Comparison of the NH2-terminal amino acid sequences of the four non-identical subunits of the NAD-linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16.
The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 mumol H2 oxidized.min-1.mg protein-1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non-identical subunits (alpha, beta, gamma, delta). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the alpha, beta, gamma, and delta subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship. The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N-terminal sequences the similarities between both alpha, beta, gamma and delta subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non-analogous subunits alpha, beta, gamma and delta.
Zaborosch
C
C
Institut für Mikrobiologie der Universität Göttingen, Federal Republic of Germany.
Schneider
K
K
Schlegel
H G
HG
Kratzin
H
H
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D005779:Immunodiffusion; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009615:Nocardia / Q000201:enzymology*; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity
D046911:Macromolecular Substances; D010088:Oxidoreductases; C021376:hydrogen dehydrogenase