A specific and saturable spermine-binding component present in bovine testis membranes.
To determine if polyamine (PA)-binding components were present in testis membranes, a membrane fraction derived from homogenates of immature bovine testes was utilized in a radioligand assay. [14C]Spermine ([14C] Sp) bound to a spermine-binding component (SpBC) in a saturable manner and radioligand-binding data were analyzed and fit to a one-site model. Estimates of the binding constant (Kd = 7.2 +/- 1.2 X 10(-6) M) and of the concentration of sites (11.6 +/- 1.6 X 10(-6) M) were determined assuming a one-to-one stoichiometry. Metal cations (Ca+2, Mg+2, Mn+2 and Na+) as chloride salts had no effect on binding of [14C]Sp to SpBC when tested at concentrations up to 10 mM. Not all polyamines tested were effective competitors for [14C]Sp binding, which had an ID50 of 21.0 +/- 2.0 microM. The inhibitory potencies for putrescine and spermidine relative to spermine were 3.1 +/- 0.43 X 10(-4) and 0.1 +/- 0.01 (nmol/nmol) respectively, illustrating the specificity of SpBC binding. In addition, acetylation of polyamines, which is generally associated with degradation and interconversion of polyamines, resulted in a reduction of potency of 25.2 +/- 2.0-fold of spermine and 31.1 +/- 10.6-fold of spermidine in the SpBC radioligand assay. Binding of SpBC was decreased by tryptic digestion of the membrane fraction, suggesting that protein may be a part of the SpBC. Neuraminidase treatment had no effect on [14C]Sp binding but phospholipase-C digestion increased [14C]Sp binding.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D013096:Spermine / Q000378:metabolism; D013737:Testis / Q000378:metabolism*
D002352:Carrier Proteins; D013096:Spermine