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A series, consisting of 52 benzamidine derivatives, was evaluated for inhibitory activity against homogeneous boar sperm acrosin. All of the compounds in the series proved to be more potent than benzamidine (Ki = 4.0 x 10(-6) M), with one of the derivatives, alpha-(4-amidino-2,6-diiodophenoxy)-3-nitrotoluene (compound 16), showing outstanding potency with a Ki value of 4.5 X 10(-8) M. Although all of the derivatives were effective acrosin inhibitors, structural specificity was observed within homologous groups of compounds. The information gained from this preliminary study should prove extremely beneficial in the design and synthesis of future acrosin inhibitors.

Aqueous dispersions of synthetic phospholipids, in the form of anionic, single bilayer vesicles, were observed to stimulate the appearance of acrosin esterase activity from its zymogen precursor, proacrosin. Enzymatic activity measurements, in parallel with polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate, indicated that the enzymatic activity produced had resulted from the conversion of proacrosin to acrosin (EC 3.4.21.10), and not from the direct stimulation of a possible proacrosin esterase activity. It is suggested that such bilayer lipid vesicles can be used as a model membrane system to study the activation of proacrosin in vitro.Acrosin (EC 3.4.21.10) is an acrosomal proteinase thought to digest a path for the spermatozoon through the zona pellucida of the ovum, a process that is required for fertilization (1). However, freshly ejaculated spermatozoa contains greater than 95% of the potential acrosin activity as proacrosin, the enzymatically inactive zymogen (2, 3). Interest in this enzyme has been enhanced by the observations that acrosin inhibitors prevented fertilization both in vitro (4, 5) and in vivo (6, 7). Since fertilization does not occur in the absence of enzymatically active acrosin, the conversion of proacrosin to acrosin is of critical importance in the reproductive process.In a previous communication (8), possible mechanisms for proacrosin activation were examined. The results suggested that for highly purified boar proacrosin, the zymogen appeared to have the capability to autocatalytically generate acrosin from proacrosin. However, factors that influence the activation of the zymogen have received only minimal attention. Calcium (9), zinc (9), and polyamines (10) have been demonstrated to inhibit the activation process in varying degrees. Immunological (11) and biochemical fractionation techniques (12, 13) indicated that enzymatically active acrosin was bound to the inner acrosomal membrane, suggesting that proacrosin was also membrane bound and that the activation of proacrosin occurred on the membrane. However, the effect of membranes on the activation process has not been evaluated. In this communication we demonstrate that aqueous phospholipid dispersions, in the form of anionic single bilayer vesicles, enhanced the autocatalytic conversion of highly purified boar proacrosin to acrosin. It appears that phospholipid bilayer vesicles may be used as a model membrane system to study the mechanism of proacrosin activation in vitro. MATERIALS AND METHODSProacrosin was isolated from ejaculated boar spermatozoa by extraction at pH 3.0 followed by pH 5.6 fractionation, ammonium sulfate and sodium chloride precipitation, and Sephadex G-75 chromatography in the presence of 8 M urea (8). TheThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 149 purity of the proacrosin preparation was es...

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichlorosocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.

Proacrosin, the zymogen precursor of acrosin, was shown to associate with anionic phospholipid membranes through apparent electrostatic charge interactions. This association was diminished by elevated cation concentrations and was dependent on membrane composition, as shown both by direct binding assays and by following the phospholipid stimulation of conversion of proacrosin into acrosin.

52 Benzamidin‐Derivate (I)‐(IV) werden teilweise dargestellt und auf Acrosin‐inhibierende Wirkung untersucht.

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