Cock seminal plasma acid phosphatase: active site directed inactivation, crystallization and in vitro denaturation-renaturation studies.
1. Seminal plasma acid phosphatase from the mini Rock cocks was purified on a Sepharose 6B column and was not homogeneous on polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Its stability in time was also determined. 2. In the same time, the enzyme was crystallized in both ethanol and ammonium sulfate and this is also an evidence that this acid phosphatase was obtained in an advanced grade of purification but is present in a complex with some quantities of other proteins. 3. It was inactivated by iodoacetate to a degree consistent with the modification of an active site residue. DTNB and thiosulfate also inhibited this enzyme. 4. The enzyme was sensitive to 6 M guanidinum hydrochloride which in the presence and absence of 0.25 M mercaptoethanol produces a deep loss of activity. After dialysis the activity was increased during the first 10 days up to 66.2% of the initial one. 5. In the presence of molar concentration of mercaptoethanol, the enzyme activity is deeply decreased, but it is partially restored after 120 hr when 1 microM CuCl2 is added.
Dumitru
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IF
Department of Enzymology and Biotechnology, University of Bucharest, Faculty of Biology, Romania.
Dinischiotu
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A
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003460:Crystallization; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D066298:In Vitro Techniques; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008297:Male; D011489:Protein Denaturation / Q000187:drug effects; D012661:Semen / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D006146:Guanidines; D007461:Iodoacetates; D013439:Sulfhydryl Reagents; D000135:Acid Phosphatase; D019791:Guanidine; D019807:Iodoacetic Acid