The Effect of Red and Far-red Light on Subsequent Enzyme Activity in Arena Coleoptiles

The effect of red light (660 nm), far-red light (730 nm) and dark treatment on the subsequent enzyme activity in homogenates of Avena coleoptiles was investigated. The activities of succinic dehydrogenase (SDH), lactic dehydro-genase (LDH) and glucose-6-P dehydrogenase (GDH) were investigated. The activity of SDH was greatest in material receiving continuous darkness. LDH and GDH activity was stimulated by both light treatments compared with the dark values. Little or no difference in enzyme activity was found using either a single 15 min flash of light or continuous light for 24 h. Admixtures of extracts from dark treated and light treated material in a 1:1 ratio gave unexpected levels of enzyme activity. In all cases such admixtures gave much less than the anticipated enzyme activity.     

Histochemical studies of enzymes of the energy metabolism in postimplantation rat embryos

Using histochemical procedures for the detection of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and cytochrome c oxidase (cytox), we investigated the levels of these enzymes of the energy metabolism in postimplantation rat embryos (9.5-12.5 days of gestation). On day 10.5 of gestation, the neural tube, somites, myocardium, and mesenchyme displayed moderate levels of LDH activity; this activity gradually increased in strength, so that, on day 12.5 of gestation, intense LDH activity was uniformly distributed in these intraembryonic tissues. In contrast to LDH, distinct regional differences in the distribution of SDH and cytox were detected. On day 10.5 of gestation, the myocardium exhibited weak to moderate SDH and cytox activity, and on day 11.5, the myocardial activity of these enzymes had become moderate to intense. However, in all other embryonic tissues, e.g., the neural tube and somites, only weak SDH and cytox activity was present. On day 12.5 of gestation, the myocardium displayed very intense SDH and cytox activity, whereas the mantle layer of the neural tube, the spinal ganglia, and the myotomes exhibited only moderate levels of SDH and cytox activity. In the matrix of the neural tube and mesenchyme, these enzyme activities remained at low levels. At electron microscopy, cytox activity was detectable in the spaces between the inner and outer membranes as well as in the intracristal spaces of mitochondria. In general, cytox activity increased in parallel with the differentiation of mitochondria (i.e., increased mitochondrial numbers and size, and the development of mitochondrial cristae), but when the distribution of the cytox activity was considered in detail, it was found to differ among mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical studies of enzymes of the energy metabolism in postimplantation rat embryos

Summary Using histochemical procedures for the detection of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and cytochrome c oxidase (cytox), we investigated the levels of these enzymes of the energy metabolism in postimplantation rat embryos (9.5–12.5 days of gestation). On day 10.5 of gestation, the neural tube, somites, myocardium, and mesenchyme displayed moderate levels of LDH activity; this activity gradually increased in strength, so that, on day 12.5 of gestation, intense LDH activity was uniformly distributed in these intraembryonic tissues. In contrast to LDH, distinet regional differences in the distribution of SDH and cytox were detected. On day 10.5 of gestation, the myocardium exhibited weak to moderate SDH and cytox activity, and on day 11.5, the myocardial activity of these enzymes had become moderate to intense. However, in all other embryonic tissues, e.g., the neural tube and somites, only weak SDH and cytox activity was present. On day 12.5 of gestation, the myocardium displayed very intense SDH and cytox activity, whereas the mantle layer of the neural tube, the spinal ganglia, and the myotomes exhibited only moderate levels of SDH and cytox activity. In the matrix of the neural tube and mesenchyme, these enzyme activities remained at low levels. At electron microscopy, cytox activity was detectable in the spaces between the inner and outer membranes as well as in the intracristal spaces of mitochondria. In general, cytox activity increased in paralled with the differentiation of mitochondria (i.e., increased mitochondrial numbers and size, and the development of mitochondrial cristae), but when the distribution of the cytox activity was considered in detail, it was found to differ among mitochondria. The relationship between, on the one hand, changes in the enzymatic patterns with a bearing on the energy-yielding metabolism and, on the other hand, cellular differentiation during major organogenesis in rat embryos is discussed.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Electron-microscopic-histochemical demonstration of succinic-dehydrogenase activity in root cells of yellow lupine

Summary Succinic dehydrogenase (SDH) activity was demonstrated in unfixed root segments from Lupinus luteus at the ultrastructural level by the use of ferricyanide as electron acceptor. The specificity of the reaction was proven by malonate inhibition. The reaction product was found to be localized in the mitochondria and to a lesser extent on the membranes of plastids. Different mitochondria of the same cell often showed different intensity of the staining reaction. Different cells of the same tissue exhibited varying degrees of enzyme activity. An increase was found in the number of cells exhibiting the SDH reaction, as well as in the intensity of the reaction itself, from the meristematic zone of the root to the more differentiated regions.     

Electron microscopic localization of lactate dehydrogenase in osteoclasts of chick embryo tibia

Synopsis Lactate dehydrogenase (LDH) was localized in osteoclasts of fixed and unfixed 19-day chick embryo tibias using a copper ferrocyanide capture reaction and osmiophilic polymer generation. This study revealed that: (1) LDH activity in fixed, briefly rinsed osteoclasts was associated principally with limiting membranes of cytoplasmic vacuoles and vesicles and with the plasma membrane; (2) LDH activity in unfixed osteoclasts was associated only with mitochondria; and (3) some mitochondria were stained in fixed tissue given a long rinse. These results indicate that: cytoplasmic LDH diffused out of unfixed tissue; mitochondrial LDH was inactivated by formaldehyde in fixed tissue; and formaldehyde-inhibited mitochondrial LDH can be reactivated by a long rinse. Although the vesicles that stained for LDH activity were found in all parts of the cell, they were concentrated near the ruffled border, and there is evidence that they contained material from the bone surface. These results suggest that the LDH associated with cytoplasmic vesicles of the osteoclast may be important in processing of material resorbed from the bone surface and that osteoclastic mitochondria may utilize lactate from the bone fluid for energy production.     

Sublethal effects of textile dye stuff effluent on selected oxidative enzymes and tissue respiration of Cyprinus carpio (Linn.).

Toxic effects of sublethal concentration of dye stuff effluent on succinic dehydrogenase (SDH) and lactic dehydrogenase (LDH) activities and tissue respiration were studied in C. carpio. While the sublethal exposure significantly reduced SDH activity and tissue respiration, LDH activity increased in gill, brain, liver, muscle and kidney. The maximum inhibition of SDH activity (74%) was recorded in gill and the minimum (38%) in liver. The percentage reduction of oxygen consumption in the tested tissues was in the order of gill greater than brain greater than liver greater than muscle greater than kidney. The muscle showed the highest level (96%) of increase in LDH activity whereas the kidney cells showed the minimum increase. Exposure to sublethal concentration suppressed the aerobic respiration and triggered the anaerobic respiration.     

In vitro effects of polyvinylpyrrolidone and sucrose on the acetylcholinesterase,succinate dehydrogenase and lactate dehydrogenase activities in the brain

Summary The supernatant prepared from the brain tissue homogenate incubated in vitro in the presence of PVP or sucrose exhibits a decrease of AChE, SDH as well as of LDH activity. A 0.75% PVP solution inhibits AChE activity by 30%, LDH activity is inhibited by 35% and SDH activity by 40%. A two hours lasting effect of a 7.5% PVP solution at 3° C on enzymatic preparations induces in AChE 20% inhibition of its activity, in LDH an inhibition of 44% and in SDH the inhibition of its activity amounts to 74%. 1 M Sucrose inhibits AChE activity by 34%, LDH activity by 41% and SDH activity is inhibited by 31%. After two hours lasting effect of 1.4 M sucrose at 3° C on the supernatant the AChE activity is inhibited by 22% and that of LDH by 30%. The SDH activity was after a two hours lasting effect of 1 M sucrose at 3° C inhibited by 34%. The inhibition of activity of the above mentioned enzymes localized in brain cortex preparations was compared with the inhibition of activity of the isolated serum cholinesterase. 0.25 M Sucrose inhibited the activity of this enzyme by 25% and 0.75% PVP by 45%. A two hours lasting effect of 7.5% PVP or 1 M sucrose at 3° C on the cholinesterase induced a 40% and 22% inhibition respectively. After double washing of the brain cortical minced tissue, prepared in a 7.5% PVP containing solution, AChE activity was constant. By triple washing of the brain cortical crude mitochondrial fraction, exposed for two hours at 3° C to the effect of 1 M sucrose, SDH activity was also constant.Abbreviations AChE acetylcholinesterase (EC 3.1.1.7.) - INT 2(p-iodophenyl)3-p-nitrophenyl-5-phenyl tetrazolium chloride - LDH lactate dehydrogenase (EC 1.1.1.27.) - PMS phenazine methosulfate - PVP polyvinylpyrrolidone - SDH succinate dehydrogenase (EC 1.3.99.1.)     

Methodological aspects of the histochemical localization and activity of some cerebellar dehydrogenases

Summary In a detailed study focused on the methodological problems in dehydrogenase histochemistry [e.g., fixation, diffusion of enzymes and of reduced inermediates, conversion of NADPH and NADP to NADH and NAD, respectively, penetration of tetrazolium salt and formazan substantivity, nothing dehydrogenase reaction, use of exogenous CoQ₁₀ and of flavoprotein substitute (PMS)], the distribution and activity of succinate dehydrogenase, NAD(P)H-tetrazolium reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase (H and M types), and of l-glutamate dehydrogenase (E.C. 1.4.1.2 and E.C. 1.4.1.3) have been investigated in the rat cerebellum.It was evident from the study that reliable results could only be obtained if all the aforementioned factors had been considered. The image of actual concentration of SDH in the neuropil of the molecular layer could only be recorded by adding CoQ₁₀, while other structures exhibited greater balance between SDH and endogenous mitochondrial CoQ. Contrary to previous studies, a reversed localization of the activity of G-6-PDH and LDH was noticed. The elements of molecular and Purkinje layers were rich in G-6-PDH, while the granular layer was nearly depleted. The actual level of LDH could only be recorded if NADH-tetrazolium reductase was bypassed with PMS. The H and M types of LDH coexisted in the three cortical layers, the H type being prevalent and the M type attaining its highest level in synaptic glomeruli followed by the structures of the molecular layer and the Purkinje cells. High activity of GDH was noticed in Bergmann glia followed by synaptic glomeruli, while most other structures showed weak to moderate activity. The two GDH types coexisted in all structures showing activity, except for Bergmann cells, which only showed presence of the E.C. 1.4.1.3 type.Furthermore, Bergmann glia was exceptional by showing no activity of SDH and LDH, but strong activity of G-6-PDH and NADPH-tetrazolium reductase. The granular cells were exceptional by showing weak or no activity of all enzymes in question.     

The molecular mechanism of menadione resistance in the <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 cyanobacterium

The effect of mutations in the genes encoding dehydrogenases and oxidases on the resistance of the Synechocystis sp. PCC 6803 cyanobacterium to menadione, an oxidative stress inducer, was studied. An enhanced sensitivity to menadione was observed in the mutants carrying inserts in the drgA gene encoding the NAD(P)H:quinone oxidoreductase (NQR) and in the ndhB gene encoding the subunit of NDH-1 complex. The menadione resistance in the mutants lacking oxidases (Ox), succinate dehydrogenase (SDH), and NDH-2 dehydrogenase do not differ from those in wild-type cells. An additional mutation in the drgA gene increased the sensitivity to menadione in the NDH-2 and Ox mutants. The double mutant that lacks both SDH and NQR was not viable. The expression of the drgA gene decreased during cell incubation in the dark but increased in the presence of glucose both in the dark and in light. Under photoautotrophic growth conditions, the dehydrogenase activity of the cells mainly depends on the NQR and NDH-1 functions. The re-reduction rate of the photosystem I reaction center (P700⁺) increased in wild-type and NDH-1 mutants after its oxidation with white light in the presence of DCMU after addition of menadione, and it decreased in the NQR mutant. The reduction of P700⁺ was accelerated in the presence of menadiol in all the strains studied. These results suggest that NQR provides defense of cyanobacterium cells from the toxic effect of menadione via its two-electron reduction to menadiol. An increased sensitivity of the NDH-1 mutant to menadione may result from the inhibition of respiration and the cyclic electron transport in photosystem I.     

Succinate dehydrogenase activity of isolated myocardial mitochondria during chronic insufficiency]

Succinic dehydrogenase (SDH) activity of the isolated mitochondria myocardial cells in chronic cardiac insufficiency was studied experimentally. The highest SDH values were found in the mitochondria of condensed type. The activity of the enzyme was low in the ordinary (orthodox) mitochondria. The medium SDH activity was registered in the intermediary type of mitochondria. The ordinary and the intermediary types of mitochondria prevailed in the fraction under study reflecting a fall of the SDH activity in the myocardial tissue. Biochemical study of the mitochondrial fraction revealed a slight dissociation between the tissue respiration and the oxidative phosphorilation. The low SDH activity values seen to indicatate the state of overstrain of the energy-producing structures.     

The ultrastructural localization of membrane ATPase in rat thin limbs of the loop of Henle

Summary Intraacinar distribution of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), NADP-dependent isocitrate dehydrogenase (IDH), glutamate dehydrogenase (GluDH), lactate dehydrogenase (LDH) and NADH-tetrazolium dehydrogenase (TR) was studied in rat liver cryostat sections by multipositional microphotometric activity determinations. By statistical evaluation, activity of individual enzymes could be related to the acinar topography. Activity was evaluated with regard to distance of measuring position either from afferent (portal) or efferent (hepatic) vessels. Two independent distribution curves were obtained for each enzyme. Acinar distribution of all the enzymes studied followed sigmoid courses with maximal activity of SDH, MDH and LDH in zone 1 (periportal) and GluDH, IDH, TR in zone 3 (pericentral). For all enzymes, maximum activity gradients were confined to zone 2 of the acinus. Data were also evaluated as ratios of activities in zone 1 and zone 3. The following ratios zone 1/zone 3 were obtained: SDH=1.9, MDH=1.7, IDH=0.5, GluDH=0.5, LDH=1.3 and TR=0.6.     

Enzyme histochemistry of the mesenteric and dorsal root ganglion cells of cat and squirrel monkey

Summary A detailed histochemical study has been made on the mesenteric ganglia of the cat, and dorsal root ganglia of the squirrel monkey by the use of appropriate histochemical techniques accompanied by appropriate controls for phosphatases, esterases, and oxidative enzymes. The different neurons of a particular ganglion show varied amounts of enzyme activity at a particular time depending upon the functional state of the neurons. SDH, CYO and LDH reaction is prominent in the cytoplasm of the neurons, gliocytes and satellite cells, whereas the MAO preparations generally show a weak reaction. The AK is prominent in the neuropil, cell membranes and peripheral part of cytoplasm, whereas ATPase activity has been observed in blood vessels as well. In AC preparations the area of lipofuscin concentration shows more intense reaction than the rest of the cytoplasm. The activity of AChE and BChE varies from mild, to moderate to strong. The TPPase preparations show morphologically different types and amounts of TPPase positive Golgi material even in the adjoining cells. The relationship between the TPPase Golgi material and various oxidative and dephosphorylating enzymes has been briefly discussed.Abbreviations used AC Acid phosphatase - AChE Acetyl-cholinesterase (specific) - AK Alkaline phosphatase - AMPase Adenosine monophosphatase (5-nucleotidase) - ATPase Adenosine triphosphatase - BChE Butyryl-cholinesterase (nonspecific) - CYO Cytochrome oxidase - DPN-D DPN-diaphorase - G6P Glucose-6-phosphatase - LDH Lactic dehydrogenase - MAO Monoamine oxidase - MDH Malic dehydrogenase - NAD-D NAD-diaphorase - SDH Succinic dehydrogenase - SE Simple esterase - TPPase thiamine pyrophosphatase T. R. Shanthaveerappa in previous publications.     

Peculiarities of metabolism of UV-irradiated lymphocytes

The influence of UV-light (240-390 nm) at a dose of 151 and 755 J/m2 on the functional properties of lymphocyte metabolism key enzymes from donors' human blood: lactate dehydrogenase (LDH), cytochrome c oxidase, succinate dehydrogenase (SDH), Ca2(+)-ATPase of plasma membranes has been investigated. It has been revealed that photoinactivation of enzymes immediately after UV-irradiation which leads to the decrease of the ATP content in lymphocytes is replaced by the increased activity of the enzymes under investigation during daily incubation of lymphocytes. As a result, the level of ATP in photo-modified lymphocytes does not differ from that in native cells before incubation. This indicates the normalization of biochemical processes in lymphocytes influenced by UV-light applied in autotransfusion of UV-irradiated blood.     

Effect of thyroidectomy on fast and slow muscle fibres of rat gastrocnemius muscle

A combined histochemical, biochemical and electrophoretic study with respect to the enzymes succnic dehydrogenase(SDH), myofibrillar adenosine triphosphatase (m-ATPase), lactate dehydrogenase (LDH) isozymes and myosin light chains was carried out to investigate the response of rat gastrocnemius muscle (medial head). Twelve weeks after thyroidectomy, the results indicated a shift from fast to slow type pattern of LDH isozymes, fibre type transformation from Type II to Type I and a decrease in SDH and m-ATPase activity. The results suggest, possible thyroidal involvement in determining the phenotypic properties of skeletal muscle.     

Regulating effect of mitochondrial lactate dehydrogenase on oxidation of cytoplasmic NADH via an "external" pathway in skeletal muscle mitochondria.

1. The specific activity of lactate dehydrogenase of skeletal muscle mitochondria was found to be 2.5 times lower than specific activity of total NADH-cytochrome c reductase. 2. The specific activity of mitochondrial LDH in skeletal muscle mitochondria was almost equal to the activity of rotenone-insensitive NADH-cytochrome c reductase. 3. Mitochondrial LDH acting as an oxidase of lactate to pyruvate may feed an "external" pathway, but the activity of the mitochondrial enzyme is a limiting factor in oxidation of lactate-derived NADH. 4. Mitochondrial LDH acting as a reductase of pyruvate to lactate successfully competes with an "external" pathway for cytoplasmic NADH. 5. Exogenous NADH oxidation via an "external" pathway was inhibited by pyruvic acid. This inhibition was overcome by addition of oxamic acid or hydrazine.     

Effect of tuftsin on enzyme activity in the energy metabolism of lymphocytes

The cytochemical technique was used to measure the activity of succinate dehydrogenase (SDH), lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) of peripheral blood lymphocytes of mice and rats given intraperitoneal injections of an endogenous immunostimulant tuftcin (Tre-Lys-Pro-Arg) in a dose of 0.3 mg/kg. A significant decrease of SDH activity was observed both in mice and rats 4 and 6 hours following injection, respectively. In mice, that activity returned to normal in 12, while in rats in 24 hours. An opposite action was produced by tuftcin on G-6-PDH, causing the maximum elevation of the enzyme activity in rat lymphocytes 6 hours after peptide administration. The decrease to the initial level was observed in 24 hours. Tuftcin did not affect the activity of LDH. The data obtained indicate that the immunological effect of tuftcin is coupled with the changes in the activity of Krebs cycle enzymes (SDH) and pentose phosphate cycle enzymes (G-6-PDH).     

The role of calcium cations in the mechanism of phytochrome-dependent regulation of the <Emphasis Type="Italic">sdh1-2</Emphasis> gene expression and succinate dehydrogenase activity in maize leaves

The regulation of succinate dehydrogenase (SDH) activity and its gene expression by the phytochrome system has been demonstrated: red light (660 nm) reduces the SDH activity and the level of expression of the sdh1-2 gene. At the same time, the content of calcium cations in the nuclear fraction increases; it seems to be one of the mechanisms of phytochrome signal transduction in plant cells. Far-red light (730 nm) had opposite effects, i.e., increased SDH activity and the level of expression of the sdh1-2 gene. The data suggest that the SDH activity can be regulated at the level of expression in the green leaves of Zea mays by the phytochrome system with the involvement of calcium ions as a signal transduction messenger.     

HISTOCHEMISTRY OF LACTIC DEHYDROGENASE IN HEART AND PECTORALIS MUSCLES OF RAT

Left-ventricular heart muscle and pectoralis major muscle of the rat were studied to determine the intracellular localization of lactic dehydrogenase (LDH) isoenzymes. Fixation of tissue for 2 hr in 2% buffered formaldehyde provided the best preservation of the ultrastructure and enzyme activity. Total LDH activity was found diffusely in the ground substance of the sarcoplasm and in the mitochondria of the heart muscle. In skeletal muscle a strong reaction was noted in the sarcoplasmic reticulum, and moderate activity was seen in the ground substance of the sarcoplasm and in the mitochondria. Differentiation of the isoenzymes of LDH was accomplished by addition of 4 M urea or application of heat. Heart-type isoenzymes were mainly localized in the mitochondria and sarcoplasm, whereas muscle-type isoenzymes were localized mainly in the sarcoplasmic reticulum of the skeletal muscle. It is speculated that the sarcoplasmic reticulum of the skeletal muscle is the site of anaerobic glycolysis and that the sarcoplasm and mitochondria are involved primarily in aerobic metabolism of pyruvate.     

Light regulation of succinate dehydrogenase expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves

A potential mechanism of light regulation of the succinate dehydrogenase (SDH) expression in Arabidopsis thaliana leaves was studied. As was shown by dot-hybridization and polymerase chain reaction in real time (RT-PCR), the SDH mRNA level in wild-type Arabidopsis thaliana plants changed depending on light conditions. The level of SDH mRNA in darkness was higher than in the light. The analysis of Arabidopsis thaliana plants carrying the mutant genes of phytochromes A and B showed that phytochrome A was involved in the regulation of the SDH enzyme activity. The active form of phytochrome A suppressed the SDHI-2 gene expression, and that resulted in decreasing activity of SDH.     

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.