Kinetic analysis of lactate dehydrogenase in situ in mouse liver determined with a quantitative histochemical technique

Summary The kinetics of lactate dehydrogenase in situ were studied in sections of unfixed liver of the male mouse using a quantitative histochemical technique. The sections were incubated on substrate-containing gel films. The absorbance of the final reaction products deposited in a single hepatocyte was measured continuously during the incubation as a function of incubation time using a scanning microdensitometer. The absorbance increased non-linearly during the first minute of incubation, but linearly for at least the next 3 min afterwards. The initial velocity (v _i ) of the dehydrogenase was calculated from two equations proposed previously by us, v _i=2.82 °A and v _i =v+2°A, where v and °A are, respectively, the gradient and intercept o linear regression line of absorbance on time for incubation times between 1 and 3 min.The dependence of v _i on lactate concentration gave the following mean kinetic constants. For periportal hepatocytes, the apparent K _m =14 mM and V _max =80 moles hydrogen equivalents formed cm^–3 hepatocyte cytoplasm min^–1. For pericentral hepatocytes, K _m =12 mM and V _max =87 moles hydrogen equivalents cm^–3 min^–1. The K _m values are very similar to those determined previously from biochemical assays. The concentrations of the enzyme in single hepatocytes calculated from the V _max values are in good agreement with those obtained by another method. These data substantiate the validity of our equations.     

Kinetic formulations for the reduction of ketomalonate by lactate dehydrogenase

Initial rate kinetic studies of lactate dehydrogenase with ketomalonate and NADH as substrates suggest that this enzymatic system is adapted to a rapid equilibrium ordered bi-bi ternary complex mechanism. The application of the reaction product inhibition method reveals the existence of the enzyme-NADH-hydroxymalonate and enzyme-NAD(+)-ketomalonate abortive complexes. This kinetic behaviour is confirmed by the differential inhibition induced by several alternate products on the pyruvate-lactate dehydrogenase-NADH and ketomalonate-lactate dehydrogenase-NADH systems.     

Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke

Summary SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2R1, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy.LDH activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of LDH activity.Supported by a research grant from the ASFC (Association Suisse des Fabricants de Cigarettes), Switzerland     

Image analysis combined with quantitative cytochemistry

Summary This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%. In LEYTAS-2, which is based on LEYTAS-1 studies, the microscope is equipped with a new type of objective, enabling the analysis of microscope fields, which are four times as large as in LEYTAS-1. The image analysis part consists of the Modular Image Analysis Computer (MIAC, Leitz) and for alarm storage an eight-bit grey value processor is used. Comparison with LEYTAS-1 shows that cell selection capacities are similar and that the speed is four times higher.In honour of Prof. P. van Duijn     

Kinetic behaviour of chicken liver mitochondrial malate dehydrogenase and lactate dehydrogenase mixtures

• 1.1. In the mitochondria of chicken liver cells there is lactate dehydrogenase activity that catalyses the reduction of the oxaloacetate by the NADH.
• 2.2. The presence of lactate dehydrogenase in the malate dehydrogenase preparations causes an apparent activation in the double-reciprocal plot at high oxaloacetate concentrations that depends on the lactate dehydrogenase/malate dehydrogenase ratio in the preparation.
• 3.3. The separation of the two molecular forms of chicken liver mitochondrial malate dehydrogenase, free from lactate dehydrogenase, is described.

     

Kinetic behaviour of soluble and mitochondrial bound lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The kinetic behaviour of mitochondrial bound enzyme fits a bibi sequential type mechanism as well as the cytosolic rabbit liver lactate dehydrogenase. The bound enzyme has greater values of Km(NADH) and Km(pyruvate) than the soluble one, suggesting that binding induces a decrease in the affinity of both substrates. The behaviour of the free and the mitochondrial-bound enzyme is of the Michaelis-Menten type, but the kinetics of a mixture of rabbit liver cytosolic and mitochondrial-bound lactate dehydrogenase is sigmoidal, suggesting that a cooperative phenomenon takes place.     

Kinetic studies on human lactate dehydrogenase isoenzyme-catalyzed lactate-to-pyruvate reaction

In order to evaluate the functional differences that may exist in human lactate dehydrogenase (LDH) isoenzymes widely used for clinical examination the kinetic and thermodynamic properties of the lactate to pyruvate reaction that they catalize were examined. Small but significant differences in the kinetic properties of the three isoenzymes were observed. The difference in the rate constants might affect the activity measurement of the individual isoenzyme as the initial velocity for the L-P reaction catalyzed will not be the same for an equal amount of enzyme. Equilibrium constants for the overall reaction in the presence and absence of pyruvate have been determined. On the basis of transition-state theory, the standard enthalpy and free-energy changes for formation of ternary activated complex were positive, while the standard entropy change was negative. Although the standard free-energy change was the same for activation by the three isoenzymes, the enthalpy and entropy changes for the LDH-3-catalyzed reaction were different from the respective values for others. A large positive value for the free-energy change and a negative value for the entropy change indicated unfavorable production of the activated complex (K _infeq. ^sup╪ =1.89×10^-16). The enzyme appears to stabilize and retain the activated complex until it dissociates into the products.     

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 formulations for the oxidation and the reduction of glyoxylate by lactate dehydrogenase.

Chicken liver lactate dehydrogenase (L-lactate : NAD+ oxidoreductase, EC 1.1.1.27) irreversibly catalyses the oxidation of glyoxylate (hydrated form) (I) to oxalate (pH = 9.6) and the reduction of (non-hydrated form) (II) to glycolate (pH = 7.4). (I) attaches to the enzyme in the pyruvate binding site and (II) attaches to the enzyme at the L-lactate binding site. The oxidation of (I) (pH = 9.6) is adapted to the following mechanism: (see book). The abortive complexes, E-NADH-I and E-NAD+-II, are responsible for the inhibition by excess substrate in the reduction and oxidation systems, respectively. When lactate dehydrogenase and NAD+ are preincubated, E-NAD+- NAD+ appears and causes inhibition by excess NAD+ in the glyoxylate-lactate dehydrogenase-NAD+ and L-lactate-lactate dehydrogenase-NAD+ systems; the second NAD+ molecule attaches to the enzyme at the L-lactate binding site.     

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.     

Intermediate electron-acceptors in quantitative cytochemistry

Summary The efficacy of Meldola Blue (MB), a new intermediate electronacceptor, has been compared with that of phenazine methosulphate (PMS) in the assay of oxidoreductase activity in cryostat sections; various tetrazolium salts have been used as the final electron-acceptors. Three enzymes: succinate dehydrogenase, glucose 6-phosphate dehydrogenase and lactate dehydrogenase were investigated, the activity in sections being quantitated by scanning and integrating microdensitometry. Phenazine methosulphate was superior to Meldola Blue in transferring reducing equivalents from reduced coenzyme to all the tetrazolium salts examined.     

Detection of metabolic changes in hepatocytes by quantitative cytochemistry

Summary Studies by means of quantitative histochemistry and cytochemistry have greatly contributed to the knowledge of metabolic changes in liver parenchymal cells. In the present paper recent work along this line is reviewed with emphasis on three topics, polyploidy as a source of metabolic heterogeneity, proteolysis in the regulation of hepatocyte cell mass and ischemic injury of hepatocytes. In all three fields, accuracy and precision of information obtained by quantitative histochemical means has been greatly enhanced by (1) a thorough knowledge of the mechanisms of histochemical reactions obtained by fundamental work on matrix chemistry, and (2) well-considered application of optical measuring tools and conditions of measurement. These are the principles put forward by van Duijn since the pioneer period of histochemistry and to whom this review is dedicated.In honour of Prof. P. van Duijn     

Detection of metabolic changes in hepatocytes by quantitative cytochemistry

Studies by means of quantitative histochemistry and cytochemistry have greatly contributed to the knowledge of metabolic changes in liver parenchymal cells. In the present paper recent work along this line is reviewed with emphasis on three topics, polyploidy as a source of metabolic heterogeneity, proteolysis in the regulation of hepatocyte cell mass and ischemic injury of hepatocytes. In all three fields, accuracy and precision of information obtained by quantitative histochemical means has been greatly enhanced by a thorough knowledge of the mechanisms of histochemical reactions obtained by fundamental work on matrix chemistry, and well-considered application of optical measuring tools and conditions of measurement. These are the principles put forward by van Duijn since the pioneer period of histochemistry and to whom this review is dedicated.     

Factors affecting the lactate dehydrogenase isoenzyme pattern of cultured kidney-cortex cells

1. The lactate dehydrogenase isoenzyme pattern of cultured calf kidney-cortex cells was correlated to growth phase, changes in oxygen supply, mean generation time and changes in nutritional supply. 2. During culture of free cells and intact explants the lactate dehydrogenase isoenzyme pattern changed towards a dominance of isoenzymes containing the M subunit. 3. Of the shift in monomer proportion, 58% occurred during the lag phase and 42% during the initial part of the exponential growth phase. During the stationary phase the shift in monomer proportion reversed slightly. It was possible to relate the observed shift in monomer proportion to the glycolytic rate. 4. Factors that depressed glycolysis decreased the shift in monomer proportion. Oxygen was found to limit the decrease in the H subunit/M subunit ratio caused by anaerobic culture in vitro. 5. The results obtained support the view that the altered lactate dehydrogenase isoenzyme pattern of urine in renal ischaemia may be explained by anaerobic changes in the lactate dehydrogenase isoenzyme pattern of cortical tubule cells.