Biospecific affinity chromatography of an adenosine 3':5'-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides.
1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3':5'-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N(6)-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP-Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50-100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N(6)-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP-Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.
D000227:Adenine Nucleotides; D000249:Adenosine Monophosphate; D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D000242:Cyclic AMP / Q000378:metabolism*; D004590:Electrophoresis, Paper; D008297:Male; D013058:Mass Spectrometry; D011479:Protamines; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013737:Testis / Q000201:enzymology; D014337:Trout; D014462:Ultrafiltration
D000227:Adenine Nucleotides; D011479:Protamines; D000249:Adenosine Monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases