l of sodium azide. The flow rate was 0.8 ml/h. The molecular masses were calculated from the K,, value [lo] and the relationship between the elution volumes (u, - u,) of proteins and their molecular masses. The isoenzymes (from 1 ml serum) were separated by ion-exchange chromatography on DEAE-Sephadex A-50 (5 X 0.8 cm bed) with g-ml portions of 50 mmol/l Tris buffer containing 100, 200 or 500 mmol/l sodium chloride
[ill. CK isoenzymes were separated electrophoretically in 0.9% agarose gel for 40 min (4.5 V/cm) with kit reagents from Sigma Chemie GmbH, Miinchen. CK-B activity was determined by use of an inhibiting antibody against the CK monomer M (Merck-l-Test, CK-MB, E. Merck, Darmstadt) [12]. Precipitating antibodies from sheep against CK-nn and CK-BB (E. Merck, Darmstadt) were used according to the vendor’s instructions [ 121. For characterisation of the CK-linked immunoglobulins the double immunodiffusion method (Ouchterlony) was performed in agarose. The precipitin lines formed by specific antisera (anti IgG, anti IgA, anti IgM) were stained for CK activity as described above. Quantitative precipitation of the immunoglobulinCK complexes was performed by adding increasing volumes of antisera against human immunoglobulins (Behring Werke, Marburg) to the patient’s serum (incubation time: 1 h at 37°C plus overnight at 4°C). After centrifugation at 7500 X g, the CK activities were determined in the supernatants. Case report
The serum studied was obtained from patient Doe. (Pat. No. 12580/6/17), a woman aged 58 years with coronary heart disease who had an acute myocardial infarction in August 1978. Diagnosis was based upon characteristic clinical signs and ECG changes. Surprisingly, for 7 months, we observed a persistently elevated total CK (130 U/l) and CK-MB isoenzyme activities, the latter being determined by an immunoinhibition test. CK-MB activity was 86% of the total CK activity on the average. Results The persistently raised activity of total CK and the atypical and contradictory ratios of total CK versus CK-MB or CK-BB found in the routine tests [ 11,121 were investigated and are explained as makro CK.
85
n/lolecular size: Fig. 1 shows the profile of Sephadex G-200 exclusion chroma~~aphy. This elution pattern suggests the presence of two different complexes of CK with relative molecular masses of about 350 000 (fraction 39) and 650000 (fraction 34), whereas normal CK (fraction 54) has a relative molecular mass of 80 000 [ 11. Ion-exchange chromatography and electrophoresis: Fig. 2 shows the elution pattern from ion-exchange chromatography. CK activity is observed in all three fractions with a large tailing between fraction B and fraction C, Evidently no CK remained on the column for recovery was 104%. However, electrophoresis of the fractions A, B, C clearly showed (Fig. 2) an atypical CK isoenzyme which was eluted as fraction A (simulating normal CK-MM) and fraction B (simulating CK-MB). Only fraction C (CK-BB) migrated to the expected position on eleetrophoresis. The amount of this atypical CK eluted during ionexchange chromato~p~y was found to be ~m~ependent. Upon treatment of the serum with DEAE-Sephadex A-50 up to 24 h at room temperature, the ratio of atypical CK : CK-BB was shifted towards CK-BB. After 24 h only a small amount of atypical CK is found in fraction A and B and almost the total CK activity is eluted as fraction C (CK-BB). After electrophoresis of the serum an atypical isoenzyme with only little electrophoretic mobility was detected (Fig. 2) between CK-MB and CK-MM bands. All these bands were absent, when creatine phosphate was omitted from the staining reagents. Immu~oZogic~l ~nuest~gat~o~s: Reacting the serum with precipitating anti CK-nn serum -did not influence CK activity in the supernatant, whereas, after incubation with anti CK-BB serum, no activity remained in the supemaht ? +
1EW-T234 CK
CU/‘lI
+
78.0 03. 0
Fig. 1. Elution pattern of exclusion chromatography on Sephadex E2OOsf (50 X O.Qcm column). Elution buffer: 50 mmoln Tris, pH 7.3; flow rate: 0.8 ml/h; fractions: 0.3 ml. + * =* * * *+. Transmission of proCK activity patient Doe.
