Isolation and purification of creatine kinase conversion factor from human serum and its identification as carboxypeptidase N

1987 ◽  
Vol 20 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Lisa Michelutti ◽  
Hermann Falter ◽  
Susan Certossi ◽  
Barbara Marcotte ◽  
Angelo Mazzuchin
Keyword(s):  
Creatine Kinase ◽  
Human Serum ◽  
Conversion Factor ◽  
Isolation And Purification ◽  
Carboxypeptidase N

scholarly journals Human ‘creatine kinase conversion factor’ identified as a carboxypeptidase

1984 ◽  
Vol 221 (2) ◽  
pp. 465-470 ◽  
Author(s):  
R J Edwards ◽  
D C Watts
Keyword(s):  
Creatine Kinase ◽  
Conversion Factor ◽  
Electrophoretic Pattern ◽  
Beta Subunits ◽  
Rabbit Muscle ◽  
Activity Profile ◽  
Muscle Creatine Kinase ◽  
Electrophoretic Patterns ◽  
Carboxypeptidase N ◽  
Low Activity

The effect of partially purified ‘creatine kinase conversion factor’ on rabbit muscle creatine kinase is shown to be that of a carboxypeptidase, removing the C-terminal lysine residue from both subunits. These changes fully explain the three-banded electrophoretic patterns of the partially and the fully modified rabbit and human enzymes. The factor also produces a similar electrophoretic pattern with haemoglobin A; comparison with the effects of carboxypeptidases A and B permits the inference that the C-terminal residues of both alpha- and beta-subunits are removed. Small synthetic peptides are poor or non-substrates. A low activity with hippuryl-L-lysine may be due to contamination of the preparation with carboxypeptidase N. The possibility has been excluded that the action of conversion factor on creatine kinase involves modification of the protein thiol groups. Mr, substrate-specificity, pH-activity profile and the effects of metal ions distinguish creatine kinase conversion factor from carboxypeptidases A, B and N. On the basis of this evidence it is proposed to give the conversion factor the provisional name of carboxypeptidase K.

Monoclonal antibody inhibiting creatine kinase MM3 but not isoform MM1

1990 ◽  
Vol 36 (1) ◽  
pp. 153-156 ◽  
Author(s):  
T Suzuki ◽  
T Shiraishi ◽  
K Tomita ◽  
M Totani ◽  
T Murachi
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Monoclonal Antibody ◽  
Creatine Kinase ◽  
Early Diagnosis ◽  
Human Serum ◽  
Carboxypeptidase N ◽  
Analytical Separation ◽  
Diagnostic Reagent ◽  
Inhibition Index

Abstract Monoclonal antibody CKM-G01 inhibited greater than 99% of the activity of porcine and human creatine kinase(CK)-MM isoenzyme purified from muscle. However, it inhibited only 54% of CK-MM in human serum. Chromatofocusing of serum CK-MM showed that CKM-G01 inhibited 100% of MM3 but not isoform MM1. CKM-G01 inhibited CK-MM2 by 57%. CKM-G01 specifically inhibited only the original CK-M subunit and not the subunit modified by removal of C-terminal lysine by carboxypeptidase N. CKM-G01 can be used for assay of CK isoforms. We devised a new diagnostic reagent involving it, which requires no analytical separation of isoforms, based on the immunoinhibition method, and applied it to early diagnosis of acute myocardial infarction. The "inhibition index," (inhibited CK activity/total CK activity) x 100, increased more rapidly than did total CK and CK-MB. Evidently this diagnostic reagent can be used for easy, early diagnosis of acute myocardial infarction.

Production and certification of secondary enzyme reference materials (ERMs). Part 1: Preparation of the sera and some of their properties.

1986 ◽  
Vol 32 (10) ◽  
pp. 1901-1905 ◽  
Author(s):  
J C Koedam ◽  
G M Steentjes ◽  
S Buitenhuis ◽  
E Schmidt ◽  
R Klauke
Keyword(s):  
Creatine Kinase ◽  
Lactate Dehydrogenase ◽  
Reference Material ◽  
Human Serum ◽  
Dehydrogenase Activity ◽  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Glutamate Dehydrogenase Activity ◽  
The Stability ◽  
Clinical Enzymology

Abstract We produced three batches of a human-serum-based enzyme reference material (ERM) enriched with human aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), creatine kinase (EC 2.7.3.2), and lactate dehydrogenase (EC 1.1.1.27). The added enzymes were not exhaustively purified; thus the final ERMs contained some enzymes as contaminants, of which only glutamate dehydrogenase activity might interfere. The stability during storage and after reconstitution was good. The commutability of the four enzymes in the three ERM batches was also good, except when German or Scandinavian methods for aminotransferases were involved. The temperature-conversion factors for the ERMs were equivalent to those for patients' sera. Reactivation after reconstitution was complete within 5 min and was independent of the temperature of the reconstitution fluid. We believe that these secondary ERMs will aid in the transfer of accuracy between well-defined reference methods and daily working methods so that clinical enzymology results will become more comparable from laboratory to laboratory.

Immunochemical determination of CK-MB isoenzyme in human serum. II. An enzymic approach.

1982 ◽  
Vol 28 (1) ◽  
pp. 54-58 ◽  
Author(s):  
R Wicks ◽  
M Usategui-Gomez ◽  
M Miller ◽  
M Warshaw
Keyword(s):  
Creatine Kinase ◽  
Human Serum ◽  
Adenylate Kinase ◽  
Specific Antibodies ◽  
B Subunit ◽  
Immunochemical Determination ◽  
Agarose Electrophoresis

Abstract A novel immunochemical technique for a specific enzymic determination of the myocardial isoenzyme of creatine kinase, CK-MB, involves determination of B-subunit activity of a specimen in which the M-subunit activity has been inhibited by specific antibodies to the M-subunit. Interfering activities from CK-BB isoenzyme, atypical forms of creatine kinase, and adenylate kinase are eliminated by using a blank tube in which all the M-subunit-containing isoenzymes have been removed by a specific immunoprecipitation step. The assay is convenient, linear, and reproducible, and results compare well with those by agarose electrophoresis.

Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme BB.

1978 ◽  
Vol 24 (3) ◽  
pp. 422-428 ◽  
Author(s):  
M H Zweig ◽  
A C Van Steirteghem ◽  
A N Schechter
Keyword(s):  
Creatine Kinase ◽  
Human Serum ◽  
Cross Reactivity ◽  
Healthy Adults ◽  
Serum Samples ◽  
Creatine Kinase Isoenzymes ◽  
Specific Radioimmunoassay ◽  
Assay Precision ◽  
Free Antigen ◽  
Healthy Persons

Abstract We describe a sensitive, specific radioimmunoassay for the BB isoenzyme of creatine kinase (CK-BB) in serum. A sequential saturation assay was used to achieve sufficient sensitivity to detect the isoenzyme in 100-microliter serum samples of all healthy persons and patients tested. Bound and free antigen were separated by a second antibody system. Large excesses of purified isoenzyme MM did not react in the assay. Cross reactivity of two preparations of CK-MB was only 1 to 7+. The 95th percentile of serum CK-BB in 208 healthy adults was 6.2 microgram/liter. Within-assay and between-assay precision ranged from 5.5 to 11.9% and 9.7 to 13.6%, respectively.

Binding of enzyme--IgG complexes in human serum to Protein-A Sepharose CL-4B.

1980 ◽  
Vol 26 (2) ◽  
pp. 297-300
Author(s):  
K Bauer ◽  
P M Bayer ◽  
E Deutsch ◽  
F Gabl
Keyword(s):  
Alkaline Phosphatase ◽  
Creatine Kinase ◽  
Human Serum ◽  
Immunoglobulin G ◽  
Protein A ◽  
The Other ◽  
Routine Method ◽  
Simple Method ◽  
Human Serum Protein ◽  
Enzyme Complexes

Abstract We describe a simple method for detecting enzyme--immunoglobulin G (IgG) complexes in human serum. Protein-A Sepharose CL-4B binds IgG and therefore also the enzyme--IgG complexes, which can then be separated easily from the serum by centrifugation. We demonstrate this separation in two patients, one with a complex of IgG and creatine kinase (EC 2.7.3.2) BB isoenzyme, the other with an IgG--alkaline phosphatase (EC 3.1.3.1) complex. Both patients had unexplainably high activities of the respective enzymes in their serum. The method we propose should be a useful, simple, routine method of detection in cases where IgG--enzyme complexes are suspected.

A labile enzyme in fresh human serum interferes with the assay of carboxypeptidase N.

1989 ◽  
Vol 35 (1) ◽  
pp. 177-177 ◽  
Author(s):  
D Hendriks ◽  
S Scharpé ◽  
M van Sande ◽  
M P Lommaert
Keyword(s):  
Human Serum ◽  
Carboxypeptidase N

Effect of creatine kinase-MM subtype composition on a CK-MB immunoinhibition assay.

1988 ◽  
Vol 34 (3) ◽  
pp. 535-538 ◽  
Author(s):  
I M Morison ◽  
K J Clayson ◽  
J S Fine
Keyword(s):  
Myocardial Infarction ◽  
Creatine Kinase ◽  
Human Serum ◽  
Time Dependent ◽  
Clinical Specimens ◽  
In Vitro Incubation ◽  
Dependent Change ◽  
Time Dependent Change

Abstract Immunoinhibition (INH) by use of polyclonal anti-human CK-M antibody may be used to measure CK-MB in serum. Previous studies have shown that inhibiting antibodies prepared against purified muscle extracts may inhibit CK-MM by greater than 99%. Using patients' sera and muscle homogenates incubated with human serum, we studied the effect of CK-MM subtype composition on an INH assay. We found that with increasing time from the CK-releasing event, e.g., myocardial infarction, or with longer in vitro incubation, the proportion of CK-MM1 increased and the proportion of uninhibited CK-MM increased from 0.2% to 0.7-0.8%. As a consequence, CK-MB activity may be overestimated by as much as 1.6% of total CK when uncorrected INH results are used. Inhibition was maximal in samples containing 100% CK-MM3, the tissue subtype. Because of the time-dependent change in CK-MM subtypes, published results for INH from studies in which CK-MM purified from muscle was used may not be directly applicable to clinical specimens.

Sign in / Sign up

Export Citation Format

Share Document