Microphotometric determination of enzymes in brain sections. V. Glycerophosphate dehydrogenases

An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM L-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.5 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent Km value at 25 degrees C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Microphotometric determination of enzymes in brain sections. III. Glutamate dehydrogenase

An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM L-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities.     

Microphotometric determination of enzymes in brain sections

Summary An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM l-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.05 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent K _m value at 25°C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Microphotometric determination of enzymes in brain sections. II. GABA transaminase

The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM alpha-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 microns and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT.     

Quantitative dehydrogenase histochemistry with exogenous electron carriers (PMS,MPMS, MB)

Summary The relative efficiencies of phenazine methosulfate (PMS), 1-methoxy-phenazine methosulfate (MPMS) and Meldola Blue (MB) as electron carriers were determined biochemically (non-enzymic NADH-tetrazolium salt-test) and by quantitative histochemistry (heart and kidney slices; succinate dehydrogenase, SDH; lactate dehydrogenase, LDH). MPMS developed the highest electron transfer velocity in biochemical assays. The reaction was independent of the pH value between 7.0–8.5. PMS and MB always showed a lower transfer ability in biochemical tests which was higher with iodonitrotetrazolium chloride (INT) than with nitro blue tetrazolium chloride (NBT). A distinct pH dependence was demonstrable with MB in this respect, preferentially using INT as tetrazolium salt.Quantitative histochemical results with electron carriers are often at variance with biochemical ones. MPMS leads to somewhat higher demonstrable activities only in the determination of the NAD-dependent LDH, whereas MB results in somewhat higher LDH activity than PMS (reaction medium with agarose). MB and PMS yielded almost equally high activities in the demonstration of the flavoprotein-dependent SDH using a reaction medium with agarose. With an aqueous reaction medium, PMS resulted in higer SDH activities than MB. MPMS always had the lowest efficiency in electron transfer ability using an aqueous or agarose containing reaction medium (SDH). With PVA in the reaction medium (SDH determination) PMS was clearly superior to MPMS. MB showed only a small transfer activity under these conditions because PVA seems to bind MB almost completely. It is concluded that in histochemistry an appropriate electron carrier and electron carrier concentration must be determined for different incubation conditions, tissues, tissue preparations and dehydrogenases studied. General statements about the efficiency or inefficiency of an electron carrier as a result of only one incubation condition does not seem to be justified.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Microphotometric determination of enzymes in brain sections

Summary An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM l-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN₃), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. — The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN₃ as a blocker of the respiratory chain was indispensible, because without NaN₃ most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres

Summary A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., nothing dehydrogenase activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and nothing dehydrogenase) when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.     

Meldola Blue: A new electron carrier for the histochemical demonstration of dehydrogenases (SDH,LDH, G-6-PDH)

Summary A new electron carrier, Meldola Blue (8-dimethylamino-2,3-benzophenoxazine; Boehringer Mannheim GmbH, Deutsche Patentschrift P 1959410) was tested for its usefulness in the histochemical demonstration of dehydrogenase activity in adrenal cortex, liver, heart muscle of guinea pig and human oviduct and compared with PMS.For demonstrating SDH activity Meldola Blue (MB) is as efficient as PMS. A decisive advantage of MB as compared with PMS is its low sensitivity to light exposure, facilitating direct visualisation of histochemical reaction processes.Generally, a high diffusion rate of reduced electron carriers (PMS and MB) from the section into the incubation medium (PVA) leads to a loss of reduction equivalents, particularly in the demonstration of NAD- or NADP-dependent dehydrogenases (LDH, G-6-PDH) with lower TNBT concentrations. However, no inhibition of SDH-, LDH- and G-6-PDH activities was observed with incubation media containing the tested concentrations of PMS and MB.     

Microphotometric determination of enzymes in brain sections. I. Hexokinase

A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM D-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN3, 10 mM MgCl2, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 microns. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.     

Improvement in Soluble Dehydrogenase Histochemistry by Nitroblue Tetrazolium Preuptake in Sections: A Qualitative and Quantitative Study

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/ acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.     

Improvement in soluble dehydrogenase histochemistry by nitroblue tetrazolium preuptake in sections: a qualitative and quantitative study

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.     

Microphotometric determination of enzymes in brain sections. II. GABA transaminase

Summary The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM α-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 μm and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique.

The dissociation rate constants for the two products of the reaction catalyzed by protein kinase A, ADP and phosphopeptide, were measured using a catalytic trapping technique to determine the role of product release in enzyme turnover. The enzyme was preequilibrated with ADP, and the reaction was initiated with a peptide substrate, LRRASLG, and ATP in a rapid quench flow instrument. At high, free magnesium concentrations (>2 mM), the large 'burst' in phosphopeptide production disappears, and, at low concentrations of free magnesium (0.5-1 mM), the kinetic transients become sigmoidal prior to the linear turnover phase. Increasing the concentrations of ATP or ADP did not influence the shape of the kinetic transients in the first 20 ms. ADP preequilibration protects the enzyme from inhibition by the covalent inactivator p-fluorosulfonylbenzoyl 5'-adenosine at 0.5 mM free magnesium, indicating that a competent E. ADP complex forms at low metal concentrations and the sigmoidal behavior in the catalytic trapping experiment is not due to free enzyme at high ATP concentrations. Simulations of the data indicate that ADP release is rate-limiting for turnover at high magnesium concentrations, but, at lower physiological levels of 0.5 and 1 mM, the off rate of ADP is 3- and 2-fold higher than k(cat), respectively. In contrast, the initial portions of the kinetic transients at 0.5 mM free magnesium were unaffected by phosphopeptide preequilibration, indicating that the release rate of this product is significantly larger than turnover. The transient kinetic data, coupled with a previous report [Shaffer and Adams (1999) Biochemistry 38, 5572-5581], support a phosphorylation mechanism under physiological magnesium concentrations that incorporates two partially rate-determining conformational changes, one prior to and one after the phosphoryl transfer step. We propose that the initial step activates the enzyme through key positioning of one or more active-site residues and the second step relaxes this conformation, a prerequisite for a subsequent catalytic cycle.     

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3-6 months old) and aged male rats (24-27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05 M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1, the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio ento-rhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.     

Magnesium deprivation inhibits the expression of differentiation-related phenotypes in human promyelocytic leukemia HL-60 cells

The role of magnesium ions in the differentiation of human promyelocytic leukemia HL-60 cells was investigated. When HL-60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1 alpha, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM beta-all-trans retinoic acid (RA), 20 nM 12-o-tetradecanoyl phorbol-13-acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM] in magnesium-deficient medium, the expression of differentiation-related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2-day treatment with 100 nM 1,25(OH)2D3 in magnesium-deficient medium, the expression of differentiation-related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL-60 differentiation-related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2-day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium-deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and alpha-amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium-requiring step in HL-60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c-myc and c-fms mRNA levels in HL-60 cells treated with 100 nM 1,25(OH)2D3 in magnesium-deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation-related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium-requiring step in HL-60 cell differentiation is different from that inhibited by cyclohexamide.     

Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions.

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than Km for MgATP2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP leads to ATP + creatine) equal to 3.5 mumol times min-1 times mg-1 is essentially higher than maximal rate of the forward reaction (0.8 mumol times min-1 times mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reactions mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be expained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP AND ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.     

NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.     

Reduction of tetrazolium salts catalyzed by soluble diaphorase in rat liver and embryos of Vicia faba

Summary The presence of two diaphorases has been shown in rat liver and embryos of Vicia fdba. One of these, the NAD(P)H tetrazolium reductase, was firmly bound in the section and was not lost into the incubation medium under conditions of histochemical assay The second diaphorase (soluble diaphorase) was lost from the section into the incubation medium during the first five minutes of incubation. This soluble diaphorase from both rat liver and embryos of V. faba is capable of transferring electrons from NAD(P)H to MTT, INT, NBT and TNBT, but not to tellurite, TTC, BT and NT. The behaviour of the soluble diaphorase in histochemical reactions involving tetrazolium salts as electron acceptors is discussed.     

Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative histochemistry of creatine kinase in rat myocardium and skeletal muscle

Summary Creatine kinase (ec 2.7.3.2) activity was demonstrated in rat myocardium using a polyvinyl alcohol-containing incubation medium and auxiliary enzymes. The activity was quantified by microdensitometry using both endpoint measurements and kinetic measurements. Control reactions were performed in the absence of creatine phosphate and ADP.The linear regression lines of the absorbances of reduced Nitro BT at the isobestic wavelength (585 nm) on incubation time were highly significant for both endpoint and kinetic measurements. The activity obtained from endpoint measurements was about 40% lower. This was caused by loss of the formazan reaction product from the tissue sections when the incubation medium was removed at the end of the reaction. The relationship between creatine kinase activity (test minus control reaction) and section thickness was not linear for either myocardium or skeletal muscle; control reactions, however, showed linear relationships with section thickness for both tissues. Limited penetration of auxiliary enzymes into the sections may be responsible for this disporportionality. Therefore, care should be taken in the interpretation of quantitative data obtained with different tissues.In conclusion, multi-step enzyme reactions can be used for quantitative histochemical purposes provided it is taken into account that the reactivity is not proportional to section thickness.