Carl S. Vestling

Rapid purification of lactate dehydrogenase from rat liver and hepatoma: A new approach

A new affinity column has been developed for the isolation of lactate dehydrogenase (EC 1.1.1.27; M 4 -LDH) from rat liver and from two Morris hepatomas (Nos. 7777 and 7793). LDH of maximum specific catalytic activity and single-component behavior during polyacrylamide electrophoresis can be obtained in high yield within a period of several hours, depending on the scale of the preparation. Blue Dextran has been coupled covalently to Sepharose-4B by the cyanogen bromide procedure. Application of a crude liver or hepatoma extract in dilute buffer pH 8.6, to a column of Blue Dextran-Sepharose-4B results in quantitive binding of the LDH. After washing with the extracting buffer to remove ultraviolet-absorbing materials, additional contaminating proteins are removed by treatment with NAD + -lactate. Elution of LDH is accomplished with NADH. Residual contaminants are removed on a column of DEAE-Sephadex, and the apparently homogeneous LDH is obtained in 60% yield after ultrafiltration.

Spectrophotometric analysis of rat liver lactate dehydrogenase-coenzyme and coenzyme analog complexes

Abstract A study of difference spectra in the range, 300–500 mμ is reported involving highly purified rat liver lactate dehydrogenase (LDH) and oxidized or reduced coenzyme or coenzyme analog (nicotinamide-adenine dinucleotide (NAD) or acetylpyridine-adenine dinucleotide (AcPyAD). The formation of the binary complexes, LDH · NAD or LDH · AcPyAD is associated with the development of spectral bands with λ max at 335 mμ and 355 mμ, respectively. The observed absorbance is proportional to coenzyme concentration at fixed LDH concentration. The spectral behavior of the LDH · oxidized coenzyme binary complexes is clearly distinguishable from that of the binary complexes involving NADH or AcPyADH. A conclusion is that the binding of oxidized coenzymes to LDH results in a significant change in the chromophoric behavior of a hybridized form of the substituted pyridine ring. The formation of abortive ternary complexes involving LDH · oxidized coenzyme /sd pyruvate or LDH · oxidized coenzyme · /ga-ketobutyrate was accompanied by slow progressive spectral changes which reached equilibrium values, dependent on the concentration of all three reactants. For LDH · NAD · pyruvate λ max , was 323 mμ; for LDH · AcPyAD · pyruvate λ max was 341 mμ. The spectral changes were fully reversible upon dialysis vs. AcPyAD-buffer in the one case but not upon dialysis vs. NAD-buffer in the other. Complete recovery of enzyme activity was noted in each case after dialysis to remove pyruvate or α-ketobutyrate. The spectral changes are considered to be those associated with the allosteric binding of pyruvate or α-ketobutyrate to binary complexes of LDH · oxidized coenzyme with resulting conformational changes in LDH which affect the active sites. No abortive ternary complex involving LDH-reduced coenzyme (or reduced coenzyme analog) -lactate could be detected by spectral means.

Determination of the C-terminal status of rat liver lactate dehydrogenase by an improved selective tritiation method.

Summary Attempts to determine the C-terminal status of rat liver lactate dehydrogenase by the procedures of (a) hydrazinolysis, (b) controlled carboxypeptidase action, and (c) selective tritiation have led to inconclusive results. However, the use of a procedure which involves improved selective tritiation followed by short-term carboxypeptidase action has led to an unequivocal demonstration that phenylalanine is the C-terminal amino acid of rat liver lactate dehydrogenase. The procedure involves: (a) dissolution of the enzyme in tritiated water, (b) sequential addition of pyridine and acetic anhydride, (c) removal of excess reagents by dialysis, (d) short-term carboxypeptidase treatment, and (e) identification of the C-terminal amino acid by chromatography and radioassay.

Beef heart H4 lactate dehydrogenase: N-terminal and C-terminal residues

Abstract N-terminal analyses of beef heart H 4 lactate dehydrogenase involving the use of fluorodinitrobenzene, phenylisothiocyanate, or 2,4,6-trinitrobenzene sulfonic acid failed to demonstrate the presence of a free amino acid at the amino terminus of the protein in contrast to an earlier report (E. A PPELA and R. Z ITO , Ann. N.Y. Acad. Sci. , 151 (1968) 568). Quantitative determination of bound acetyl groups on several batches of enzyme gave values of 3.2–4.6 moles/mole of protein (molecular weight 131 000). The N-terminal peptide, characterized as N -acetylalanyl-threonine, was isolated from pronase digests of the protein, and quantitative recovery experiments using 3 H-labeled N -acetylalanylthreonine as an internal standard showed that all of the acetyl residues in commercial preparations are found at the amino termini. Leucine was demonstrated to be the C-terminal amino acid in the H 4 isozyme using a tritium exchange method while both leucine and phenylalanine were present at the C-terminus in a mixture of H 4 and H 3 M isozymes.

Comparison of purified lactate dehydrogenases from normal rat liver and Morris hepatomas in rats and in culture.

Abstract Lactate dehydrogenase (EC 1.1.1.27) from Morris rat hepatomas grown in vivo and from Morris rat Hepatoma 7288c grown in cell culture has been maximally purified for comparison with lactate dehydrogenase from normal rat liver. A rabbit antiserum directed against normal rat liver lactate dehydrogenase has been produced to allow immunochemical characterization. Other studies included heat inactivation, oxamate inhibition, ethanol sensitivity, starch gel electrophoresis, and polyacrylamide gel electrophoresis. By each criterion the comparisons revealed enzyme identity.

LACTATE DEHYDROGENASE FROM LIVER, MORRIS HEPATOMAS, AND HTC CELLS

ABSTRACT: Lactate Dehydrogenase (LDH) from adult rat liver was isolated in the authors laboratory as a pure enzyme and characterized in 1953. Since that time many studies dealing with catalytic function and with the structure of the enzyme have been reported. The present report concerns three advances: (1) An improved isolation procedure involving the use of a Sepharose-Blue Dextran affinity column makes it possible to obtain 100-mg quantities of pure enzyme within 24 hours; (2) Comparison of pure LDH from liver and from Morris hepatomas grown in vivo and in cell culture (HTC cells) reveals no structural or functional differences when catalytic function, immunochemical properties, heat inactivation, oxamate inhibition, ethanol sensitivity, and electrophoretic behavior are studied; (3) Further experiments involving ultraviolet difference spectrophotometry have been directed toward an understanding of the slow changes in NAD + and NADH chromophoric behavior which result when binary complexes with LDH are formed and full catalytic activity of the enzyme is retained.