Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. I. Experimental conditions to quantitate membrane-bound adenylyl cyclase activity.
Although cyclic adenosine monophosphate (cAMP) is an important regulator of motility and metabolism in human spermatozoa, little is known on the cellular system responsible for its synthesis. Here, we investigated the experimental conditions directly to quantitate adenylyl cyclase (AC) activity synthesizing cAMP in human ejaculated spermatozoa and analysed the general properties of the enzyme. A 10,000 g membrane fraction was prepared from washed sperm cells homogenized by sonication. AC activity was monitored by the direct conversion of [alpha-32P]adenosine triphosphate (ATP) into [32P]cAMP. Using a nucleoside triphosphate regenerating system to ensure availability of ATP substrate, the human sperm AC showed a steady production of cAMP for at least 1 h. The assay was optimized for pH, buffer concentration, membrane protein and substrate concentration. Activity was dependent upon the presence of Mn2+ as a divalent cation and showed a pH optimum between 7.0 and 8.5. Optimal activity required 5 mM ATP, 1 mM ethylenediamine tetraacetic acid (EDTA) and 20-40 mM total MnCl2. Dependence on Mn2+ was not mandatory; Mg2+ at 5-40 mM also supported significant activity, but the activity was 4-6 times lower than that with Mn2+. Regardless of the presence of Mn2+ or Mg2+ as divalent cation in the assay, human sperm AC was insensitive to the regulatory ligands NaF, guanine nucleotide or forskolin. Insensitivity to these ligands supports the proposal that this enzyme system does not contain a stimulatory guanine nucleotide-binding regulatory protein and that its catalytic component is unique and different from that of somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Rojas
F J
FJ
Department of Obstetrics and Gynecology, University of California, Irvine, Orange.
Bruzzone
M E
ME
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000328:Adult; D002021:Buffers; D002462:Cell Membrane / Q000201:enzymology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis*; D004492:Edetic Acid / Q000494:pharmacology; D004542:Ejaculation; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013094:Spermatozoa / Q000201:enzymology*
D002021:Buffers; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D004492:Edetic Acid; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D008274:Magnesium