Human cytosolic adenylate kinase allelozymes; purification and characterization.
Cytosolic adenylate kinase (ATP + AMP in equilibrium 2 ADP:AMP phosphotransferase, EC 2.7.4.3) shows a balanced polymorphism of AK1*1 (95%) and AK1*2 (5%). AK1*2 occurs only in Caucasian populations and is common among hemophilia-A patients (László, A., Kósa, F., Zimányi, I. and Egyed, A. (1985) Acta Paediatr. Hung. 26, 87-96). 1. From 350 g heterozygous human muscle we purified 7.5 mg AK1*2 and 8.5 mg AK1*1. The final step was the separation of the two allelozymes by preparative cellulose acetate gel electrophoresis. Throughout the preparation, the AK1 allelozymes were stabilized by adding diadenosine pentaphosphate. When necessary the proteins were freed from this ligand using phosphocellulose chromatography at pH 5.2. The specific activity of AK1*2 (420 U/mg protein at 25 degrees C) is 3.5-times lower than that of AK1*1, whereas the Michaelis constants do not differ for the allelozymes. The molecular mass of both proteins was found to be 21.7 kDa. 3. High-performance liquid chromatography of tryptic peptides revealed no differences between AK1*1 and AK1*2 except for the peptide t(109-127). Sequence analysis showed that Glu-123 in AK1*1 is exchanged for Gln-123 in AK1*2. In light of the known three-dimensional structure of AK1*1, this substitution alone might account for the different properties of the allelozymes.
Luz
C M
CM
Institut für Biochemie II der Universität, Heidelberg, F.R.G.
König
I
I
Schirmer
R H
RH
Frank
R
R
D000263:Adenylate Kinase / Q000302:isolation & purification*; D000483:Alleles; D003600:Cytosol / Q000201:enzymology; D004586:Electrophoresis / Q000379:methods; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D005973:Glutamine / Q000032:analysis; D006801:Humans; D007527:Isoenzymes / Q000032:analysis; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010770:Phosphotransferases / Q000302:isolation & purification*; D011487:Protein Conformation
D005971:Glutamates; D007527:Isoenzymes; D010446:Peptide Fragments; D005973:Glutamine; D018698:Glutamic Acid; D010770:Phosphotransferases; D000263:Adenylate Kinase