The amino acid composition of soya lactate dehydrogenase

Lactate dehydrogenase from germinating soya plants was prepared in an electrophoretically homogeneous form by affinity chromatography on AMP-Sepharose.     

Subcellular localization of branched-chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse spermatozoa.

Spermatozoa isolated from rat and mouse epididymes show a relatively high branched-chain amino acid aminotransferase (leucine aminotransferase, EC 2.6.1.6) activity. There is a significant reduction of leucine aminotransferase and of the isoenzyme C4 of lactate dehydrogenase (EC 1.1.1.27) in the gametes during their epididymal transit. Studies of patterns of liberation of the leucine aminotransferase and of the lactate dehydrogenase C4 from intact spermatozoa, treated with increasing concentrations of digitonin, indicate that both enzymes have the same dual subcellular location, i.e. in the cytosol and in the mitochondria.     

The amino acid sequence of the tryptic peptides isolated from dogfish M4 lactate dehydrogenase.

The peptides which result from treatment of the S-[14C]carboxymethyl derivative of dogfish M4 lactate dehydrogenase (EC 1.1.1.27) with trypsin have been isolated and their sequences have been elucidated. Each identical subunit has a molecular weight of 36,000 and on the basis of the amino acid composition 40 unique tryptic peptides are anticipated. Thirty-seven of these peptides have been isolated and have been completely characterized. The amino acid sequences of these peptides are presented.     

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.     

The structure of mouse testicular lactate dehydrogenase isoenzyme C4 at 2.9 A resolution.

The structure of lactate dehydrogenase isoenzyme C4 from mouse testes was solved at 2.9 A resolution using the technique of molecular replacement. The electron density map revealed a ternary-like configuration of the flexible loop peptide although density corresponding to the coenzyme and substrate molecules was not present. Apparently the apo-lactate dehydrogenase molecule in solution is in a dynamic equilibrium between the O (loop open as found in dogfish apo-lactate dehydrogenase M4) and C (loop closed as found in a variety of ternary complexes) conformations. During crystallization of the apoenzyme one or the other conformers is selected. The apparent stability of the closed conformation for the apo-lactate dehydrogenase C4 molecule may in part explain the low catalytic turnover number of the C isoenzyme. A possible substitution of an arginine residue at position 30 may also be a contributing factor as well as allowing NADP to act as coenzyme.     

Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes

Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.     

Crystallization and preliminary X-ray diffraction analysis of staphylococcal enterotoxin type C.

The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 A, respectively. The triclinic crystals have unit cell parameters a = 38.5 A, b = 43.7 A, c = 36.9 A, and interaxial angles alpha = 99.9 degrees, beta = 95.8 degrees, and gamma = 98.5 degrees. The tetragonal crystals are of space group P4(1)22 with unit cell parameters a = 43.4 A and c = 278.0 A.     

Separation of rat lactate dehydrogenase isoenzyme C4 from other isoenzymes by affinity and ion-exchange chromatography.

Lactate dehydrogenase C, an isoenzyme composed of C polypeptide subunits and found only in mature testes and spermatozoa, differs kinetically, chemically and immunologically from the five common isoenzymes of lactate dehydrogenase, each of which is a tetramer of A and/or B subunits. In the rat lactate dehydrogenase C exists in two molecular forms, isoenzymes C4 and A1C3. In addition to these two forms of lactate dehydrogenase C, rat testicular homogenate contains all the five isoenzymes of A and B type. Purification of isoenzyme C4 requires its separation from the other six isoenzymes, of which isoenzymes A1C3 and A3B1 are the most difficult ones to separate. In the present study isoenzyme A3B1, along with other enzymes, was separated from isoenzyme C4 by AMP-Sepharose chromatography by using a gradient of increasing concentration of NAD+-pyruvate adduct. In the next step, isoenzyme A1C3 was separated from isoenzyme C4 by DEAD-cellulose chromatography, resulting in a pure lactate dehydrogenase isoenzyme C4 preparation.     

Cloning and characterization of lactate dehydrogenase C4 from Ochotona curzoniae

Lactate dehydrogenase C4 (LDH-C4) is considered to be a good target protein for the development of contraceptive drugs. To develop contraceptive rodenticide against pika (Ochotona curzoniae) LDH-C4, the pika LDH-C gene was cloned and expressed in Escherichia coli. The recombinant protein was purified and characterized. The cDNA of pika LDH-C gene was cloned by the RACE method. The cDNA was 1498 bp in length and contained an ORF of 996 bp, which encoded a polypeptide of 332 amino acids. The ORF of pika LDH-C was introduced in E. coli and expressed with no fusion tags added. The recombinant LDH-C4 protein was purified by heating, affinity chromatography and ion-exchange chromatography. The recombinant pika LDH-C4 was a tetramer with a molecular weight of approximately 140 kDa, and it had temperature-dependent catalytic activity, as it was thermally stable up to 60°C. The optimal pH values in the forward and backward reactions were around 7.48 and 10.28, respectively. The apparent Michaelis constants for pyruvate and lactate were 51.2 ± 3.8 and 8568.8 ± 409 μM respectively. The inhibition constant for oxalic acid was 11.8 ± 3.5 mM. This study laid a solid foundation for contraceptive rodenticide development against pika LDH-C4.     

Characterization of structural unit of phospholamban by amino acid sequencing and electrophoretic analysis

The partial amino acid sequence of phospholamban from canine cardiac sarcoplasmic reticulum was determined by sequence analysis of the peptides obtained from the protein cleaved by cyanogen bromide and with TPCK-trypsin. The sequence determined initiated with N alpha-acetylated methionine followed by 44 amino acid residues intervening two unidentified residues. This polypeptide would represent a structural unit (protomer) of phospholamban. Analysis of temperature-dependent conversion of phospholamban from 26 kDa to lower molecular weight form (6 kDa) suggested that phospholamban holoprotein is composed of five identical protomers.     

The disproportionation of glyoxylate by lactate dehydrogenase

Lactate dehydrogenase (EC 1.1.1.27) catalyzes the NAD-dependent oxidation to (oxalate) and reduction (to glycollate) of glyoxylate. The kinetics of this disproportionation are in accord with the usual reaction pathway of lactate dehydrogenase:substrate inhibition with appropriate pH dependence occurs; a steady state in the ratio of NADH to NAD⁺ is set up during the reaction, has the expected dependence on pH, and is independent of the initial glyoxylate, coenzyme, and enzyme concentration. At pH 7 the lactate dehydrogenase-NADH complex is about fivefold more likely to react with and reduce glyoxylate (at a concentration of 100 mm) than to dissociate to produce free NADH, and the ratio of the fraction of the enzyme-NADH complex which dissociates to the fraction which reacts with and reduces glyoxylate varies with glyoxylate concentration and with pH in a manner in agreement with the normal reaction pathway of the enzyme. With all concentrations of glyoxylate and over the pH range 7–9.6 both free (not enzyme bound) NAD⁺ and free NADH are formed in the steady state of the disproportionation. From these results it is apparent that lactate dehydrogenase, like alcohol dehydrogenase (EC 1.1.1.1), catalyzes a disproportionation within the bounds of its normal kinetic reaction pathway.     

X-ray diffraction analysis of crystals of bovine platelet factor 4

Bovine platelet factor 4 has been crystallized by “vapor dilution” in space group P2₁2₁2₁, $\text{a = 63.7}\text{A}\text{?}\text{, b = 66.7}\text{A}\text{?}\text{, c = 80.5}\text{A}\text{?}$, with four molecules, each 9505 M_r, in the asymmetric unit. The crystals diffract X-rays to better than 2.8 Å resolution.     

Inhibition kinetics of lactate dehydrogenase isoenzymes by gossypol acetic acid

The effect of gossypol acetic acid, a potent male sterilent was studied on LDH from goat liver (LDH-A4), heart (LDH-B4) and testis (LDH-C4) in vitro. All the preparations of LDH were inhibited by gossypol when the reaction was carried out in pyruvate-lactate (direct) or lactate to pyruvate (reverse) directions. The IC50 of gossypol for the pyruvate oxidation by LDH isozymes varied between 16 and 42 microM in presence of 0.27 mM pyruvate and 0.15 mM NADH at 25 degrees C and pH 7.4 whereas for the lactate oxidation, IC50 was 125 microM in a system containing 3.3 mM lactic acid and 1.8 mM NAD at 25 degrees C and pH 9.0. Reciprocal plots due to Lineweaver-Burk showed that these isozymes are inhibited in a non-competitive manner with respect to pyruvate and lactate, and in a competitive fashion when NAD and NADH were varied as substrates. Ki values of LDH-A4, -B4 and -C4 isozymes in presence of gossypol were 20, 34 and 29 microM against pyruvate; 33, 43 and 45 microM against NADH; 85, 85 and 125 microM against lactate and 94, 108 and 83 microM against NAD respectively.     

Molecular symmetry of fructose-1,6-diphosphatase by X-ray diffraction analysis.

Large single crystals of rabbit liver fructose-1,6-diphosphatase suitable for a high resolution structure analysis have been grown from polyethylene glycol. The space group of these crystals is I222 with a = 75 A, b = 81 A, and c = 132 A and there are 2 tetrameric molecules in the unit cell. These crystals have one protein subunit as the crystallographic asymmetric unit and establish point group symmetry 222 as the molecular symmetry.