RESUMO
The sarcoplasmic proteome of beef Longissimus lumborum demonstrating animal-to-animal variation in color stability was examined to correlate proteome profile with color. Longissimus lumborum (36 h post-mortem) muscles were obtained from 73 beef carcasses, aged for 13 days, and fabricated to 2.5-cm steaks. One steak was allotted to retail display, and another was immediately vacuum packaged and frozen at -80°C. Aerobically packaged steaks were stored under display, and color was evaluated on days 0 and 11. The steaks were ranked based on redness and color stability on day 11, and ten color-stable and ten color-labile carcasses were identified. Sarcoplasmic proteome of frozen steaks from the selected carcasses was analyzed. Nine proteins were differentially abundant in color-stable and color-labile steaks. Three glycolytic enzymes (phosphoglucomutase-1, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase M2) were over-abundant in color-stable steaks and positively correlated (P<0.05) to redness and color stability. These results indicated that animal variations in proteome contribute to differences in beef color.
Assuntos
Qualidade dos Alimentos , Carne/análise , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Pigmentos Biológicos/biossíntese , Retículo Sarcoplasmático/metabolismo , Matadouros , Animais , Bovinos , Armazenamento de Alimentos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/biossíntese , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Proteínas Musculares/análise , Músculo Esquelético/enzimologia , Músculo Esquelético/crescimento & desenvolvimento , Fosfoglucomutase/biossíntese , Fosfoglucomutase/metabolismo , Pigmentos Biológicos/análise , Estabilidade Proteica , Piruvato Quinase/biossíntese , Piruvato Quinase/metabolismo , Reprodutibilidade dos Testes , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/enzimologia , Eletroforese em Gel Diferencial BidimensionalRESUMO
We have cloned and characterized the pykA gene from Bacillus subtilis which codes for a pyruvate kinase (PK) enzyme. This gene has been located downstream a putative phosphofructokinase gene, suggesting that they are part of the same operon. The deduced amino acid sequence of this PK showed a strong similarity to other PKs from different sources; however, as it has been found in other bacilli, the B. subtilis pykA enzyme had an extra C-terminal sequence consisting of about 112 amino acid residues. This gene was insertionally inactivated at the chromosomal level, with an antibiotic resistance marker. The analysis of this mutation in wild type and pts- backgrounds, indicated that B. subtilis has no other pyruvate kinase activity capable of complementing the absence of PykA.
Assuntos
Bacillus subtilis/genética , Genes Bacterianos , Piruvato Quinase/genética , Sequência de Aminoácidos , Bacillus subtilis/fisiologia , Sequência de Bases , Clonagem Molecular , Biblioteca Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Filogenia , Piruvato Quinase/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Esporos BacterianosRESUMO
Leishmania mexicana mexicana contains two tandemly arranged genes for pyruvate kinase (PYK). The 5' located gene codes for a polypeptide with a molecular mass of 54,370. The calculated net charge and isoelectric point of the polypeptide are -6 and 6.5, respectively. Its amino-acid sequence is 73.7% identical to that of the Trypanosoma brucei PYK and 46.4-49.8% to the enzyme of mammalian cells. The second gene appears not to be functional, because its 5' and 3' extremities have undergone recombinations. L. m. mexicana PYK has been overexpressed in Escherichia coli, using a T7 expression system. Approximately 30% of the protein was detected in the soluble cell fraction. It has been highly purified by chromatography over DEAE-Sephacel and Affigel Blue. From a 1-1 culture 6 mg enzyme was obtained with a specific activity of 224 units mg-1. The protein has a subunit molecular mass of 59,000, as determined by SDS/PAGE, and an isoelectric point of 5.9. Some kinetic properties of the enzyme have been measured and compared with those reported for the T. brucei enzyme. The kinetics of both enzymes are very similar, the most important aspect being their activation by fructose 2,6-bisphosphate. Nevertheless, some differences were observed; the T. brucei enzyme is activated by the effector in a cooperative manner, whereas the activation of the L. m. mexicana enzyme is not cooperative.