ISOLATION OF A 43 KDA PROTEIN FROM MYCOBACTERIUM-TUBERCULOSIS H(37)RVAND ITS IDENTIFICATION AS A PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE

A 43 kDa protein (TB43) was isolated from the cell sonicate (CS) of My cobacterium tuberculosis H(37)Rv with immobilized metal affinity chrom atography (IMAC) on a Ni-nitrilotriacetic acid column. Two-dimensional electrophoresis of the IMAC fraction showed a major spot with an M(r) of 43 000 and a pI of similar to 6.0. The N-terminal amino acid seque nce of TB43 was g-val-gly-ile-pro-asn-glu-thr-lys-asn-asn-glu-phe- arg -val-ala-ile-thr-pro-ala. It showed 86% homology with the N-terminal e nd of the alanine dehydrogenase of Myco. tuberculosis and 65% homology with the N-terminal end of the alpha-subunit of the Escherichia coli pyridine nucleotide transhydrogenase (Tsh). TB43 did not show any alan ine dehydrogenase activity and did not react with monoclonal antibody (MAb) HBT10, which is known to recognize the 40 kDa alanine dehydrogen ase of Myco. tuberculosis. It was also not recognized by MAb F29-29 wh ich is known to react with a 43 kDa protein of Myco. tuberculosis comp lex. This protein exhibited strong Tsh activity. A similar 43 kDa prot ein showing Tsh activity was also isolated by IMAC from Myco. bovis CS . However, the pI of the protein was similar to 7.0. A similar protein could not be isolated from the CS or culture filtrate of Myco. bovis BCG and Myco. tuberculosis H37Ra. TB43 is a cell-associated pyridine n ucleotide transhydrogenase and is distinct from the 40/44 kDa secreted alanine dehydrogenase of Myco. tuberculosis.