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)     

Intermediate electron-acceptors in quantitative cytochemistry

Summary The efficacy of Meldola Blue (MB), a new intermediate electronacceptor, has been compared with that of phenazine methosulphate (PMS) in the assay of oxidoreductase activity in cryostat sections; various tetrazolium salts have been used as the final electron-acceptors. Three enzymes: succinate dehydrogenase, glucose 6-phosphate dehydrogenase and lactate dehydrogenase were investigated, the activity in sections being quantitated by scanning and integrating microdensitometry. Phenazine methosulphate was superior to Meldola Blue in transferring reducing equivalents from reduced coenzyme to all the tetrazolium salts examined.     

Delta 5-3 beta-hydroxysteroid dehydrogenase, succinate dehydrogenase and glucose-6-phosphate dehydrogenase in the bovine corpus luteum of estrous cycle: a quantitative histochemical study

Genital organs and blood were obtained from dairy cows at a local abattoir. 3 recently ovulated follicles and 20 corpora lutea of estrous cycle (CLC) were used for the quantitative enzyme histochemical demonstration of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-OHSDH), succinate dehydrogenase (SDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activity, employing a computerized microscope photometer. Progesterone was determined in blood serum by radioimmunoassay. Luteal tissue was grouped into several stages of development according to micromorphological criteria. Activities per volume unit of 3 beta-OHSDH and SDH in large luteal cells (LLC), as well as in small luteal cells (SLC), and luteal tissue (LT), relative amounts of the 3 beta-OHSDH-positive tissue fraction (PLCC), and progesterone concentrations in blood serum exhibited a significant pattern corresponding to the morphological development of the endocrine gland. G-6-PDH showed an increase in activity per volume unit during tissue development lasting until the beginning of regressive changes, and as significant in LLC and LT. Activities per volume unit of 3 beta-OHSDH (p less than or equal to 0.001) and SDH (p less than or equal to 0.01) were higher in LLC than in SLC, indicating superior steroidogenic capacities, while G-6-PDH activity was distinctly higher in the latter (p less than or equal to 0.001). Almost all parameters tested were correlated positively. 3 beta-OHSDH and SDH exhibited a significantly positive correlation in LLC (p less than or equal to 0.01) and LT (p less than or equal to 0.001) during periods of measureable progesterone secretion. In SLC this correlation was nonsignificant (p greater than 0.05). G-6-PDH showed a relative poor correlation to 3 beta-OHSDH (LLC, p less than or equal to 0.05; LT, p less than or equal to 0.01) and SDH (LT, p less than or equal to 0.05). Enzyme activities in LLC as well as in SLC were generally positively correlated (p less than or equal to 0.001). All enzymes tested exhibited a significantly positive correlation with progesterone concentrations in blood serum. This was significant for SDH only during measurable progesterone secretion, and less marked for G-6-PDH.     

Histochemical study on the localization of glucose-6-phosphate dehydrogenase induced by androgen or thyroxine in the convoluted tubules of mouse submandibular gland.

The effects of 5 alpha-dihydrotestosterone (DHT) and thyroxine (T4) on glucose-6-phosphate dehydrogenase (G-6-PDH) activity in mouse submandibular gland were investigated histochemically. A strong positive histochemical reaction for G-6-PDH was observed in the excretory ducts of untreated male and female mice, with a slight reaction in the basal portion of the convoluted tubules (striated ducts) of males. Administration of DHT to female mice increased G-6-PDH activity specifically in the convoluted tubules. T4 increased the enzyme activity in the tubules more than DHT. The induction of G-6-PDH activity by T4 in adrenalectomized mice suggests that T4 has a direct effect on the submandibular gland.     

Red blood cell dysfunction in septic glucose-6-phosphate dehydrogenase-deficient mice

Glucose-6-phosphate dehydrogenase (G-6-PDH) deficiency is the most common known human genetic polymorphism. This study tested the hypothesis that G-6-PDH deficiency worsens sepsis-induced erythrocyte dysfunction. Sepsis (24 h) was induced by cecal ligation and puncture in wild-type (WT) and G-6-PDH-deficient (G-6-PDH activity 15% of WT) mice. Erythrocyte responses were tested in whole blood as well as in subpopulations of circulating erythrocytes. Whereas erythrocyte deformability was similar in unchallenged deficient and WT animals, sepsis decreased erythrocyte deformability that was more pronounced in deficient than WT animals. Sepsis also resulted in anemia and hemolysis in deficient compared with WT animals. Mean corpuscular hemoglobin content and erythrocyte deformability decreased in younger erythrocyte subpopulations from septic deficient compared with WT animals. Sepsis decreased the reduced-to-oxidized glutathione ratio in erythrocytes from both deficient and WT animals; however, plasma glutathione increased more in deficient than in WT animals. Erythrocyte content of band 3 associated with the cytoskeleton was elevated in deficient compared with WT erythrocytes. The antioxidant N-acetyl-l-cysteine in vivo alleviated the sepsis-induced decrease in erythrocyte deformability in deficient animals compared with sham-operated control animals. This study demonstrates that a mild degree of G-6-PDH deficiency (comparable to the human class III G-6-PDH deficiencies) worsens erythrocyte dysfunction during sepsis. Increased erythrocyte rigidity and tendency for hemolysis together with alterations in band 3-spectrin interactions may contribute to the immunomodulatory effects of G-6-PDH deficiency observed after major trauma and infections in humans.     

Histochemical study on the localization of glucose-6-phosphate dehydrogenase induced by androgen or thyroxine in the convoluted tubules of mouse submandibular gland

Summary The effects of 5-dihydrotestosterone (DHT) and thyroxine (T₄) on glucose-6-phosphate dehydrogenase (G-6-PDH) activity in mouse submandibular gland were investigated histochemically. A strong positive histochemical reaction for G-6-PDH was observed in the excretory ducts of untreated male and female mice, with a slight reaction in the basal portion of the convoluted tubules (striated ducts) of males. Administraition of DHT to female mice increased G-6-PDH activity specifically in the convoluted tubules. T₄ increased the enzyme activity in the tubules more than DHT. The induction of G-6-PDH activity by T₄ in adrenalectomized mice suggests that T₄ has a direct effect on the submandibular gland.     

砷对兰州鲇组织中代谢酶活性及RNA和蛋白质含量影响

采用毒性试验方法,研究了安全浓度（1.288 mg/L）条件下亚砷酸钠（NaAsO2）对兰州鲇（Silurus lanzhouensis）脑、鳃、肝脏、肌肉4种组织中6-磷酸葡萄糖脱氢酶（G-6-PDH）和乳酸脱氢酶（LDH）活性,以及RNA和蛋白质含量的影响。结果表明,染毒21d时,As（Ⅲ）可显著降低4种组织中G-6-PDH和LDH活性、RNA和蛋白质含量（P0.05）。撤毒后21d,除脑和肝组织中蛋白质含量未恢复到对照组水平（P0.05）,肝脏中G-6-PDH活性超过了对照组水平（P0.05）外,其余各组织中G-6-PDH和LDH活性、RNA和蛋白质含量均可恢复到对照组水平（P0.05）。以上结果表明,As（Ⅲ）对兰州鲇组织中代谢酶活性具有明显的抑制作用,可致组织细胞RNA损伤和可溶性蛋白质减少,但这种影响是可逆的,撤毒后一定时间内可恢复到正常水平。       

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.     

Glucose-6-phosphate Dehydrogenase Activity under Conditions of Water Limitation: a Possible Model System for Enzyme Reactions in Unimbibed Resting Seeds and its Relevance to Seed Viability

A model system has been used to measure glucose-6-phosphatedehydrogenase (G-6-PDH) activity when the water contents ofthe reactants are comparable to the water contents of dry restingseeds. The activity of G-6-PDH is reduced by 10²–10³ whenthe water content is limited to between 1.5 and 25 per cent.G-6-PDH activity is affected by temperature and by the proteincontent of the model system. The glucose 6-phosphate (7.03 nmolg^–1 embryo) and the NADP⁺ (25.0 nmol g^–1 embryo)contents of barley embryos were measured. Using these measurements,together with the measurements in the model system of G-6-PDHactivity at low water concentrations, an estimate is made ofthe G-6-PDH activity in resting barley embryo. A cor-relationbetween estimated G-6-PDH activity at different water contentsand the periods for which seeds remain viable is indicated.The limitations of the model system are discussed.     

Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots

The pivotal role of glucose-6-phosphate dehydrogenase (G-6-PDH)-mediated nitric oxide (NO) production in the tolerance to oxidative stress induced by 100 mM NaCl in red kidney bean (Phaseolus vulgaris) roots was investigated. The results show that the G-6-PDH activity was enhanced rapidly in the presence of NaCl and reached a maximum at 100 mM. Western blot analysis indicated that the increase of G-6-PDH activity in the red kidney bean roots under 100 mM NaCl was mainly due to the increased content of the G-6-PDH protein. NO production and nitrate reductase (NR) activity were also induced by 100 mM NaCl. The NO production was reduced by NaN(3) (an NR inhibitor), but not affected by N(omega)-nitro-L-arginine (L-NNA) (an NOS inhibitor). Application of 2.5 mM Na(3)PO(4), an inhibitor of G-6-PDH, blocked the increase of G-6-PDH and NR activity, as well as NO production in red kidney bean roots under 100 mM NaCl. The activities of antioxidant enzymes in red kidney bean roots increased in the presence of 100 mM NaCl or sodium nitroprusside (SNP), an NO donor. The increased activities of all antioxidant enzymes tested at 100 mM NaCl were completely inhibited by 2.5 mM Na(3)PO(4). Based on these results, we conclude that G-6-PDH plays a pivotal role in NR-dependent NO production, and in establishing tolerance of red kidney bean roots to salt stress.     

Activités de quelques enzymes associés à la conidiogenèse du Neurospora crassa

Summary Enzyme activities have been measured and compared at several stages of the development of Neurospora crassa i.e. from free conidia (inoculum) to conidiated mycelia grown on sucrose versus acetate (poor versus highly conidiogenous) media.Glucose-6-phosphate dehydrogenase (G-6-PDH) and NADP nucleotidase (NADPase) show inverse activity-time curves, both on sucrose and acetate media. G-6-PDH has its higher activity at the preconidiating stage while NADPase progressively increases to reach its maximal value in the mature conidia.Malate dehydrogenase (MDH) has a higher activity in extracts from acetate compared to sucrose cultures; in both conditions, maximal MDH activity corresponds with the initiation of conidiation. Malate synthetase (MS) has a delayed activity which is much higher in acetate extracts.Succinate dehydrogenase (SDH) is more active in sucrose than acetate extract with a sharp activity peak just preceding conidiation proper.Isozymes of G-6-PDH and MDH, as well as total soluble proteins from extracts of sucrose versus acetate cultures have been compared after their separation on polyacrylamide columns.     

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.     

Adaptogenic effect of the vitamin D3 containing supplement "videchol" on glucose-6-phosphate dehydrogenase activity in erythrone of irradiated rats

Two fast migrating, major, multiple molecular forms (MMF) of glucose-6-phosphate dehydrogenase [EC:1.1.1.49]: G-6-PDH-1 and G-6-PDH-2, and two minor forms: G-6-PDH-3 and G-6-PDH-4 were revealed in the electrophoregrams of both erythrocytes haemolisates as well in the homogenates of bone marrow cellular lines of rats at control conditions. Daily 1 cGy irradiation of rats up to a cumulative dose of 20 cGy led to a drop of G-6-PDH total activity and it caused a redistribution of the MMF of the enzyme in bone marrow cellular populations. However, G-6-PDH activity in erythrocytes exceeded the control means in all the experimental terms. The calculation of the local redistribution coefficient (l(G-6-FDH-i)) showed that these changes are mainly determined by the increase of the activity of the isoform G-6-PDH-3. Vitamin D3 administration to rats generated a correction of G-6-PDH activity in all studied cellular populations. Meanwhile, the MMF profiles were characterized by multidirectional rearrangements in the bone marrow erythroid and granulocyte-monocyte cells and in erythrocytes. The specificity of changes in the distribution of the MMF of G-6-PDH in the three studied cellular populations depends on the particularities of their energetic metabolism at irradiation conditions and on the modifying action of the natural adaptogen 1,25-dihydroxicholecalciferol.     

A gel-sandwich technique for the qualitative and quantitative determination of dehydrogenases in the enzyme histochemistry. I. Development of the new methods on the example of LDH (E.C. 1.1.1.27).

A gel-sandwich technique for the histochemical demonstration of dehydrogenase is introduced with LDH set up as an example. Especially suitable, of the gels examined, for this technique is 1.5% W/V agar-agar low gel strength. In it several reaction ingredients for the histochemical reaction are dissolved. Considering LDH the following gel composition showed good results: 1.5% W/V agar-agar low gel strength, 5 mM TNBT in 150 microliter DMF, 120 mM L-lactate, 3--5 mM NAD+, 10 mM amytal, 22,4--32 X 10(-5) M Meldola Blue, 160 mM soldium phosphate buffer pH 7.6 (total solution of 1 ml). After the solidification of the gel, gel-bars were frozen with CO2-snow. The 40--80 micrometer thick gel slices were gained in the cryostat. Of the three different arrangement possibilities of the gel slices and the tissue-sections a sandwich arrangement (cover-gel slice--tissue section--ground-gel slice) produced the best results. The enzyme reaction is started by thawing of the gel slices (together with the tissue sections) and by putting them between the hotplate and the evaporator-head-piece, especially developed for this technique. The gel slices also remain in combination with the tissue sections after the reaction. The influence of the gel in combination with the electron carrier Meldola Blue on the spontaneous reduction rates of ditetrazolium salts in day light, were examined as well as the diffusion rates of TNBT and NADH out of gel slices and the influence of DMF and DMSO on the LDH activity. This technique prevents both, the loss of enzymes and the loss of reduction equivalents. There are given presuppositions for qualitative and quantitative histochemical investigations as well. The advantages of the new gel technique are discussed.     

The biochemical and morphological changes in the myocardium of patients with an acute surgical vascular pathology (based on data from early autopsies)]

Biochemical and morphological disorders of glucose 6-phosphate dehydrogenase (G-6-PDH) were studied in the myocardium of 9 patients who had died from different vascular surgical diseases. The inhibition of G-6-PDH activity most prominent in lung artery thromboembolism was shown biochemically. Histological and histoenzymological findings demonstrate low G-6-PDH activity in different myocardial regions and solitary defects of cardiomyocytes. The data obtained evidence significant sensibility of the myocardium in surgical patients to different influences.     

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.     

Glucose-6-phosphate dehydrogenase in tobacco callus strains differing in their growth and their requirement for auxin and cytokinin

The cytokinin-autonomous strain (A_s) of tobacco callus differs from the original cytokinin-dependent strain (D) and from the cytokinin- and auxin-autonomous strain (A₄) by a significantly lower activity of glucose-6-phosphate dehydrogenase (G-6-PDH). Changes in the total G-6-PDH activity were associated with differences in the number of G-6-PDH isozymes. The A_s strain contained only one isozyme, four isozymes were found in D and A₄ strains.     

A gel-sandwich technique for the qualitative and quantitative determination of dehydrogenases in the enzyme histochemistry

Summary A gel-sandwich technique for the histochemical demonstration of dehydrogenases is introduced with LDH set up as an example. Especially suitable, of the gels examined, for this technique is 1.5% W/V agar-agar low gel strength. In it several reaction ingredients for the histochemical reaction are dissolved. Considering LDH the following gel composition showed good results: 1.5% W/V agar-agar low gel strength, 5 mM TNBT in 150 l DMF, 120 mM L-lactate, 3–5 mM NAD⁺, 10 mM amytal, 22,4–32×10^–5 M Meldola Blue, 160 mM soldium phosphate buffer pH 7.6 (total solution of 1 ml). After the solidification of the gel, gel-bars were frozen with CO₂-snow. The 40–80 m thick gel slices were gained in the cryostat. Of the three different arrangement possibilities of the gel slices and the tissue-sections a sandwich arrangement (cover-gel slice — tissue section — ground-gel slice) produced the best results. The enzyme reaction is started by thawing of the gel slices (together with the tissue sections) and by putting them between the hotplate and the evaporator-head-piece, especially developed for this technique. The gel slices also remain in combination with the tissue sections after the reaction.The influence of the gel in combination with the electron carrier Meldola Blue on the spontaneous reduction rates of ditetrazolium salts in day light, were examined as well as the diffusion rates of TNBT and NADH out of gel slices and the influence of DMF and DMSO on the LDH activity.This technique prevents both, the loss of enzymes and the loss of reduction equivalents. There are given presuppositions for qualitative and quantitative histochemical investigations as well. The advantages of the new gel technique are discussed.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections.

The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study show that the enzyme histochemical technique--except for some limitations--is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.