Catalytic properties of the lactate dehydrogenase isozyme “X” from mouse testis

Battellino, Luis José; Blanco, Antonio

doi:10.1002/jez.1401740207

Cited by 48 publications

(23 citation statements)

References 21 publications

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“…As we have suggested previously, these variable values of a may be related to the different composition of LDH isozymes in cells of different types. It has been known for many years that skeletal muscle and liver contain principally the M-type LDH isozyme but cardiac muscle the H-type (e.g., Battellino and Blanco 1970). However, there are two other interpretations for the observed initial nonlinearity of the intrinsic LDH reaction.…”

Section: Discussionmentioning

confidence: 99%

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Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells

Nakae

Stoward

1997

J Histochem Cytochem.

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SUMMARYWe studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on a kinetic parameter of lactate dehydrogenase LDH that is assumed to be related to the extent of diffusion of the enzyme out of tissue sections during its histochemical assay. The kinetics of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and periportal hepatocytes were determined in unfixed sections incubated either on substrate ( L -lactate)-containing agarose gel films or in aqueous assay media in the presence or absence of 18% PVA. The absorbances of the formazan final reaction products at their isobestic point were measured continuously in the cytoplasm of individual cells as a function of incubation time, using a real-time image analysis system. Whichever incubation medium was used, the absorbances in the two cell types increased nonlinearly during the first minute of incubation but linearly for incubation times between 1 and 3 min. The nonlinearity of the LDH reaction was analyzed using the equation v i Ϫ v ϭ a Њ A , where v i is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and Њ A are respectively the gradient and intercept on the absorbance axis of the linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed ( v i Ϫ v ) against Њ A were linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12-43% lower than those for hepatocytes. The a value for hepatocytes obtained with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by the two methods were almost the same. Addition of excess pyruvate to the aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide gels containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to the extent to which the enzyme diffuses out of sections during incubation but not to product inhibition of LDH by pyruvate. PVA was more effective for protecting diffusion of LDH from hepatocytes than from skeletal muscle fibers, possibly because hepatocytes contain a greater proportion of diffusable (unbound) LDH than skeletal muscle fibers.

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Section: Discussionmentioning

confidence: 99%

“…Mouse liver and skeletal muscle both contain predominantly muscle-type (M) isozymes (e.g., Battellino and Blanco 1970). However, the cytoskeletal organizations in skeletal muscle fibers and hepatocytes differ, especially concerning F-actin and microtubules.…”

mentioning

confidence: 99%

Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells

Nakae

Stoward

1997

J Histochem Cytochem.

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“…(iMi) The forward reaction lactate-to-pyruvate is inhibited to a far greater extent by high concentrations of pyruvate in the H4 than in the M4 isozyme (5,(7)(8)(9). (iv) Both the enzymic activity and the affinity of the substrate for enzyme are reduced to a greater extent in the M4 isozyme as the length of the aliphatic chain of the a-keto acid increases (10)(11)(12). (v) The H4 isozyme binds oxidized or reduced coenzyme better than the M4 isozyme (13)(14)(15)(16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

Structural adaptations of lactate dehydrogenase isozymes.

Eventoff¹,

Rossmann²,

Taylor³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

276

139

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The three-dimensional structures of dogfish M4 (muscle) and pig H4 (heart) lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) have been determined and correlated with the amino acid sequences of the dogfish M4, pig M4, pig H4, chicken M4, and chicken H4 lactate dehydrogenase isozymes. These results have been related to the known differences of physicochemical properties between the M and H lactate dehydrogenase isozymes. By far the largest structural alterations occur in the transition between the "apo" and "ternary complex" conformational states of the enzyme rather than between species or isozymes. The major catalytic difference can be explained by a replacement of alanine (in the M chain) with a glutamine (in the H chain) in the vicinity of the binding site of the coenzyme phosphates. The known immunological differentiation of the M and H isozymes is consistent with the differences in their amino acid sequences.The subunits of lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) exist as two major structural forms, usually referred to as M (muscle) and H (heart) or A and B, respectively, which give rise to five different isozymes of the tetrameric molecule (1, 2) in higher vertebrates. The differences in the properties of the LDH isozymes are dependent on their subunit composition and are most exaggerated between the homotetramers M4 (LDH-5) and H4 (LDH-1). The most important of these differences are (independent of species): (i) The turnover number of the M4 isozyme is generally about twice that for the H4 isozyme (3-5).(ii) The activity of the H4 isozyme is more readily diminished by modification of the carboxyamide group on the 3 position of the nicotinamide (6). For instance, acetylpyridine adenine dinucleotide reduces the rate of catalysis by at least five when compared to NAD in the H4 enzyme, whereas the M4 isozyme is not especially sensitive to this difference of the NAD molecule (7). (iMi) The forward reaction lactate-to-pyruvate is inhibited to a far greater extent by high concentrations of pyruvate in the H4 than in the M4 isozyme (5, 7-9). (iv) Both the enzymic activity and the affinity of the substrate for enzyme are reduced to a greater extent in the M4 isozyme as the length of the aliphatic chain of the a-keto acid increases (10-12). (v) The H4 isozyme binds oxidized or reduced coenzyme better than the M4 isozyme (13-20). (vi) An antiserum induced in rabbit against the M4 isozyme of one species will crossreact with the M4 isozyme from several closely related species but not with the H4 isozyme of the same species (4,(21)(22)(23)(24)(25). Some appreciation of these differences is now possible from a correlation of recent studies on three-dimensional and primary structures of the H4 and M4 LDH isozymes.The tertiary structure of dogfish M4 LDH has been determined independently for the apo-enzyme and for a complex of LDH, coenzyme, and substrate analogue. The apo-enzyme has been studied at 2.0-A resolution, while a series of isomorphous ternary complexes h...

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“…Evidence for the lack of correspondence of a~ and a2 and isozyme composition is the similar low values of az and a2 in skeletal muscle fibres and cardiac muscle fibres. Skeletal muscle fibres contain principally M-type LDH (Markert & Ursprung, 1962;Costello & Kaplan, 1963;Ohashi, 1966;Battellino & Blanco, 1970;Lluis, 1985;Reid & Masters, 1985) but cardiac muscle H-type LDH (Markert & Ursprung, 1962;Costello & Kaplan, 1963;Battellino & Blanco, 1970;Hori et al, 1970) and yet their az values are both low. Conversely, mouse and human hepatocytes which have high a2 values also mostly contain M-type LDH (Wr6blewski & Gregory, 1961;Markert & Ursprung, 1962;Costello & Kaplan, 1963;Ohashi, 1966;Shaw, 1969;Battellino & Blanco, 1970;Hori et al, 1970;Reid & Masters, 1985) like skeletal muscle fibres.…”

Section: Discussionmentioning

confidence: 99%

“…In this paper we report that the equations are also applicable to LDH in other types of mouse cell, such as cardiac muscle fibres, skeletal muscle fibres, gastric parietal cells, oocytes and ductal epithelial and acinar cells of the parotid gland, in which the mix of LDH isozymes differs from that in liver (Markert & Ursprung, 1962;Costello & Kaplan, 1963;Ohashi, 1966;Shaw, 1969;Battellino & Blanco, 1970;Hori et al, 1970;Lluis, 1985;Reid & Masters, 1985). They also apply to LDH in human hepatocytes (Wr6blewski & Gregory, 1961).…”

Section: Introductionmentioning

confidence: 94%

The initial reaction velocities of lactate dehydrogenase in various cell types

Nakae

Stoward

1994

Histochem J

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The initial reaction velocities (vi) of lactate dehydrogenase in hepatocytes, cardiac muscle fibres, skeletal (gastrocnemius) muscle fibres, gastric parietal cells, ductal epithelial and acinar cells of the parotid gland, and oocytes were determined, by computer-assisted image analysis, in unfixed sections of these tissues incubated at 37 degrees C on substrate-containing agarose gel films. They were found to fit the equations vi = a1 zero A (equation 1) and vi-v = a2 zero A (equation 2) reported previously for mouse hepatocytes (Nakae & Stoward, 1993a, b), where v and zero A are, respectively, the gradients (or steady-state velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) of the final reaction product on incubation times between 1 and 3 min, and a1 and a2 are constants. Both equations 1 and 2 fitted the observed vi closely for mouse (a1 = 2.7, a2 = 2.2) and human (a1 = 3.0, a2 = 1.9) hepatocytes. However, equation 2 fitted the observed vi better than equation 1 for mouse cardiac muscle fibres (a1 = 1.5), skeletal muscle fibres (a2 = 1.2), gastric parietal cells (a2 = 1.7), acinar (a2 = 1.4) and striated ductal (a2 = 2.2) epithelial cells of the parotid gland, and oocytes (a2 = 1.6). The values of vi calculated from the two equations agreed with the observed vi to within about 11%. They ranged from 105 mumole hydrogen equivalents/cm3 cell/min units in hepatocytes to 24 units in parotid acinar cells, but for other cell types they were between 46 and 61 units. These are all considerably higher than values reported previously.

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Catalytic properties of the lactate dehydrogenase isozyme “X” from mouse testis

Cited by 48 publications

References 21 publications

Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells

Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells

Structural adaptations of lactate dehydrogenase isozymes.

The initial reaction velocities of lactate dehydrogenase in various cell types

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