Ian T. Forrester

A sodium—calcium exchange mechanism in plasma membrane vesicles isolated from ram sperm flagella

The ability of mature mammalian spermatozoa to maintain a co-ordinated and forward motility is dependent upon intracellular free Ca2+ being maintained within anarrow concentration range [l]. At the level of the flagellar plasma membrane this could be achieved by two systems: (a) an ATP requiring Ca2’pump; or (b) a Na+/Ca2+ antiporter. Although such plasma membrane Ca2+extrusion mechanisms have been found in a variety of cell types [2] they have only recently been experimentally determined in spermatozoa. Using a procedure to selectively fractionate mammalian sperm we have been able to show that there exists in the flagellar plasma membrane of ram sperm a Ca 2+-dependent Mg2+ATPase or Ca2+pump [3,4]. The report demonstrating the presence of both a Ca2+-pump and a Na+/Ca’+ antiporter in membranes isolated from dog sarcolemma [5], has now prompted us to examine sperm plasma membranes for the existence of such an antiporter mechanism. From these studies we hope to obtain a more detailed understanding of Ca” regulation in mammalian spermatozoa.

Evidence for the occurrence of calmodulin in the yeasts Candidas albicans and Saccharomyces cerevisiae

Calmodulin was originally characterised as the calcium-dependent activator of brain cyclic nucleotide phosphodiesterase. Subsequently it was shown to be the modulator of a variety of calcium-dependent cellular activities (reviews [l-3]). Calmodulin is now recognised as being a highly conserved and widely distributed protein, having been demonstrated in many vertebrates and invertebrates [3,4], plants and higher fungi [5,6] as well as several unicellular eukaryotes; e.g., Dictyostelium discoideum [7], Blastocladiella emersonii [8], Euglena gracilis and Amoeba proteus [9]. Prokaryotes, in contrast,appear to lack calmodulin [6,7] although (exogenous) calmodulin has been shown to activate the adenylate cyclase of Bordetella pertussis [lo]. The observed distribution of calmodulin has led to the suggestion that it is ubiquitous in eukaryotes [3,5]. However, the inability of several laboratories to detect calmodulin in yeast [6,7,1 l] is in conflict with this assertion. This paper reports the presence of a calmodulln-like protein in extracts of the yeasts Candida albicans and Saccharomyces cerevisiae. The demonstration of this protein appears to be dependent on the use of the protease inhibitor, PMSF.

A [Ca2+ + Mg2+]-ATPase and active Ca2+ transport in the plasma membranes isolated from ram sperm flagella

Abstract Plasma membranes isolated from the flagella of ram ejaculated sperm were found to contain a [Ca 2+ + Mg 2+ ]-ATPase. Freeze-fracture electron microscopy showed the membranes occur as vesicles. The membrane vesicles actively accumulate Ca 2+ , uptake was reversed by the ionophore A23187 and inhibited by either ruthenium red or La 3+ . The plasma membranes contain two major proteins, designated proteins A and B, with molecular weights of 109,000 and 18,300 daltons, respectively. Protein B is not detected in plasma membranes isolated from ram epididymal sperm. The plasma membrane Ca 2+ pump may be modulated by protein factors present in seminal plasma.

Identification of calmodulin-like activity in human seminal plasma.

Abstract Human seminal plasma was found to contain relatively high levels of a heat stable proteinaceous factor with properties similar to that of the calcium-binding protein calmodulin. The seminal plasma factor increases the (Ca 2+ + Mg 2+ )-ATPase activity found in human red blood cell plasma membranes by 370% and the activation was completely abolished by chlorpromazine, amitriptyline and theophylline. A similar calmodulin-activated Ca 2+ pump, has been found in the plasma membrane of ram sperm tails. The existence of calmodulin in mammalian seminal plasma may be responsible for some of the metabolic changes associated with sperm maturation.

Calmodulin-like activity in a mineralising tissue: The rat molar tooth germ

SummaryCalmodulin, a calcium binding protein, has been implicated in the regulation of many calcium-dependent biological processes. Since calcium has an important role in hard tissue genesis, both at intra- and extracellular levels, we anticipate that calcium binding proteins may modulate this process. The present study investigated a mineralising tissue, the rat molar tooth germ, to determine the presence of calmodulin-like activity. A heat-treated cell-free extract of tooth germs provided enhancement of Ca2+-dependent Mg2+-ATPase and 3′:5′-nucleotide phosphodiesterase activity. No enhancement occurred in the absence of calcium or in the presence of trifluoperazine. SDS-polyacrylamide gel electrophoresis of this extract revealed a protein band of approximately 18,000 mol. wt. These findings indicate the presence of calmodulin-like activity in rat molar tooth germs and support the proposal that calcium and calcium binding proteins, in particular calmodulin, have a major regulatory role in the biology of mineralising tissues.

Identification of a male-specific (H-Y) antigen on the flagellar plasma membrane of ram epididymal spermatozoa

SummaryH-Y (male-specific) antigen has been detected on the plasma membranes of both caput and caudal ram spermatozoa using both immunoperoxidase and immunofluorescence labelling techniques. In these spermatozoa the distribution of H-Y antigen appears to be confined to both the posterior region of the head and the mid-piece region of the flagellum. In addition, caput spermatozoa also exhibit intense immunoperoxidase staining of the cytoplasmic droplet which is situated on the flagellum at the base of the head. Western blot analyses of purified plasma membranes from the flagella of caudal spermatozoa have revealed the presence of a malespecific protein with an estimated molecular weight of 25,000–27,000.