Purification of bicarbonate-sensitive sperm adenylylcyclase by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-affinity chromatography.
Anion transport inhibitors, such as SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) and heparin, inhibit reversibly the bicarbonate-sensitive adenylylcyclase of porcine sperm plasma membrane. In the light of this, SITS- and heparin-affinity chromatographies were applied in order to purify sperm adenylylcyclase. SITS-Affi-Gel 102 binds proteins extracted from the porcine cauda epididymal sperm plasma membrane by Lubrol-PX, more selectively than heparin-agarose. However, recovery of adenylylcyclase activity is higher when heparin-agarose is used. The hormone-sensitive liver adenylylcyclase, which is less sensitive to bicarbonate than sperm enzyme, has less affinity for these affinity resins than sperm enzyme. Adenylylcyclase can be purified to apparent homogeneity on two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) from the Lubrol-PX extract of the purified sperm plasma membrane by using SITS-affinity chromatography at the first step of the purification followed by preparative isoelectric focusing and gel filtration. The molecular weight and pI of the purified enzyme are 46,300 and 6.9, respectively. The purified enzyme activity is highly dependent on Mn2+. Bicarbonate activates even the purified enzyme both by decreasing Km and by increasing Vmax.
Okamura
N
N
Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki, Japan.
Tajima
Y
Y
Onoe
S
S
Sugita
Y
Y
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000737:chemistry; D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D012964:Sodium / Q000494:pharmacology; D017693:Sodium Bicarbonate; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology*; D013552:Swine
D001639:Bicarbonates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017693:Sodium Bicarbonate; D012964:Sodium; D000262:Adenylyl Cyclases