Radiation Therapy Reduced Blood Levels of LDH, HIF-1α, and miR-210 in OSCC.

Radiation Therapy (RT) is a treatment option for a large number of neoplasias. However, the effect of RT on the level of hypoxia markers is poorly understood. The present study aimed to investigate the effect of RT on the levels of hypoxic markers in Oral squamous cell carcinoma (OSCC). Evaluation of HIF-1α and miR-210 levels in OSCC was performed. Then a proteomic analysis was performed to identify candidate hypoxic targets of RT. To validate proteomic studies, the effect of RT on HIF-1α, miR-210, PDH-A and LDH-A levels under hypoxia was assessed by qRT-PCR. The impact of RT in hypoxia markers was evaluated in patients to confirm in vitro results. An increase in the HIF-1α levels was observed in OSCC. RT reduced OSCC cell proliferation and migration. Interestingly, hypoxia could revert the effect of radiation on OSCC phenotype. However, proteomics analyses suggested that LDH is one of the critical targets of RT even in hypoxia. Moreover, RT decreased HIF-1α, miR-210, and LDH even in hypoxia. The current study demonstrated that hypoxia could revert the effects of RT in the OSCC context. However, RT reduces the levels HIF-1α, miR-210 and LDH in vivo and in vitro. The consequences of RT in blood should be carefully investigated.

Cytotoxicity of gamma-ray in rat immature hippocampal neurons.

This in vitro study evaluated the detrimental effect of acute gamma (γ)-irradiation on rat immature hippocampal neurons. Rat immature hippocampal neurons (0.5 day in vitro) were irradiated with 0~4 Gy γ-rays. Cytotoxicity was analyzed using a lactate dehydrogenase release assay at 24 h after γ-irradiation. Radiation-induced cytotoxicity in immature hippocampal neurons increased in a dose-dependent manner. Pre-treatments of pro-apoptotic caspase inhibitors and anti-oxidative substances significantly blocked γ-irradiation-induced cytotoxicity in immature hippocampal neurons. The results suggest that the caspase-dependent cytotoxicity of γ-rays in immature hippocampal cultured neurons may be caused by oxidative stress.

Low intensity microwave radiation as modulator of the L-lactate dehydrogenase activity.

In this study, we investigated experimentally the possibility of modulating protein activity by low intensity microwaves by measuring alternations of L: -Lactate Dehydrogenase enzyme (LDH) activity. The LDH enzyme solutions were irradiated by microwaves of the selected frequencies and powers using the Transverse Electro-Magnetic (TEM) cell. The kinetics of the irradiated LDH was measured by continuous monitoring of nicotine adenine dinucleotide, reduced (NADH) absorbance at 340 nm. A comparative analysis of changes in the activity of the irradiated LDH enzyme versus the non-radiated enzyme was performed for the selected frequencies and powers. It was found that LDH activity can be selectively increased only by irradiation at the particular frequencies of 500 MHz [electric field: 0.02 V/m (1.2 × 10⁻6 W/m²)-2.1 V/m (1.2 × 10⁻² W/m²)] and 900 MHz [electric field: 0.021-0.21 V/m (1.2 × 10⁻4 W/m²)]. Based on results obtained it was concluded that LDH enzyme activity can be modulated by specific frequencies of low power microwave radiation. This finding can serve to support the hypothesis that low intensity microwaves can induce non-thermal effects in bio-molecules.

Influence of irradiation on in vitro red-blood-cell (RBC) storage variables of leucoreduced RBCs in additive solution PAGGS-M.

BACKGROUND: The effect of gamma irradiation on leucoreduced red-blood-cells (RBCs) stored in an additive solution (AS) containing phosphate, adenine, glucose, guanosine, saline and mannitol (PAGGS-M) has not yet been studied, and there are different recommendations about storage time of leucoreduced RBCs after irradiation. STUDY DESIGN AND METHODS: We studied 63 leucoreduced RBC units. All RBCs were stored in AS PAGGS-M and leucoreduced on the collection day. Twenty-one components were irradiated on Day +14 with 30 Gy and 22 served as non-irradiated controls. Samples were drawn and analysed from these 43 units on Day +7, +14, +21, +28, +35, +42 and +49 from the collection day. From 20 units, no samples were taken earlier than on Day +49. Of these, 10 components had been irradiated on Day +14 with 30 Gy and 10 served as non-irradiated controls. RESULTS: Gamma irradiation induced an enhanced in vitro haemolysis rate in the irradiated components. One of the irradiated units showed a haemolysis rate over the recommended limit of 0·8% on Day +42 and four on Day +49. The leakage of potassium ions from irradiated RBCs started to increase faster than that of unirradiated RBCs from the day of irradiation. Lactate dehydrogenase levels increased faster in irradiated units 3 weeks after irradiation. We showed that taking samples weekly does not affect the final result. CONCLUSIONS: Our findings show that the European recommendations should not be changed in regard to the limitation of the storageability after irradiation of leucoreduced RBCs. The damage after irradiation and storage cannot be prevented by using the high-quality AS PAGGS-M.

In vitro effects on platelets irradiated with short-wave ultraviolet light without any additional photoactive reagent using the THERAFLEX UV-Platelets method.

BACKGROUND: A novel short-wave ultraviolet light (UVC) pathogen reduction technology (THERAFLEX UV-Platelets; MacoPharma, Mouvaux, France) without the need of any additional photoactive reagent has recently been evaluated for various bacteria and virus infectivity assays. The use of UVC alone has on the one hand been shown to reduce pathogens but may, on the other hand, have some impact on the platelet (PLT) quality. The purpose of this study was to determine the potential effects on PLT quality of pathogen inactivation treatment using the novel UVC method for PLT concentrates. STUDY DESIGN AND METHODS: Buffy-coat-derived PLTs suspended in SSP+ were irradiated with UVC light in plastic bags (MacoPharma) made of ethyl vinyl acetate, considered to be highly permeable to UVC light. The UVC-treated (test, n=8) as well as the untreated (reference, n=8) PLT units were stored in PLT storage bags composed of n-butyryl, tri n-hexyl citrate-plasticized polyvinyl chloride (MacoPharma) on a flat bed agitator for in vitro testing during 7 days of storage. RESULTS: No significant difference in PLT counts and lactate dehydrogenase between the groups was detected. During storage, glucose decreased more and lactate increased more in the test units. Statistically significant differences were found for glucose (P<0·01) and lactate (P<0·05) on day 7. ATP levels were higher (P<0·01 from day 5) in the reference units. With exception of day 7 (P<0·01 reference vs. test), hypotonic shock response reactivity was not different between groups. Extent of shape change was lower (P<0·01), and CD62P (P<0·05 day 5) was higher in the test units. CD42b and CD41/61 showed similar trends throughout storage, without any significant difference between the units. pH was maintained at >6·8 (day 7) and swirling remained at the highest level (score = 2) for all units throughout storage. CONCLUSION: Our results suggest that irradiation with UVC light has a slight impact on PLT in vitro quality and appears to be insignificant with regard to current in vitro standards.

Cytotoxicity of gamma-ray in rat immature hippocampal neurons

This in vitro study evaluated the detrimental effect of acute gamma (gamma)-irradiation on rat immature hippocampal neurons. Rat immature hippocampal neurons (0.5 day in vitro) were irradiated with 0~4 Gy gamma-rays. Cytotoxicity was analyzed using a lactate dehydrogenase release assay at 24 h after gamma-irradiation. Radiation-induced cytotoxicity in immature hippocampal neurons increased in a dose-dependent manner. Pre-treatments of pro-apoptotic caspase inhibitors and anti-oxidative substances significantly blocked gamma-irradiation-induced cytotoxicity in immature hippocampal neurons. The results suggest that the caspase-dependent cytotoxicity of gamma-rays in immature hippocampal cultured neurons may be caused by oxidative stress.

Effect of same-dose single or dual field irradiation on damage to miniature pig parotid glands.

AIM: To evaluate the effect of single or dual field irradiation (IR) with the same dose on damage to miniature pig parotid glands. METHODOLOGY: Sixteen miniature pigs were divided into two IR groups (n=6) and a control group (n=4). The irradiation groups were subjected to 20 Gy X-radiation to one parotid gland using single-field or dual-field modality by linear accelerator. The dose-volume distributions between two IR groups were compared. Saliva from parotid glands and blood were collected at 0, 4, 8 and 16 weeks after irradiation. Parotid glands were removed at 16 weeks to evaluate tissue morphology. RESULTS: The irradiation dose volume distributions were significantly different between single and dual field irradiation groups (t=4.177, P=0.002), although dose volume histogramin (DVH) indicated the equal maximal dose in parotid glands. Saliva flow rates from IR side decreased dramatically at all time points in IR groups, especially in dual field irradiation group. The radiation caused changes of white blood cell count in blood, lactate dehydrogenase and amylase in serum, calcium, potassium and amylase in saliva. Morphologically, more severe radiation damage was found in irradiated parotid glands from dual field irradiation group than that from single field irradiation group. CONCLUSION: Data from this large animal model demonstrated that the radiation damage from the dual field irradiation was more severe than that of the single field irradiation at the same dose, suggesting that dose-volume distribution is an important factor in evaluation of the radiobiology of parotid glands.

Probing the role of dynamics in hydride transfer catalyzed by lactate dehydrogenase.

The dynamic nature of the interconversion of pyruvate to lactate as catalyzed by lactate dehydrogenase (LDH) is characterized by laser-induced temperature jump relaxation spectroscopy with a resolution of 20 ns. An equilibrium system of LDH.NADH plus pyruvate and LDH.NAD+ plus lactate is perturbed by a sudden T-jump, and the relaxation of the system is monitored by NADH emission and absorption changes. The substrate binding pathway is observed to be similar, although not identical, to previous work on substrate mimics: an encounter complex is formed between LDH.NADH and pyruvate, which collapses to the active Michaelis complex. The previously unresolved hydride transfer event is characterized and separated from other unimolecular isomerizations of the protein important for the catalytic mechanism, such as loop closure, a slower step, and faster events on the nanosecond-microsecond timescales whose structural basis is not understood. The results of this study show that this approach can be applied quite generally to enzyme systems and report on the dynamic nature of proteins over a very wide time range.

Inactivation of chosen dehydrogenases by the products of water radiolysis and secondary albumin and haemoglobin radicals.

PURPOSE: Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) by products of water radiolysis and by secondary radicals localized on haemoglobin (Hb) and human albumin (HSA) was studied. MATERIALS AND METHODS: Aqueous solutions of ADH, GAPDH and LDH were irradiated under air and under nitrous oxide (N2O) in the absence and in the presence of Hb or HSA. In order to determine the effectiveness of inactivation of the enzymes by radicals localized on Hb and HSA, the inactivation efficiency determined experimentally was compared with that calculated under assumption that only hydroxyl radicals are responsible for the enzyme inactivation. RESULTS: In the absence of other proteins, under air, GAPDH showed the highest radiation sensitivity, followed by ADH and LDH. The sequence was reverse under anaerobic atmosphere. Oxygen increased considerably the inactivation of GAPDH and ADH. Secondary albumin and haemoglobin radicals brought about considerable inactivation of GAPGH and ADH. Albumin radicals (HSA) generated under N2O inactivated GAPDH and ADH more effectively than haemoglobin radicals (Hb). Under air, however, inactivation of GAPDH and ADH by haemoglobin peroxyl radicals was higher than by albumin peroxyl radicals. LDH was resistant to inactivation by haemoglobin and albumin radicals, and peroxides of these proteins. CONCLUSIONS: In the light of these results and literature data, the observed differences in the effectiveness of inactivation of the dehydrogenases studied by secondary protein radicals depend on the amino acid residues present at the active site and in its close neighborhood and on the number of amino acid residues available on the protein surface.

The effect of electromagnetic radiation (550-850 nm) on 1-lactate dehydrogenase kinetics.

PURPOSE: This work is based on our earlier research of the Resonant Recognition Model (RRM), where we have proposed that protein activation is electromagnetic in its nature. In this study we investigated experimentally the possibility of modulating the protein activity by the electromagnetic radiation of the specific frequency. The concept is studied here by applying a visible light radiation to example of 1-Lactate Dehydrogenase enzyme (LDH). MATERIALS AND METHODS: The selected LDH example is radiated by monochromatic visible light in a frequency range predicted computationally by the RRM. The kinetics of the irradiated LDH is measured by continuous monitoring of the NADH absorption at 340 nm. RESULTS: A comparative analysis of the LDH enzyme activity before and after the electromagnetic field (EMF) exposures is performed. It was found that the LDH activity is selectively increased only by the radiation at the particular wavelengths of 595 nm and 828 nm. These experimentally determined wavelengths of the applied EMF are within the range predicted by the RRM. CONCLUSIONS: Results reveal the LDH activity was modulated by the EMF exposures at the computationally predicted frequencies. The RRM concept presented provides new insights into proteins susceptibility to perturbation by electromagnetic radiation and possibility to program, predict, design and modify proteins and their bioactivity.

Self-sensitized photodegradation of membrane-bound protoporphyrin mediated by chain lipid peroxidation: inhibition by nitric oxide with sustained singlet oxygen damage.

In the presence of exciting light, iron and reductants, the singlet oxygen (1O2)-generating sensitizer protoporphyrin IX (PpIX) induces free radical lipid peroxidation in membranes, but gradually degrades in the process. We postulated that NO, acting as a chain-breaking antioxidant, would protect PpIX against degradation and consequently prolong its ability to produce 1O2. This idea was tested by irradiating PpIX-containing liposomes (LUVs) in the presence of iron and ascorbate, and monitoring the cholesterol hydroperoxides 5alpha-OOH and 7alpha/beta-OOH as respective 1O2 and free radical reporters. 5alpha-OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7alpha/beta-OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7alpha/beta-OOH buildup as well as 5alpha-OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5alpha-OOH slowdown. Accompanying the 5alpha-OOH effect was a steady decrease in 1O2 quantum yield and PpIX fluorescence at 632 nm, both of which were inhibited by NO. An NO-inhibitable decay of PpIX fluorescence was also observed during dark incubation of 5alpha-OOH-bearing LUVs with iron and ascorbate, confirming a link between chain peroxidation and PpIX loss. By protecting PpIX in irradiated membranes, NO might select for and prolong purely 1O2-mediated damage. Supporting this was our observation that 1O2-mediated photoinactivation of a nonmembrane target, lactate dehydrogenase, slowed concurrently with 5alpha-OOH accumulation and that spermine NONOate prevented this. Thus, NO not only protected membrane lipids against PpIX-sensitized free radical damage, but PpIX itself, thereby extending its 1O2-generating lifetime. Consistent findings were obtained using porphyrin-sensitized COH-BR1 cells. These previously unrecognized effects of NO could have important bearing on 5-aminolevulinate-based photodynamic therapy in which PpIX is metabolically deposited in tumor cells.

[Photochemical transformation of lactate dehydrogenase isoforms: UV-sensitivity in the presence of biogenous amines]. / Zakonomernosti i osobennosti fotokhimicheskikh prevrashchenii izoform laktatdegidrogenazy: UF-chuvstvitel'nost' ikh v prisutstvii biogennykh aminov.

It has been all round investigation of UV-irradiation influence in a wide dose range on the structural and functional properties of human blood lactate dehydrogenase (LDH) isoenzymes. The photoprotective action of biogenous amines on the functional activity of different enzyme isoforms was found. It has been established that the protective action of biogenous amines is caused by the formation of complex LDH--biogenous amine and by the acception of the active oxygen forms by the molecules of lactate dehydrogenase. Under the conditions of exogenous singlet oxygen generation in the presence of methylene blue, the inactivation of immobilized LDH tetramers and subunits was observed, that shows participation of this active intermediate in the processes of UV-modification of the enzyme in soluble and immobilized states. The scheme of processes of LDH molecules phototransformations in the presence of biogenous amines has been suggested.

Effects of infrared and low-power laser irradiation on cell viability, glutathione and glutathione-related enzyme activities in primary rat hepatocytes.

BACKGROUND AND PURPOSE: Both infrared and low-power laser have been applied to improve circulation, wound repair, and pain control. Infrared and low-power laser therapies have the potential for stimulating enzyme activities which might contribute to increased glutathione (GSH) concentration and provide protection against oxidative damage. This study investigated cell viability, and GSH and its related enzyme activities in rat hepatocytes after irradiation. METHODS: Hepatocytes were isolated from 8-week-old male Sprague-Dawley rats and the cultures were divided into infrared, laser, and control groups. The cells were treated with infrared and low-power laser at a distance of 35 cm for 20 minutes. The cell morphology, lactate dehydrogenase (LDH) leakage, lipid peroxidation, GSH concentration, GSH peroxidase, GSH reductase (GRd), and GSH S-transferase activities were measured after irradiation. RESULTS: The morphology and LDH leakage of hepatocytes in the irradiation groups did not differ significantly from those of the control group. After infrared irradiation, a significant decrease in thiobarbituric acid-reactive substances and an increase in GSH concentration were found after 48 hours of incubation compared to the control group (p < 0.05). Furthermore, laser irradiation resulted in a significant increase in GRd activity after 48 hours of incubation compared to the control group (p < 0.05). A 48-hour incubation period produced greater GRd activity in all groups compared to a 24-hour period (p < 0.05). CONCLUSIONS: Irradiation did not damage rat hepatocytes in this study. Infrared was shown to stimulate GSH production, while laser irradiation increased GRd activity.

Gamma knife irradiation increases cerebral endothelial expression of intercellular adhesion molecule 1 and E-selectin.

OBJECTIVE: Alterations in multiple functions of the microvasculature occur in response to gamma irradiation and are thought to contribute to radiation-induced end organ damage by inducing inflammatory responses, particularly leukocyte infiltration into the affected area. Endothelial cell adhesion molecules (ECAMs) mediate leukocyte adhesion and migration. Here, we validate a method to study the effect of Leksell gamma knife stereotactic radiosurgery on the expression of ECAMs on human cerebral endothelium at 0, 24, 48, and 72 hours after irradiation. METHODS: A human brain endothelial cell line (IHEC) was cultured on 12-mm coverslips and subjected to 50 Gy of collimated gamma irradiation with the Leksell gamma knife (Elekta Instruments, Inc., Atlanta, GA). Lactate dehydrogenase release was measured at 24, 48, and 72 hours after irradiation and caspase-3 at 24, 48, 72, 96, and 120 hours. ECAM expression was measured at postirradiation intervals of 0, 24, 48, and 72 hours by cell enzyme-linked immunoabsorbent assay. We used a cell irradiator composed of two chambers. The upper chamber holds the coverslips firmly in place while they are immersed in media. The lower chamber is connected to a peristaltic pump, which pumps water into the chamber and maintains the media in the upper chamber at 37 degrees C through convection. RESULTS: None of the ECAMs tested was significantly elevated compared with the control basally. Twenty-four hours after irradiation, intercellular adhesion molecule 1 was significantly elevated on brain endothelial cells but there was no significant elevation of E-selectin. Vascular cell adhesion molecule 1 was increased slightly but not significantly and decreased at 48 hours. At 72 hours, E-selectin expression was significantly increased; intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were not altered relative to sham controls. CONCLUSION: Increased ECAM expression and lactate dehydrogenase release support the idea that the cerebral microvasculature undergoes an inflammatory response after Leksell gamma knife stereotactic radiosurgery.

[Structure-functional modification of lactate dehydrogenase isoforms under the effect of ultraviolet radiation and active forms of oxygen]. / Strukturno-funktsional'nye modifikatsii izoform laktatdegidrogenazy pod vliianiem UF-oblucheniia i aktivnykh form kisloroda.

The investigation results on the UV-induced changes of lactate dehydrogenase structural and functional properties in the presence of some chemical substances, which are able to interact with the oxygen active forms have been summarized. As well the kinetic characteristics of the photoinactivation processes for heart and muscle LDH types in free state and in the complex with the exogenous agents have been studied. Singlet molecular oxygen has been shown to play an important role in the process of UV-modification of different protein isoforms. The scheme of probable physical and chemical processes, leading to the inactivation of lactate dehydrogenase molecules, has been suggested.

Altered intracellular purine nucleotides in gamma-irradiated red blood cell concentrates.

BACKGROUND AND OBJECTIVES: Gamma irradiation at a dose of 30 Gy induces deterioration of erythrocytes, resulting in storage lesions that significantly shorten the shelf-life of packed red cell concentrates (RCCs). The aim of the present study was to investigate the effect of gamma irradiation on intracellular purine nucleotides of red blood cells during storage. MATERIALS AND METHODS: Three-day-old leucocyte-depleted saline-adenine-glucose-mannitol (SAGM)-preserved RCCs, obtained from the Blood Service of the Austrian Red Cross, were gamma irradiated with 30 Gy. Samples were taken on days 1, 2, 3 and 7 after irradiation and subsequently at weekly intervals up to the end the of shelf-life (day 39 after irradiation) and were investigated for the K+ and Na+ content in the supernatant, for intracellular concentrations of ATP, ADP, ITP, IDP, GTP and GDP of erythrocytes, and for haemolysis. RESULTS: Within the first 24 h after gamma irradiation, no metabolic or biochemical changes were detectable in the RCCs. The K+ concentration in the supernatant increased after 24 h, while the Na+ concentration decreased in irradiated units and this ion disequilibrium persisted until the end of the shelf-life. After an initial increase of intracellular ATP, ADP and GTP during the first week of storage, the intracellular concentrations of ATP, ADP, GTP and ITP decreased, while IDP increased. The decrease of ATP and ADP was found to be more pronounced in irradiated units. At the end of the shelf-life, the ATP, GTP and ITP concentrations of irradiated RCCs had decreased to < 10% of the initial level and the critical threshold of 0.8% haemolysis was reached. CONCLUSION: Gamma irradiation of SAGM-preserved RCCs leads to serious deterioration of the purine nucleotide metabolism of erythrocytes during storage, which can reduce the in vivo recovery of the transfused red cells.

Effects of radiation on frozen lactate dehydrogenase.

Results concerning the influence of 6-MeV electron beam irradiation, of 2.45-GHz, 565-W microwaves, and of the combined electron and microwave irradiation, at -21 degrees C and -196 degrees C, on lactate dehydrogenase activity are presented. The microwave-irradiated samples exhibited a non-linear behaviour (successive activation and inactivation of the enzyme molecules), suggesting the major influence of the non-thermal component of microwave radiation. The combined electron and microwave irradiation led to a decrease of activity similar to the one caused by electron beam irradiation, which seemed to prove that microwave influence was insignificant in the dose, power and time ranges used. The radiation target analysis of the enzymatic decrease due to electron irradiation indicated a very large aggregation of the enzyme molecules. Our data suggest that radiation target analysis is not suitable to measure the molecular mass of lactate dehydrogenase, when frozen enzyme suspensions are irradiated. The D2O-protected enzyme, when exposed to electron irradiation, showed an even larger aggregation according to radiation target analysis, while the microwave irradiation of the protected enzyme led to a similar, though lesser, non-linear behaviour of the frozen enzyme molecules.

Effect of azidothymidine on the radiation-induced LDH release in HeLa cells.

Treatment of HeLa cells with various concentrations of 0, 0.01, 0.1, 1, 10, and 100 microM AZT resulted in a concentration dependent elevation in the LDH release at 0, 0.5, 1, 2 and 4 h post-treatment. An elevation of 1.7 to 9.2 fold in LDH content was observed at 1 h post-treatment depending on the drug concentration. Similarly, treatment of HeLa cells with 0.1 microM AZT before irradiation caused an irradiation dose dependent increase in the LDH release in AZT + irradiation groups. This increase in LDH release was approximately two fold greater at 0 h post-irradiation in AZT + irradiation group, when compared with the PBS + irradiation group. This trend of elevation in LDH release continued up to 2 h, except 2 and 3 Gy, where it was 1.7 fold in the former group when compared with the latter. However, a peak level of LDH release was observed at 0 h post-irradiation.

Molecular and kinetic properties of sperm specific LDH after radiation inactivation.

Radiation inactivation of sperm specific lactate dehydrogenase-C4 (LDH-C4) has been studied and compared with the somatic LDH in aqueous solution. D37 of C isozyme was 470 Gy and that of B isozyme was 520 Gy. Semi-log plots of log N/No versus dose suggested that the inactivation of two LDH isozymes in presence of normal saline follows a single hit kinetics. Target molecular weight calculated by radiation analysis was found as 1.52 x 10(5) gm/mole for LDH-C4 and 1.38 x 10(5) gm/mole for LDH-B4. SDS-PAGE of irradiated enzymes showed a band of 35 kDa but did not indicate the presence of any other extra band, when compared with sham-irradiated enzymes. Chemical kinetics of residual activity following irradiation at D37 showed decrease in Vmax with coenzymes and primary substrates. However, decrease in Km was seen with pyruvate as increasing substrate. Nevertheless, K did not change when NAD+ was the leading substrate for LDH-B4 or LDH-C4. A hyperchromicity in intrinsic fluorescence and a blue shift in lambdamax over sham-irradiated LDH-C4 revealed the exposure of buried tryptophan residues to the surface after radiation inactivation. Results suggest that inspite of presence of variant amino acids, the conformations of two isozymes are stabilized by similar forces which behave in a similar way for radiation inactivation in aqueous phase.

[Regularities in the kinetic of photoactivation of lactate dehydrogenase by the action of UV light in different microenvironment]. / Kineticheskie zakonomernosti fotoaktivatsii laktatdegidrogenazy pod deistviem UF-izlucheniia v usloviiakh razlichnogo mikrookruzheniia.

The kinetics of photoinactivation of cardiac (H4) and muscular (M4) isoforms of lactate dehydrogenase irradiated by UV light (240-390 nm) in the free form and in the presence of sodium azide, D-mannitol, and serotonin was studied. It was shown that the decrease in the catalytic activity of both enzymes can be described by the kinetics of the first-order monomolecular reaction. The inactivation rate constant of lactate dehydrogenase M4 is considerably higher than that of lactate dehydrogenase H4, indicating a greater photochemical lability of the isoform M4. It was shown that sodium azide has a different protective action on the proteins studied. The irradiation of the muscular isoform in the presence of serotonin and D-mannitol did not change the character of the "dose-effect" curve and only led to a decrease in the photoinactivation rate constant of the protein.