RESUMEN
The catalytical properties and thermostability of free leucyl-, glutamyl- and lysyl-tRNA synthetases and of the same synthetases in codosomes are compared. The stability of different aminoacyl-tRNA synthetases in highly purified preparations and in codosomes did not submit to any common regularities. Km for all substrates both for purified and assembled ARSases are values of the same order. It is shown in some model systems that the aminoacyl-tRNA synthetase activity in codosomes depends on the presence of pyrophosphatase. Other important components of codosomes are protein kinases and phospholipids which are able to influence the aminoacyl-tRNA synthetase activity and structural organization.
Asunto(s)
Aminoacil-ARNt Sintetasas/análisis , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Animales , Electroforesis en Gel de Poliacrilamida , Células Eucariotas , Cinética , Hígado/enzimología , Sustancias Macromoleculares , Miocardio/enzimología , Conejos , Ratas , Ratas Endogámicas , Porcinos , TemperaturaRESUMEN
Titration of metal-freed bovine alpha-lactalbumin with Mg2+ ions causes a two-stepped decrease in the fluorescence quantum yield and a pronounced spectral shift towards shorter wavelengths. It seems to reflect conformational changes induced by the binding of two Mg2+ ions to the protein molecule which results in a transfer of some tryptophan residues from the protein surface into an interior part of the protein in rigid unpolar environment. The Mg2+ association constants evaluated from the fluorimetric Mg2+-titration are 2x10(3) and 2x10(2) M-1. Mg2+ ions in millimolar concentrations almost do not influence the binding of Ca2+ ions to protein. It is assumed that Ca2+ and Mg2+ ions are bound to different sites on alpha-lactalbumin. Mono-calcium, mono-magnesium, bi-magnesium and apo-forms of alpha-lactalbumin are distinct in their fluorescence properties which suggests the difference in the conformation of these forms. The binding of Ca2+ and Mg2+ ions to alpha-lactalbumin has to modulate its function.
Asunto(s)
Lactalbúmina/metabolismo , Magnesio , Animales , Calcio , Bovinos , Unión Proteica , Conformación Proteica , Espectrometría de Fluorescencia/métodosRESUMEN
Binding of Ca2+ ion to bovine alpha-lactalbumin molecule causes a conformational change reflected in an almost two-fold decrease of the fluorescence quantum yield and a rather pronounced spectral shift towards shorter wavelengths (by ca. 20 nm). The changes could be interpreted as a result of a transfer of highly exposed tryptophan residue(s) into a rigid internal part of the protein molecule containing effective quenching groups (probably, vicinal disulphide bridges). The Ca2+ binding constant evaluated from EGTA--and pH-titrations is (3-6) X 10(8) M-1. The results of the spectrofluorometric pH-titration of alpha-lactalbumin in the presence of various Ca2+ concentrations suggest that the well-known acid conformational change in alpha-lactalbumin is due in fact to a competitive replacement of the bound Ca2+ by three H+ ions (pK = 5.0 +/- 0.1) in the Ca2+ binding site.
Asunto(s)
Calcio , Lactalbúmina , Animales , Bovinos , Ácido Egtácico , Concentración de Iones de Hidrógeno , Unión Proteica , Conformación Proteica , Espectrometría de FluorescenciaRESUMEN
The binding of Ca+ to whiting parvalbumin has been studied by intrinsic (tryptophan) protein fluorescence. It has been shown that parvalbumin can exist in three states, which are interpreted as the protein without Ca2+ (P), with one bound Ca2+ (PC) and with two bound Ca2+ (CPC). The spectra of the states have been identified and the protein fluorescence spectra at different Ca2+ concentration have been decomposed on the components corresponding to these states. Relative populations of the P, PC and CPC states have been plotted against Ca2+ concentration. On the basis of a scheme of the successive Ca2+ binding; P+Ck1 in equilibrium PC; PC+Ck2 in equilibrium CPC, corresponding theoretical dependences have been computed and fitted to the experimental ones. The fitted equilibrium constants K1 and K2, are 5.10(8) and 6.10(6)M-1, correspondingly. The schemes are in a good agreement with the experimental data on EGTA-titration of Ca2+-saturated parvalbumin and on pH-titration of parvalbumin in the presence of EGTA.