A re-evaluation of the molecular size of duck epsilon-crystallin and its comparison with avian lactate dehydrogenases

A biochemical comparison of epsilon-crystallin isolated from the duck lens and lactate dehydrogenases of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of epsilon-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that epsilon-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and epsilon-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck epsilon-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50 degrees C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.     

Characterization and comparison of epsilon-crystallin and lactate dehydrogenases in the lenses of vertebrates and invertebrates

Screening of lens homogenates for the identification of lactate dehydrogenases was undertaken for the representative species from five major classes of vertebrates plus the cephalopod of invertebrates. The duck and caiman lenses appeared to contain the highest enzymatic activity of this glycolytic enzyme among all species examined. Biochemical isolation and characterization of epsilon-crystallins from the duck and caiman lenses revealed differences between these structural crystallins and the authentic lactate dehydrogenase of the avian heart regarding some of the kinetic properties. This is in contrast with the claim that duck epsilon-crystallin is identical to heart-type lactate dehydrogenase.     

A re-evaluation of the molecular size of duck ε-crystallin and its comparison with avian lactate dehydrogenases

A biochemical comparison of ε-crystallin isolated from the duck lens and lactate dehydrogenase of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of ε-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that ε-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and ε-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck ε-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50°C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.     

Kinetic analysis of duck epsilon-crystallin with L-lactate dehydrogenase activity: determination of kinetic constants and comparison of substrate specificity.

A systematic analysis of the kinetic properties of duck lens epsilon-crystallin with lactate dehydrogenase [LDH, (E.C. 1.1.1.27)] activity was carried out by employing some 19 different alpha-keto acids as substrates for this NADH-dependent LDH-catalyzed reaction. The steady-state Michaelis and catalytic constants (Km, kcat) were determined for a broad range of organic compounds. The results provide important insights regarding the binding and affinity of substrates to active sites of this enzyme crystallin and indicate a great potential for the application of the stable epsilon-crystallin as a catalyst to the synthesis of some important chiral alpha-hydroxyacids in a convenient and efficient way. It is also demonstrated for the first time that in addition to the enzymatic activity of lactate dehydrogenase, duck epsilon-crystallin also possesses the enzymatic activity of malate dehydrogenase.     

Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.     

Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.     

Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin

Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.     

Tryptophanyl fluorescence of sodium dodecyl sulfate treated and 2-mercaptoethanol reduced proteins: a simple assay for tryptophan

Relative tryptophanyl fluorescence intensities of eleven different proteins (bovine liver glutamate dehydrogenase, bovine pancreas trypsin and α-chymotrypsinogen, egg white lysozyme, ovalbumin, bovine serum albumin and γ-globulin, bovine heart and rabbit muscle lactate dehydrogenases, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, and yeast alcohol dehydrogenase) were evaluated as a function of the physical state of the protein, i.e., native, denatured with intact disulfide bonds, and denatured with reduced disulfide bonds.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

epsilon-Crystallin, a novel avian and reptilian eye lens protein

Gel filtration of Peking duck eye lens proteins reveals a component eluting just behind delta-crystallin and comprising approximately 10% of the total soluble protein. The native Mr of this additional component is estimated to be 120000; it appears to be composed of three identical chains of Mr 38000 and pI 7.5. Circular dichroic spectroscopy showed a relatively high alpha-helical content. No immunological cross-reactivity is found with alpha-, beta-, gamma- or delta-crystallins, and partial amino acid sequence determinations likewise failed to reveal any similarity with other known crystallins. We conclude that this protein represents another and novel family of eye lens proteins, for which we propose the designation epsilon-crystallin. epsilon-Crystallin is translated from a 1450-base mRNA, which has been partially purified. epsilon-Crystallin is found scattered among avian and reptilian taxa, but not in other vertebrates. Its rate of evolutionary change seems to be as slow as that of alpha- and beta-crystallins.     

A comparative study of the regulation of Ca2+ of the activities of the 2-oxoglutarate dehydrogenase complex and NAD+-isocitrate dehydrogenase from a variety of sources.

Ca2+ was shown to activate oxoglutarate dehydrogenase and NAD+-isocitrate dehydrogenase from heart and other rat tissues by markedly decreasing the Km values of the enzymes for their respective substrates [see Denton & McCormack (1980) FEBS Lett. 119, 1-8]. Similar effects of Ca2+ were observed in the present study with both enzymes from other vertebrate sources (pigeon, trout, frog and human heart), but not with the enzymes from blowfly or locust flight muscle, or potato or Escherichia coli. In contrast, the Km values of the oxoglutarate dehydrogenases were affected by ADP, ATP and H+ to a similar extent in every case, except for the enzyme from E. coli, which was not sensitive to regulation by these agents.     

Diphosphopyridine nucleotide-linked D-lactate dehydrogenases from the horseshoe crab, Limulus polyphemus, and the seaworm, Nereis virens. II. Catalytic properties

The catalytic properties of the purified horseshoe crab and seaworm d-lactate dehydrogenases were determined and compared with those of several l-lactate dehydrogenases. Apparent K_m's and degrees of substrate inhibition have been determined for both enzymes for pyruvate, d-lactate, NAD⁺ and NADH. They are similar to those found for l-lactate dehydrogenases. The Limulus “muscle”-type lactate dehydrogenase is notably different from the “heart”-type lactate dehydrogenase of this organism in a number of properties.The Limulus heart and muscle enzymes have been shown by several criteria to be stereospecific for d-lactate. They also stereospecifically transfer the 4-α hydrogen of NADH to pyruvate. The turnover number for purified Limulus muscle lactate dehydrogenase is 38,000 moles NADH oxidized per mole of enzyme, per minute. Limulus and Nereis lactate dehydrogenases are inhibited by oxamate and the reduced NAD-pyruvate adduct.Limulus muscle lactate dehydrogenase is stoichiometrically inhibited by para-hydroxymercuribenzoate. Extrapolation to two moles parahydroxymercuribenzoate bound to one mole of enzyme yields 100% inhibition. Alkylation by iodoacetamide or iodoacetate occurs even in the absence of urea or guanidine-HCl. Evidence suggests that the reactive sulfhydryl group may not be located at the coenzyme binding site.Reduced coenzyme (NADH or the 3-acetyl-pyridine analogue of NADH) stoichiometrically binds to Limulus muscle lactate dehydrogenase (two moles per mole of enzyme).Several pieces of physical and catalytic evidence suggest that the d- and l-lactate dehydrogenase are products of homologous genes. A consideration of a possible “active site” shows that as few as one or two key conservative amino acid changes could lead to a reversal of the lactate stereospecificity.     

The lactate dehydrogenase of the icefish heart: biochemical adaptations to hypoxia tolerance

Cardiac lactate dehydrogenase from the hemoglobin- and myoglobin-free antarctic icefish has been purified by affinity chromatography. Structural and kinetic properties of the enzyme were found close or identical to those of its skeletal muscle counterpart and other M-type lactate dehydrogenases. A model involving a dual oxidative-anaerobic metabolism of the icefish heart is proposed.     

A comparative study of NAD+ binding sites in dehydrogenases by circular polarization of fluorescence.

The spectra of the circular polarization of luminescence of a number of dehydrogenases with the fluorescent coenzyme nicotinamide-1,-N⁶-ethenoadenine dinucleotide were measured. By use of this technique it is demonstrated that there is a difference in structure between the adenine subsite in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase on the one hand and pig heart lactate dehydrogenase, horse liver alcohol dehydrogenase, beef liver glutamate dehydrogenase, and pig heart malate dehydrogenase on the other hand. It is concluded that the non-co-operative dehydrogenases have similar, if not identical, adenine subsites whereas in glyceraldehyde-3-phosphate dehydrogenase, a strongly co-operative enzyme, a different structure of the adenine subsite has evolved.     

Histochemical studies of some myocardial oxido-reductive enzymes after experimental beta-adrenergic blockade with propranolol

Summary Histochemical studies of some myocardial oxido-reductive enzymes after a beta-adrenergic blockade with propranolol have been carried out. Succinate, isocitrate and glucose-6-phosphate dehydrogenases did not indicate any changes in activity, whereas the changes in reaction intensities concerning NADH and NADPH tetrazole reductases and glutamate dehydrogenase have rather a transitory and reversible character. Only lactate dehydrogenase showed an increase in the enzymatic activity which speaks for an increase in the glycolysis process in the heart muscle. In the light of our own presented research results we assume that the experimental beta-adrenergic blockade of the heart muscle in rats does not evoke more important enzymatic changes which are noticeable in histochemical microscope examination.     

Histochemical studies of some myocardial oxido-reductive enzymes after experimental beta-adrenergic blockade with propranolol.

Histochemical studies of some myocardial oxido--reductive enzymes after a beta--adrenergic blockade with propranolol have been carried out. Succinate, isocitrate and glucose-6-phosphate dehydrogenases did not indicate any changes in activity, whereas the changes in reaction intensities concerning NADH and NADPH tetrazole reductases and glutamate dehydrogenase have rather a transitory and reversible character. Only lactate dehydrogenase showed an increase in the enzymatic activity which speaks for an increase in the glycolysis process in the heart muscle. In the light of our own presented research results we assume that the experimental beta--adrenergic blockade of the heart muscle in rats does not evoke more important enzymatic changes which are noticeable in histochemical microscope examination.     

Strongly bound nucleotide derivatives in lactate dehydrogenase from the sixgill shark

Chemical analyses on lactate dehydrogenases from diverse sources reveal the presence of substoichiometric quantities of strongly bound phosphorus, pentose, and nicotinamide. The quantities of these apparent coenzyme residuals vary among species, with a relatively high incidence in the flank muscle lactate dehydrogenase of the sixgill shark. The pentose is completely released by drastic procedures such as acid precipitation or heat denaturation of the enzyme. However, it cannot be extracted by charcoal treatment of the enzyme or exchanged with reduced 3-acetylpyridine-adenine dinucleotide. The tenacity of binding suggests that the attachment of the fragments to the enzyme is covalent and that they are somehow involved in the stabilization of the enzyme.     

Temperature stability of lactate dehydrogenase in complex with anionic polyelectrolyte poly(styrenesulfonate)

The temperature stability of the cytoplasmic enzyme of glycolysis, lactate dehydrogenase from pig muscle (isoenzyme M4) in complex with anionic polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, own protein fluorescence, and circular dichroism. Calorimetric investigations of the complex of lactate dehydrogenase with poly(styrenesulfonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulfonate)/lactate dehydrogenase, though at 20°C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulfonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

A comparison of lactate dehydrogenase from an ectothermic and an endothermic animal.

Adaptation to environmental temperature is examined in beef heart, beef muscle, and flounder muscle lactate dehydrogenases (EC 1.1.1.27). Low temperature adaptation in the ectothermic (flounder) enzyme is indicated by a reduced enthalpy of activation for kcat (enzyme turnover number, s-1) and increased catalytic efficiency. Also, the reaction rate at low substrate concentrations has a maximum at a lower temperature than in the endothermic enzymes. This is a result of altered bonding in the enzyme-substrate complexes. Adaptation to higher temperatures in the endothermic (beef) enzymes is suggested by a decreased sensitivity to heat denaturation, especially in the presence of substrates. A direct correlation is found between the degree of bonding in the enzyme-substrate complexes and the decrease in rate of heat denaturation caused by addition of substrates.     

Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of beta-lactoglobulin

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.