RESUMO
A procedure for isolation and purification of aspartate aminotransferase from wheat grain includes chromatography on DEAE cellulose, acidification-alkalization, precipitation with protamine sulfate, fractionation with ammonium sulfate, and chromatography on hydroxyapatite. The yield of protein was 27% with 95% purity. Crystals of the enzyme (0.05 x 0.025 x 0.015 mm3) were obtained from ammonium sulfate solution.
Assuntos
Aspartato Aminotransferases/química , Aspartato Aminotransferases/isolamento & purificação , Triticum/enzimologia , Cristalização , Eletroforese em Gel de PoliacrilamidaAssuntos
Aspartato Aminotransferases/química , Animais , Catálise , Galinhas , Cristalização , Dimerização , Miocárdio/enzimologia , SoluçõesRESUMO
A new crystal form of chicken cytosolic aspartate aminotransferase (EC 2.6.1.1) has been grown using a mixture of ammonium sulfate with ethanol as a precipitant. Crystals of the enzyme belong to the space group P 2(1)2(1)2(1) having the following unit cell dimensions: a = 62.38 A, b = 117.41 A, c = 124.34 A. There is one molecule of the enzyme in the asymmetric unit. The crystals diffract at 1.8 A resolution.
Assuntos
Aspartato Aminotransferases/química , Animais , Galinhas , Cristalização , Citosol/enzimologia , Difração de Raios XRESUMO
We report here the x-ray studies of the complex cytosolic aspartate aminotransferase from chicken heart with D-aspartate at 2,7 A resolution. Crystals of the complex was prepared by diffusing D-aspartate into free enzyme crystals; their space group is P 2(1)2(1)2(1) with cell dimensions (A): a = 62.59; b = 117.83; c = 124.38. They contain one dimeric molecule in the asymmetric unit. The x-ray crystallographic analysis proves that the connection of the D-aspartate induces small conformational changes in the active site of two subunits of the enzyme: considerable conformational changes are determined for His 189, Phe 360, Tyr 70, Arg 292, Phe 18 and Glu 141.
Assuntos
Aspartato Aminotransferases/química , Ácido Aspártico/química , Animais , Sítios de Ligação , Galinhas , Cristalografia por Raios X , Miocárdio/enzimologia , Conformação ProteicaRESUMO
We report here the first X-ray studies of the complex of cytosolic aspartate aminotransferase from chicken heart with D-aspartate at 2.5 A resolution. Crystals of the complex were grown by cocrystallization (space group is P2(1)2(1)2(1), parameters: a = 62.48 A, b = 117.71 A, c = 124.38 A). They contain one dimeric molecule in the asymmetric unit. The X-ray analysis proves that attachment of D-aspartate induces considerable conformational changes in the active sites of two subunits of the enzyme: both subunits of the complex are in the closed conformation, the interaction of the enzyme with D-aspartate induces a substantial turn (about 90 degrees) of the coenzyme in one subunit, the coenzyme ring is deformed, considerable conformational changes are determined for Phe-18 and Glu-141. Apparently, the amino group of the substrate is a trigger of the conformational changes in the active site of the enzyme.
Assuntos
Aspartato Aminotransferases/química , Ácido Aspártico/química , Animais , Sítios de Ligação , Galinhas , Cristalografia por Raios X , Citosol/enzimologia , Miocárdio/enzimologia , Conformação ProteicaRESUMO
The crystals of free cytosolic chicken aspartate aminotransferase were subjected to X-ray investigation at 2.7 A. One subunit of the dimeric molecule crystalline enzyme is in the open conformation and the other is in the closed conformation.
Assuntos
Aspartato Aminotransferases/química , Animais , Aspartato Aminotransferases/metabolismo , Sítios de Ligação , Galinhas , Cristalização , Miocárdio/enzimologia , Conformação Proteica , Difração de Raios XRESUMO
Amylose isomerase (AI) preparations were isolated from rabbit muscles after Petrova et al., as well as by the additional fractionation steps. Their homogeneity, enzymatic activity and RNA, isolated from those preparations, were characterized. AI preparations, as described by Petrova et al., proved to be heterogeneous in respect to the protein and RNA; by using additional fractionation methods RNA and protein have been separated from each other, which proves that a homogeneous stable ribonucleoprotein complex, exerting AI activity, does not exist. It was shown by three independent methods that AI preparations isolated after Petrova do not display branching, but have amylolytic activity. RNA, isolated along with the AI preparations, proved to be mainly total tRNA degraded to different degrees. No RNA corresponding to the previously sequenced 2.5S RNA could be detected in these preparations. RNA preparations do not manifest neither branching, nor amylolytic activity. Our data prove that there is no ribozyme, whose existence has been suggested previously.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Músculos/enzimologia , RNA Catalítico/metabolismo , Ribonucleoproteínas/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Animais , Sequência de Bases , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , RNA/genética , CoelhosRESUMO
Homogeneous aspartate aminotransferase (purity--99%, yield--70%) has been prepared from chicken heart cytosol. The purification procedure included fractionation with ammonium sulfate and ethanol and crystallization. Crystals (0.3 x 0.5 x 2 mm) of the free enzyme were prepared from ammonium sulfate solution and studied by X-ray analysis at 2.5 A resolution.
Assuntos
Aspartato Aminotransferases/metabolismo , Citosol/enzimologia , Miocárdio/enzimologia , Sulfato de Amônio/química , Animais , Galinhas , Cristalização , Eletroforese em Gel de Poliacrilamida , Etanol/química , Focalização Isoelétrica , Difração de Raios XRESUMO
Two soybean insulin-binding proteins were isolated using affinity chromatography on insulin-Sepharose. Both proteins have molecular mass about 39 kDa and consist of two subunits linked by disulphide bonds. According to the amino acid composition and N-terminal sequences of the subunits, these proteins, characterized by the absence of free thiol groups and sugar residues, are variants of the previously described soybean basic 7S globulin. The blotted proteins as well as their subunits were shown to bind 125I-labelled bovine insulin. For one of the proteins and insulin, dissociation constant of 4.10(-9) M was measured. The existence of plant insulin-binding proteins suggests the insulin-like regulation in the plant metabolism.
Assuntos
Glycine max/metabolismo , Receptor de Insulina/isolamento & purificação , Sequência de Aminoácidos , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Receptor de Insulina/químicaRESUMO
Analysis of Michaelis--Menten kinetics revealed that the enzyme in solution and the crystalline cytosolic aspartate aminotransferase (EC 2.6.1.1) possess a functional nonequivalence of active sites of the enzyme dimer for two substrates--aspartate and 2-oxoglutarate.
Assuntos
Aspartato Aminotransferases/metabolismo , Miocárdio/enzimologia , Animais , Sítios de Ligação , Catálise , Galinhas , Cristalização , Cinética , Especificidade por SubstratoRESUMO
For the first time an interaction between aspartate transaminase (EC 2.6.1.1.) from chicken heart cytosol and the substrates and their analogues has been investigated by means of circular dichroism and absorption spectra (at pH 5,0-8,0 range). The asymmetry factor of the native enzyme and the enzymes--substrate intermediates was found. The results obtained were explained in terms of changes of the enzyme's active site conformation.
Assuntos
Aspartato Aminotransferases/análise , Miocárdio/enzimologia , Animais , Galinhas , Dicroísmo Circular , Coenzimas/análise , Citosol/enzimologia , Espectrofotometria , Especificidade por SubstratoRESUMO
The spatial structure of cytosolic chicken aspartate aminotransferase (AAT) has been determined by X-ray crystallographic analysis at 2.8 A resolution. AAT consists of two chemically identical subunits. Each subunit can be subdivided into the large pyridoxal phosphate (PLP) binding domain and the small domain. The two active sites of AAT are situated in deep clefts at the subunit interface. The binding of PLP and 2-oxoglutarate is described. Conformations of the following enzyme forms have been compared by difference Fourier syntheses: the nonliganded PLP-form in phosphate and acetate buffers; the non-liganded pyridoxamine phosphate (PMP) form; complexes of the PLP-form with glutarate and 2-oxoglutarate. Lattice-induced dynamic asymmetry of the dimeric AAT molecules was revealed. In one subunit the small domain is mobile and shifted either toward the active site ("closed" conformation) or in the opposite direction ("open" conformation). The closed conformation is induced by the binding of dicarboxylate anions. In the second subunit the small domain is immobile and shifted toward the active site in all enzyme forms or complexes studied. In this subunit, there occurs a rotation of the PLP ring by approximately 20 degrees toward the substrate site. The rotation is observed when crystals are soaked in 0.6 saturated (NH4)2SO4 solution buffered with 0.3 M potassium phosphate, pH 7.5; it was explained by formation of an external aldimine between PLP and NH3. This aldimine is not formed in the presence of dicarboxylates or acetate. It was inferred that dicarboxylate or acetate anions stabilize the internal PLP-lysine aldimine and prevent its reaction with ammonia. Conversion of AAT from the PLP- to PMP-form is accompanied by rotation of the coenzyme ring by approximately 20 degrees; the rotation occurs in both subunits.