Association of bovine sperm aldolase with sperm subcellular components.

The nature of the association of the glycolytic enzyme, aldolase, with mature bovine spermatozoa was investigated in comparison with bovine muscle aldolase. Bovine muscle aldolase (BMA) was optimally solubilized by 0.1% deoxycholate and purified to homogeneity by ammonium sulfate fractionation, gel-filtration chromatography and phosphocellulose affinity chromatography. Bovine sperm aldolase (BSpA) was solubilized with optimal specific activity by 0.1% Triton X-100 and 50 mM sodium phosphate. Soluble BSpA represented 10% of the total aldolase activity in bovine spermatozoa. It could not be purified from other sperm components by standard procedures. The association of BSpA with sperm components involved noncovalent, ionic and hydrophobic interactions and did not involve disulfide bonds or covalent bonds. The stability of the BSpA association with intracellular substructure implies that very specific multiple-ligand bonding is involved. The Km for fructose-1-phosphate (1.7 X 10(-1) M) was higher and the activity with fructose-1,6-biphosphate relative to fructose-1-phosphate (Vmax FBP/Vmax F-1-P = 0.038) was much lower than for either liver or muscle aldolase. Kinetic analysis and subcellular associations indicated that sperm aldolase is different from other isozymes of aldolase.

A bovine seminal plasma inhibitor of actin-stimulated myosin adenosine triphosphatase.

During attempts to isolate bovine sperm actin, persistent low molecular weight proteinaceous (LMWP) contaminants were found. A LMWP fraction was prepared by gel filtration chromatography on Sephadex G150. The LMWP was found in extracts of washed bovine ejaculated spermatozoa and in clarified bovine seminal plasma. It was substantially reduced in amount in bovine epididymal spermatozoa, indicating that it originated from secondary sex gland secretions. The LMWP inhibited rabbit muscle actin-stimulated myosin adenosine triphosphatase (actin-myosin ATPase) activity. The LMWP:actin ratio for 50% inhibition of actin-myosin ATPase was 2.6 +/- 0.12 mg LMWP per mg actin. The LMWP interfered with actin inhibition of deoxyribonuclease, indicating that LMWP interacted with actin. The LMWP from seminal plasma had an estimated molecular weight of 8300 and consisted of several acidic components. It had negligible protease activity and its inhibition of actin-myosin ATPase was independent of divalent cations. The LMWP appears to readily aggregate with itself and other proteins, which may be related to its physiological role in semen.

Purification and properties of carnitine acetyltransferases from bovine spermatozoa and heart.

To investigate the physical and kinetic properties of sperm carnitine acetyltransferase, the enzyme was purified from bovine spermatozoa and heart muscle. Carnitine acetyltransferase was purified 580-fold from ejaculated bovine spermatozoa to a specific activity of 85 units/mg protein (95% homogeneity). Sperm carnitine acetyltransferase was characterized as a single polypeptide of Mr 62,000 and pI 8.2. Heart carnitine acetyltransferase was purified 650-fold by the same procedure to a final specific activity of 71 units/mg protein. The kinetic properties of purified bovine sperm carnitine acetyltransferase were consistent with the proposed function of this enzyme in acetylcarnitine pool formation. Product inhibition by either acetyl-L-carnitine or CoASH was not sufficient to predict significant in vivo inhibition of acetyl transfer. At high concentrations of L-carnitine, bovine sperm and heart carnitine acetyltransferases were most active with propionyl- and butyryl-CoA substrates, although octanoyl-, iso-butyryl-, and iso-valeryl-CoA were acceptable substrates. Binding of one substrate was enhanced by the presence of the second substrate. Carnitine analogs that have significance in reproduction, such as phosphorylcholine and taurine, did not inhibit carnitine acetyltransferase. Bovine sperm and heart carnitine acetyltransferases were indistinguishable on the basis of purification behavior, pI, pH optima, kinetic properties, acyl-CoA specificity, and sensitivity to sulfhydryl reagents and divalent cations; thus there was no indication that bovine sperm carnitine acetyltransferase is a sperm-specific isozyme.

Comparative ability of RNA and DNA to prime DNA synthesis in vitro: role of sequence, sugar, and structure of template-primer.

The priming efficiency of oligo(RNA) vs. oligo(DNA) in a homopolymer template-homooligomer primer system was compared using four DNA polymerases. The templates included (dT)n, (dA)n, (dC)n, and (dI)n. Primers were the oligomers of the complementary DNA OR RNA with chain lengths of 6 to 23. The DNA polymerases used were from Micrococcus luteus, avian myeloblastosis virus (AMV), and Escherichia coli (polymerase I and polymerase III). The polymerases demonstrated a preference for the DNA primers with (dC)n, (dA)n, and (dI)n templates. However, when (dT)n was the template, all but the AMV polymerase preferred (rA)11 more than 200-fold better than (dA)12. This preference was due to the physical structure of the initiation complex. The structures of the oligo-polymer complexes were characterized by mixing curves, melting curves, and analytical bouyant density analyses. (rA)11 + (dT)n formed predominatly a duplex structure, whereas (dA)12 + (dT)n formed the three- stranded structure, (dA12-2(dT)n. The Km of the duplex with E. coli Pol III was 2.9 mugM (rA)11. The Ki of the triplex was 2.2 mugM (dA)12, indicating that Pol III could bind to the triplex but would not elongate the (dA)12 primer. The influence of structure on priming also was demonstrated with longer oligomers, (dA)23 and (rA)23, where the (dA)23 formed more duplex-like structures and primed more than the (dA)12.(dT)10 + (dA)n complexes also were shown to form triplex structures that inhibited priming. These results show that template-primer structure has more influence on priming than the sugar moiety or the sequence of the nucleic acid.