During spermatogenesis, the various classes of germ cells synthesize proteins necessary for their own functioning and for regulation of the Sertoli cells. However, the nature of these proteins has been little studied, especially in spermatogonia, the germ stem cells. In this study, the electrophoretic patterns of high-resolution, silver-stained, two-dimensional polyacrylamide gels of intracellular spermatogonial protein extracts were studied by computerized gel image analysis. We detected 675 individual spots, some of which we identified by mass spectrometry and database searching. We present here a first set of 53 proteins identified. They include housekeeping proteins never before detected in spermatogonia, ten proteins previously detected in the reproductive tract but not in spermatogonia, including stathmin, a protein previously shown to be involved in cell proliferation and differentiation, and one new testicular protein named translationally controlled tumor protein (TCTP), also known as a growth-related protein. Immunohistochemistry demonstrated that the two latter proteins were indeed highly expressed in spermatogonia in situ, and their possible involvement in spermatogonial division and proliferation is currently under investigation in our laboratory. We conclude that this type of experimental strategy, known as proteomics, is a very powerful way to analyze germ cell proteins comprehensively and should rapidly greatly improve our understanding of spermatogenesis.
a-Chymotrypsin is rapidly and irreversibly inactivated by 3,4-dihydro-3,4-dibromo-6-bromomethylcoumarin (VII) a t neutral pH. The inactivation is pH dependent between pH 5 and 7 and is delayed when the active site of the enzyme is protected by acetylation of the active serine-195 or by binding of a competitive inhibitor.This dihydrocoumarin VII and its 6-methyl analogue are substrates for a-chymotrypsin, but when the ester bond in VII is broken during formation of the acyl-enzyme, a reactive p-hydroxybenzylbromide group is generated in situ within the active site. This newly formed group then alkylates one histidine residue (probably histidine-57), as shown by amino acid analysis of the modified enzyme on acid hydrolysis. A model non-enzymic reaction shows that the dihydrocoumarin VII i s able to alkylate imidazole in aqueous solution at pH 7, yielding the N-substituted imidazole, 3-bromo-6-(imidazol-l-yl-methyl)coumarin (XI).The modified enzyme has practically no activity against a specific substrate and seems no longer to have its intact active site. However its binding site is at least partly free for it is still able to bind proflavin. a-Chymotrypsin is also inactivated by 6-bromomethylcoumarin, the ester bond of which is stable, but the mode of inactivation and the properties of the modified enzyme are different from those found in the study with the dihydrocoumarin VII.The selective modification of amino acids present in the active site of enzymes has often been facilitated by the use of "active-site-directed reagents" [l-31. Some of these reagents have substrate-like structural features which promote the formation of an adsorption complex with the enzyme; a chemical group present in the reagent structure may then achieve a covalent modification within the active center. An example of this reagent type is the chloromethylketone from tosylphenylalanine which was shown to alkylate histidine-57 in a-chymotrypsin [a].As numerous enzymatic reactions proceed with the formation of a covalently bound intermediate The occurrence of this second covalent change can be improved if a reactive functional group is generated in situ after formation of the covalently bound intermediate [7-91. For example a-chymotrypsin catalyzes the hydrolysis of 2-acetoxy-5-nitrobenzyl chloride, releasing the tryptophan-modifying reagent, 2-hydroxy-5-introbenyl chloride at the active site [8] ; this reagent then preferentially reacts with tryptophan residues near the active site, but the enzyme is not inactivated. The mechanism of this reactive chloride or bromide (Koshland's
Resonance Raman spectra of twenty parasubstituted azobenzene derivatives are compared, enabling a more complete assignment of bands to be made. Since all the spectra are rather similar, the various ring substituents are not resonance Raman active, and all the observed bands can be assigned to vibrational modes of the aromatic rings and to the azo group itself. Frequency shifts in the PhN stretching region are opposite to those expected based on delocalization along the PhNNPh moiety. From the complexity of band pattern around the NN stretching region and from the above described frequency shifts, several types of vibrational coupling are evident. On the basis of the band assignments, some useful conclusions can be reached for interpreting the observed spectral changes when azobenzene derivatives are used as chromophore probes in biochemical system studies.
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