LDH-C4-substrate binary complexes studied by intrinsic fluorescence method.

Lactate dehydrogenase-C4 (LDH-C4) has been studied in presence of substrates using intrinsic fluorescence measurements. Excitation maximum of LDH-C4 occurred at 282 nm whereas fluorescence emission maximum was obtained at 340 nm. Fluorescence intensities at 340 nm showed that ligands viz. NAD+, NADH, pyruvate and lactate quench the relative fluorescence intensities of LDH-C4 in a concentration dependent manner. NAD+ and NADH produced a maximum quenching between 92-93% while pyruvate and lactate quenched the fluorescence of LDH up to 29% and 21% respectively. Association constants (Ka) based on fluorescence measurements were 6.05 x 10(4)M-1, 20 x 10(4)M-1, 0.113 x 10(4)M-1 and 0.3 x 10(4)M-1, for NAD+, NADH, lactate and pyruvate respectively. Stern-Volmer constants (Ksv) show that NAD+ and NADH have single Ksv of 4.07 x 10(4)M-1 and 1.47 x 10(5)M-1, whereas lactate and pyruvate indicated quenching reaction to be made up of two components. Ksv at low and high concentration of lactate respectively were 0.645 x 10(2)M-1 and 0.05 x 10(2)M-1, whereas corresponding Ksv with pyruvate were 1.008 x 10(3)M-1 and 0.408 x 10(3)M-1. Low Ksv at higher concentrations suggested that the aromatic chromophores are located within a hydrophobic environment. Red shift in fluorescence maximum (lambda max) by 2nm with lactate and 6nm with pyruvate showed that interaction of these ligands with LDH-C4 exposes some buried chromophores of the enzyme to the surface.

Molecular inactivation of testicular hyaluronidase in solid state after proton irradiation: a study based on target size, substrate binding and thermodynamic analysis of heat denaturation.

Dose response activity curve of testicular hyaluronidase (HDase) following proton irradiation in dry state follows complicated mechanisms which may involve multiple hits and multiple targets of variable sizes giving a constant G value of 1.66. Target analysis appears to be modified by slow recovery of activity when irradiated enzyme is brought to aqueous phase. However, pattern of irradiation at a dose of 1 x 10(5) to 8 x 10(5) Gy reveals that though binding affinity of enzyme to the substrate (hyaluronic acid) increases as shown by declining Km from 500 mg/l to 300-70 mg/l, the reaction rate of catalysis by irradiated HDase is decreased due to decrease in reaction velocity (Vmax: 266 versus 76 units at 8 x 10(5) Gy). Activation analysis of heat denaturation of nonirradiated HDase suggested the involvement of 78 kcal/mole of energy of activation (E*a) which declined to 63-52 k cal/mole after irradiation at 1 x 10(5) to 8 x 10(5) Gy for residual enzyme. The corresponding change in entropy of activation (delta S*) increased from a control value of -291 eu to -236 eu at 8 x 10(5) Gy. From thermodynamic analysis in association with recovery in aqueous phase, it is concluded that HDase is inactivated due to dissipation of proton energy among weak forces including H bonds associated with secondary/tertiary structure of molecules.

Autoimmune-like activity of sperm specific LDH: a pathophysiological and electron microscopic study of atrophied testis and epididymis.

Hyper-immunization of male mice with human LDH-C4 evoked autoimmune reactions illustrated by the loss of LDH activity, associated histopathological changes in testes and epididymis and induction of sterility in mice. This was substantiated by the altered morphology of sperm mitochondria and plasma membrane, and by reduced number of cytoplasmic droplets as observed by electron microscopy. However, the presence of lymphoblasts and other lymphoid cells in testes indicated that the testicular damage is accentuated by activated T lymphocytes. It is concluded that immunization with human LDH-C4 produces lesions in mouse testis and epididymis, which are similar to experimentally induced autoimmune orchitis.

Circadian rhythms of renal phosphohydrolases analysed by Cosinor method.

Rhythmometric analysis of hydrolytic enzymes of mouse kidney has been performed on circadian time scale using the F test. Significant rhythms were detected in glucose-6-phosphatase (G6Pase), inorganic pyrophosphatase (InPPase) and alkaline phosphatase (AlPase) on protein and fresh weight basis. Acrophase (time for peak activity) of G6Pase, InPPase and AlPase per mg protein was at 9.9 degrees (1.0 hr), 88.5 degrees (6.0 hr) and at 342.3 degrees (20 hr) respectively. ATPase, which did not show significant rhythm (mean +/- SD = 4.51 +/- 0.30), had a peak value at 32.1 degrees (2.14 hr) with an amplitude of 0.31 units on protein basis. However, G6Pase and AlPase oscillated with high amplitudes (0.18 and 0.71) across the mean value (mesor) of 0.68 +/- 0.3 and 1.43 +/- 0.46 units respectively and with a phase shift of 5 hr. Since G6Pase is a multicomponent and multifunctional enzyme having several overlapping enzyme activities (viz. InPPase), coordinated events of G6Pase, InPPase and ATPase in the regulation of daily renal functions have been mapped in the intact animals, along a physiologic time scale.

Effects of immunization with sperm specific lactate dehydrogenase with & without muramyl dipeptide as adjuvant.

The effect of sperm specific lactate dehydrogenase-C4 (LDH-C4) alone or with muramyl dipeptide (MDP) and Freund's complete adjuvant (FCA) on immune responses and breeding capacity have been studied in isogeneic C57 BI/Ks (H-2d) mice. Results per se suggested that LDH-C4 generates isoantibodies even in absence of adjuvant. Though MDP could be substituted for FCA as adjuvant, amplification by MDP of the antibody levels is reduced to half that obtained with FCA. LDH-B4 from kidney did not produce any antibody response under similar conditions. Systemic immunization with LDH-C4 did not reduce the overall pregnancy rate but reduced the frequency of embryo resorption and increased litter size significantly. However, mothers with foetal non-resorption had in general, lower antibody titres than the mothers showing foetal resorptions. The popliteal lymph node (PLN) assay for local graft versus host (LGVH) reaction revealed that the maternal lymphocyte cell competence was depressed by the sperm specific isozyme during gestation; depression in stimulation index was associated with the low antibody titre group. MDP, like LDH-C4 alone did not modify LGVH reaction significantly although it exerted a similar effect as LDH-C4 in embryo protection.

Circadian rhythmometric analysis of hepatic phosphohydrolases with special reference to glucose-6-phosphatase and inorganic pyrophosphatase.

Rhythmometric analysis of a group of phosphohydrolases in mouse liver has been performed along a single 24 hr time scale. The presence of the rhythm was conducted by F test. Statistically significant circadian rhythm was detected in glucose-6-phosphatase (G6Pase) and inorganic pyrophosphatase (InPPase) activity expressed on fresh weight and protein basis. Both G6Pase and InPPase oscillated with a high amplitude of 0.44 U and 1.15 U respectively across the mean value (mesor) of 0.40 +/- 0.42 U and 2.81 +/- 1.14 U per mg protein and with a phase shift of 80 degrees (5.34 hr) among them. On the other hand, alkaline phosphatase (AlPase) did not show any rhythm whereas adenosine triphosphatase (ATPase) showed rhythmic activity on protein basis and oscillated across mesor of 1.84 +/- 0.5 U with an amplitude of 0.52. Acrophase (time for peak activity/mg protein) of G6Pase, InPPase and ATPase was found at 194.2 degrees (13.34 hr), 114.1 degrees (8.0 hr) and at 306.1 degrees (20.4 hr) respectively. AlPase, though did not show significant rhythm, had peak value at 231.8 degrees. Since hepatic G6Pase is a multicomponent and multifunctional enzyme with several overlapping activities (viz. InPPase), coordinated action of G6Pase and InPPase in the regulation of hepatic cell functions has been suggested.

Regulation of fertility in female mice after immunization with human sperm specific LDH: role in conception and contraception.

Effects on fertility of female mice after hyper-immunization with human lactate dehydrogenase-C4 have been studied at (50 + 25 x 4) and (50 + 50 x 4) micrograms doses of protein for two consecutive cycles of pregnancy. Results of heterologous immunization are described in relation to the role of LDH-C4 in conception and contraception. (i) Low dose of Immunization produced higher antibody (Ab) titer as tested before mating followed by higher infertility (70%) than by a higher dose of immunogen (46%), taking pregnancy as the end point. (ii) However, the results were reversed after second mating. For example, low dose of immunogen produced insignificant infertility, whereas high dose of immunogen gave 80% infertility. Similar order was shown by humoral Ab titer before mating i.e., high infertility was associated with high Ab titer and viceversa. (iii) It shows that the LDH-C4 induced infertility is reversible as is evidenced by low dose of immunization. (iv) In contrary to infertile dams, immunized fertile dams delivered a significantly higher litter size as compared to non-immunized control dams. It is concluded that immunity due to LDH-C4 is beneficial for embryo survival suggesting an immuno-suppressive nature of LDH-C4. Nonetheless, infertility ensues when immune-suppression is overcome by its own immune-activation.

Histomorphological changes in lung of rats following exposure to wood smoke.

Rats were exposed to repeated, intermittent exposure to smoke generated from combustion of 1g wood/15 min, total period for 75 min daily under dynamic exposure conditions, over a period of 15, 30 and 45 days. First 15 days exposure caused mild bronchiolitis, hyperplasia and hypertrophy of bronchiolar epithelial lining cells, some necrosed lining cells desquamated into lumens, congestion of parenchymatous blood vessels, oedema, hyperplasia of lymphoid follicles, peribronchiolar and perivascular infiltration of polymorphonuclear cells, and mild emphysema. These lesions progressed further during 30 and 45 days of exposure, though emphysematous changes remain constant. By 30 days and 45 days, hyperplastic and hypertrophic changes of bronchioles become quite marked, with mononuclear cells infiltration and alveolar septa thickening. Hematological studies show marginal alterations in hemoglobin levels, ESR, PCV and TLCS during 15 days, where as significant changes in eosinophil were observed during 30 and 45 days, and ESR during 45 days only. The results indicate progressive pathomorphological pulmonary lesions with subsequent exposure to wood smoke in controlled conditions.

Modulation of T cell functions by sperm-specific lactate dehydrogenase: fluorescence analysis of immune competent cells and local graft versus host reaction.

Immune responses to a well-defined sperm-specific isogenic lactate dehydrogenase-C4 (LDH-C4) have been studied in C57Bl/Ks (H-2d) mice after immunization through intra-rectal route. Presence of anti-LDH-C4-antibodies in the sera of females immunized in presence or absence of adjuvant suggested that the immune system of mice becomes exposed to sperm antigens following intrarectal insemination. LDH-C4 primed lymphocytes from both males and females, when transferred in F1 hybrids, suppressed stimulation index of local graft versus host reaction. However, contrary to females, male counterparts which did not elicit measurable anti-LDH-C4-antibody titer, showed the presence of a higher proportion of Ly2+ and Ia+ fluorescence labelled cells in the spleen of LDH-C4 administered mice. Results suggest that males are more susceptible for immune suppression of T cell functions through generation of T suppressor cells. Sex differences in relation to immune deviation by intra-rectal administration of sperm-specific LDH-C4 in mice and their consequences in AIDS and AIDS-related complex diseases are described.

Assessment of lipid peroxidation inhibitory potential of bronchoalveolar lavage fluid from rat lungs.

Studies were carried out to further localize pulmonary tissue lipid peroxidation inhibitor reported earlier to be present in whole lung homogenate in rat. A major portion of the inhibitory material could be lavaged from bronchoalveolar region using various wash media. The material was heat labile and exhibited higher degree of inhibition when recovered from the lungs of mature and aged rats.