Biochimica et Biophysica Acta, 1022 (1990) 27-32 27 Elsevier BBAMEM 74732 Inhibition by caltrin of calcium transport into spermatozoa, liver and heart mitochondria Haim Breitbart 2, Robert S. Wehbie 1, Jovenal San Agustin 1 and Henry A. Lardy 1Institute for Enzyme Research and Department of Biochemistry, University of Wisconsin, Madison, WI (U.S.A.) and 2 Department of Life Sciences, Bar-Ilan University, Ramat-Gan (Israel) (Received25 July 1989) Key words: Caltrin; Calcium ion transport; Mitochondrion The calcium-transport inhibitor, caltrin, isolated from bovine seminal fluid inhibits calcium accumulation by bovine epididymal spermatozoa, spermatozoal mitochondria, rat liver mitochondria and beef heart mitochondria. Respiration studies demonstrate a marked stimulation of oxygen consumption by caltrin in filipin-treated spermatozoa and rat liver mitochondria. A biphasic effect of caltrin on rat liver mitochondrial respiration was noted, with stimulation at low caltrin concentrations and inhibition as the concentration of caltrin is increased. The ability of caltrin to uncouple and/or inhibit respiration in filipin-treated spermatozoa and isolated liver mitochondria indicates that inhibition of mitochon- drial calcium accumulation by caltrin results from one of these mechanisms. Only a marginal effect of caltrin on respiration of intact spermatozoa was observed; indicating that the plasma membrane is impermeable to this protein. The differential effect of caltrin on respiration of intact and permeabilized spermatozoa indicates that caltrin inhibition of Ca 2 + uptake into spermatozoa in vivo occurs at the level of the plasma membrane. Introduction Caltrin is a 5411 dalton basic protein from bovine seminal plasma that inhibits bovine epididymal sper- matozoa calcium accumulation [1-3]. Previously, we proposed that caltrin inhibits spermatozoa calcium up- take by a mechanism which involves the plasma mem- brane Na+/Ca 2+ exchanger [4]. An integral aspect of this proposal is that caltrin exert its inhibitory effect at the level of the plasma membrane. Recent work from our laboratory demonstrated that ejaculated sperma- tozoa bind anticaltrin IgG over the acrosome and prin- cipal tail regions, but not to the midpiece [5]. In contrast to ejaculated spermatozoa, epididymal spermatozoa readily accumulate calcium and contain no caltrin as is demonstrated by their inability to bind anticaltrin IgG [5]. However, upon incubation with caltrin these cells adopt the regional acrosome/tail dis- tribution of caltrin and calcium transport in inhibited [5]. Babcock et al. [6] have demonstrated that the major- Abbreviation: CCCP, carbonyl cyanide M-chlorophenylhydrazone. Correspondence: H. Breitbart, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52100, Israel. 0005-2736/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical ity of calcium accumulated by bovine spermatozoa is taken up by the mitochondria of the midpiece structure. Thus, the plasma membrane calcium flux inhibited by caltrin apparently operates in the acrosome and/or tail domains, and supplies the midpiece cytosol with calcium. In the present work, we provide further evidence that caltrin inhibits spermatozoa calcium accumulation by interacting with the plasma membrane. In addition, we demonstrate that in permeabilized spermatozoa and in mitochondria from several sources, caltrin inhibits mito- chondrial calcium accumulation by uncoupling and/or inhibiting the mitochondrial electron transport chain. Materials and Methods Purification of caltrin. Bovine semen was the generous gift of American Breeder's Service, DeForest, WI. Caltrin was purified as described earlier [3]. The puri- fied caltrin was stored in a solution composed of 35% (v/v) glycerol, 90 mM NaCI and 10 mM sodium mor- pholinepropane sulfonate (pH 7.4) (Caltrin Storage Buffer). All experiments reported in this paper were conducted with the inhibitory form of caltrin [3]. Preparation of spermatozoa. Bovine epididymides were obtained from local slaughter houses. Epididymal spermatozoa were collected and washed as described previoulsy [7] in a solution (NKM) of 110 mM NaCI, 5 Division)