Abstract
An apparently unique isozyme of lactate dehydrogenase has been reported associated with transformation by Kirsten sarcoma virus, which was also expressed in human cancer. This isozyme was designated LDHk (Anderson, G.R., and Kovacik, W.P., Jr., (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3209-3213; Anderson, G. R., Kovacik, W. P., Jr., and Marotti, K. R. (1981) J. Biol. Chem. 256, 10583-10591). However, preparations of LDH5 from human placenta and from HeLa cells were later shown to exhibit some of the properties ascribed to LDHk9 and the identify of LDHk as a unique isozyme was questioned (Morin, M. E., and Hance, A. J., (1983) J. Biol. Chem. 258, 2864-2869). Saavadra and Anderson (Saavedra, R. A., and Anderson, G. R. (1983) Science (Wash. D.C.) 221, 291-292) refuted the arguments of Morin and Hance (Morin, M. E., and Hance, A. J. (1983) J. Biol. Chem. 258, 2864-2869) by claiming that commercial preparations of human placental LDH5 were contaminated with LDHk. Re-evaluation of the unique properties which distinguish LDHk from conventional LDH5 indicates that the two isozymes may not be different. Highly purified preparations of LDHk exhibit a single Mr = 34,000 polypeptide subunit on sodium dodecyl sulfate-acrylamide gels, yet retain activity detectable as both LDHk and LDH5. Attempts to separate LDHk and LDH5 by column chromatography or by continuous electrophoresis on a variety of solid support matrices were unsuccessful. Enzyme activity identified as LDHk in imidazole-borate-buffered gels migrating toward the cathode was detected as LDH5 activity on re-electrophoresis. LDH5 activity identified by electrophoretic migration toward the anode in Tris-glycine-buffered gels also recorded as LDHk when re-electrophoresed toward the cathode in imidazole-borate-buffered gels. Quantitative assays of enzyme activity recovered from the two-gel assay systems, as well as re-electrophoresis of isozyme-enriched preparations, indicated that cross-contamination of isozymes was not responsible for the results obtained.
Highlights
An apparently unique isozyme of lactate dehydro- [1] and has been found at elevated levels in some human genase hasbeen reported associated with transforma- carcinomas [2] and in the serum of colon carcinoma patients, tion by Kirstensarcomavirus,whichwasalsoexwhere it appears to correlate with metastasis [3].l pressed in human cancerT. his isozyme was designated This isozyme,LDHk,’ is detected by electrophoresis on 5.8%
The M, = 56,000 subunit of LDHk which is reported tocleave to 35,000 and 22,000 without loss of enzyme activity has not been reported for LDH, [1, 2, 8]
The enzymatically active form of LDH, has clearly been shown t o be a tetramer of M, = 33-37,000 subunits [9], while the tertiary structure of an LDHk containing a M, = 56,000 subunit has not been described. Both LDHk and LDH5 have beoebnserved migrating toward the cathode inspecific electrophoretic systems [2, 10, 11]
Summary
Affinity chromatography onAffi-Gel Blue-Sepharose [1,6], Procion Red-agarose [24], andOxymate-agarose [25] employing elution protocols reported to separate LDHisozymes 1-5 or to separate LDHk from LDH, were unsuccessful. The chromatpresence of LDHk activity in tLhDe H, region of Tris-glycinebuffered acrylamide gels, as well as (f) the presence of LDHs activityintheLDHk region of imidazole-borate-buffered acrylamide gels; and (g) the failuroef preliminary electrophoresis in either gel system to enrichfor a specific isozyme. Our conclusions from these data are that the assays currently in use for the differentiation of LDHk and LDH, do not distinguish between two isozymes of lactate dehydrogen-. Request Document No 83M 3415, cite the authors, and includea check or money order for $5.60 perset of photocopies.Full size photocopies are included in themicrofilm edition of the Journal that is available from Waverly Press
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