Dana Čechová

Isolation and biochemical characterization of a zona pellucida-binding glycoprotein of boar spermatozoa

Lectin‐like molecules on the sperm surface are implicated in the process of gamete recognition and adhesion. We have isolated and biochemically characterized a 15 kDa glycoprotein from ejaculated boar sperm which possess zona pellucida‐binding‐ and haemagglutinating‐activity. The zona/ 15 kDa protein interaction is inhibited by fucoldan, suggesting that the glycoprotein is one of the sperm components which participate in the initial gamete interaction. N‐Terminal sequence analysis of the isolated 15 kDa glycoprotein showed that it may belong to the same sperm/egg recognition‐mediating protein family as the sea urchin sperm protein binding.

Separation of α- and β-trypsin by hydrophobic interaction chromatography

Abstract The separation of α- and β-trypsin by means of hydrophobic chromatography on Spheron P 300 was investigated with respect to the separation conditions, i . e ., salt concentration, pH, temperature, sample loading, flow-rate and support particle size. The optimal conditions have been selected at low pH (3.0) where the autodigestion of trypsin is suppressed. This method based on different exposures of hydrophobic amino acid residues of α- and β-trypsin is rapid, simple and effective for both analytical- and preparative-scale separations.

Model study of hydrophobic interactions of α- and β-trypsin and α-chymotrypsin

Abstract The hydrophobic interactions of α- and β-trypsin as a function of ionic strength and pH were studied by hydrophobic chromatography. Evidence was obtained that in spite of the identical specificities and similar activities of α- and β-trypsin, the cleavage of the Lys—Ser bond induces conformational changes in the neighbourhood of the active site. Over a wide range of pH and salt concentration the non-polar residues on the surface of the molecule of β-trypsin are more exposed to an external environment than on the molecule of α-trypsin. In the trypsin(chymotrypsin)—inhibitor complexes the majority of hydrophobic amino acids are buried; other hydrophobic residues localized on the surface contribute only very slightly to the interaction with the chromatographic support. The retention of trypsin, chymotrypsin and their diisopropylphosphoryl derivatives on a support with flexible hydrophobic ligands bonded to the matrix through a spacer (octyl-Sepharose) was correlated with the retention on a support with hydrophobic binding sites incorporated into the rigid matrix of the resin (Spheron). The native enzymes are always more retained; this indicates that the substitution results in the shielding of the non-polar residues in the neighbourhood of the active site. The differences in the slope of individual proteins, resulting from the correlation of the retention values obtained with both supports at several sodium chloride concentrations are explained by differences in the accessibility of the surface non-polar residues in the individual proteins. In experiments with model peptides the contribution of the individual hydrophobic amino acids to the retention was investigated.

A Kunitz Type of Proteinase Inhibitor Isolated from Boar Seminal Vesicle Fluid

Summary:  Using immunoaffinity chromatography on a Sepharose 4B column with adsorbed antibodies to the basic inhibitor in bovine organs (Kunitz‐type), a proteinase inhibitor was isolated from boar seminal vesicle fluid. The isolated protein inhibited acrosin, trypsin, plasmin and chymotrypsin, but not kallikrein. Its molecular weight determined by gel filtration on Sephadex G‐50 was 9,500 (± 500) and by SDS electrophoresis in polyacrylamide gel 12,000 (± 500) daltons. The protein was demonstrated by immunoprecipitation only in boar seminal vesicle fluid and seminal plasma, and by indirect immunofluorescence on ejaculated spermatozoa and in the epithelium of boar seminal vesicles. This inhibitor is the first acrosin inhibitor specific for the genital organs, which evidently belongs to the group of Kunitz type inhibitors, to be described.

Similarities in primary structures of cow colostrum trypsin inhibitor and bovine basic pancreatic trypsin inhibitor

During the past few years the primary structure of the basic trypsin inhibitor from bovine pancreas (BPTI) [l-3] and of the kallikrein inhibitor from bovine lungs and parotid glands [4,5] were determined. Notwithstanding the fact that these inhibitors differ in the site of their origin in the organism and also in molecular weight, their primary structures are identical. This brought us to the interesting question as to whether the remaining bovine trypsin inhibitors share at least a part of their structure in common or whether they are derived from the same precursor. The cow colostrum trypsin inhibitor (CTI) is especially interesting from this viewpoint since it differs markedly from BPTI and KI in its isoelectric point (4.2.) [6] , low resistance toward pepsin [7] , considerable content of a nonprotein component, and amino acid composition. In this study, CT1 which had been obtained before only in heterogeneous form [6], was resolved into three electrophoretically homogeneous inhibitors [9] . Of the latter, the one present in the highest amount was investigated in detail.

Cell biology of acrosomal proteins: Zellbiologie akrosomaler Proteine

Summary Acrosin is a multifunctional enzyme combining several functional properties within a single molecule: the catalytic triad of the proteinase, hydrophobic domains responsible for the special membrane‐associating character of the enzyme and the carbohydrate binding sites by which the molecule can bind to the zona pellucida. Acrosin occurs in the sperm acrosome as an inactive precursor, proacrosin, with a molecular mass of 53–55 kDa. Proacrosin is activated by a single proteolytic clip between Arg23 and Val24 generating the high molecular mass acrosin. The activation of proacrosin to the biologically active enzyme which occurs concomitantly with the acrosome reaction appears to be regulated on and by the zona pellucida. It is hypothesized that alternating cycles of binding to the zona, digestion of the zona and release from the zona together with the forward motility of the spermatozoon would be required to achieve penetration.

[59] Bull seminal plasma proteinase inhibitors

Publisher Summary This chapter describes procedures for the isolation and characterization of two proteinase inhibitors isolated from bull seminal plasma that differ in molecular weight. Bull seminal plasma inhibitor I (BUSI I), MW 8900, exists as three strongly acidic isoinhibitors s A, B 1, and B2; BUSI II, MW 6200, is an extremely basic protein whose amino acid sequence has been determined. In view of the finding that the low-molecular-weight inhibitors from the seminal plasma of mammals are temporary inhibitors, that is, slowly digested in the presence of excess trypsin, the use of affinity chromatography with immobilized trypsin columns is not recommended for their isolation. For this reason conventional methods, such as gel filtration and ion-exchange chromatography are preferred. The assay of the inhibitory activity of the proteinase inhibitors is based on the reduction of enzymatic activity obtained by preincubation of the enzyme with the inhibitor for 10 min. before the addition of the substrate. Enzyme activity is conveniently measured by standard spectrophotometric methods using synthetic ester or amide substrates.

Preliminary structural comparison of the proteinase isoinhibitors IIA and IIB from bull seminal plasma based on individual assignments of the 1H nuclear magnetic resonance spectra by two-dimensional nuclear magnetic resonance at 500 MHz

By combined use of amino acid analysis, chemical sequence determination for the N-terminal decapeptide and two-dimensional 1H nuclear magnetic resonance at 500 MHz the amino acid sequence of bull seminal inhibitor IIB was found to coincide with that of the isoinhibitor IIA, except that the N-terminal tripeptide Pyrl-Gly2-Ala3- in HA is replaced by the dipeptide H-Leu2-Phe3- in IIB. Nearly complete, individual proton assignments were obtained for the isoinhibitor IIB, and comparison with the previously obtained corresponding nuclear magnetic resonance data for the isoinhibitor IIA showed that the two proteins must adopt closely similar secondary and tertiary structures in aqueous solution. The individual resonance assignments provide a basis for future, more detailed investigations of the influence of the local primary structure differences on the protein conformation.

Isolation of Acidic Acrosin Isoinhibitors (BUSI I A, BUSI I B1 and BUSI I B2) from Bull Seminal Plasma

Three natural proteinase isoinhibitors with low isoelectric points BUSI I A (pI = 3.9), BUSI I B1 (pI = 3.4 and BUSI I B2 (pI = 3.7) were isolated from bull seminal plasma by gel filtration on Sephadex G-50 and ion exchange chromatography on DEAE-Sephadex and SE-Sephadex. Isoinhibitors Bl and B2 have identical amino acid composition. Isoinhibitor A contains six amino acid residues less than isoinhibitors B1 and B2. Since sugars have been detected in the isoinhibitors, heterogeneity may also be due to the sugar component. The isoinhibitors show the same inhibitory properties; all of them inhibit acrosin, trypsin and chymotrypsin. Glandular kallikrein is also inhibited, but to a very low extent only. The molecular weight (Mr approximately 8 900) was determined by gel filtration.

First attempt to characterize 3H-fucose acrosomal label in spermatozoa

SummaryAcrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-Nalpha-benzoyl-l-arginine ethyl ester splitting), akin to that exhibited by acrosin.