Dehydrogenase enzyme histochemistry on freezedried or fixed resin-embedded tissue

Summary A method has been developed for the histochemical demonstration of a variety of dehydrogenases in freeze-dried or fixed resin-embedded tissue. Seven dehydrogenases were studied. Lactate dehydrogenase, NADH dehydrogenase and NADPH tetrazolium reductase were all demonstrable in sections of paraformaldehyde-fixed resin-embedded tissue. Freeze-dried specimens were embedded, without fixation, in glycol methacrylate resin or LR Gold resin at either 4°C or –20°C. All the dehydrogenases except succinate dehydrogenase retained their activity in freeze-dried, resin-embedded tissue. Enzyme activity was maximally preserved by embedding the freeze-dried tissue specimens in glycol methacrylate resin at –20°C. The dehydrogenases were accurately localized without any diffusion when the tissue sections were incubated in aqueous media. Addition of a colloid stabilizer to the incubating medium was not required. Freeze-drying combined with low-temperature resin embedding permits accurate enzyme localization without diffusion, maintenance of enzyme activity and excellent tissue morphology.     

Enzyme histochemistry on freeze-dried, resin-embedded tissue

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.     

A novel tight cylindrical mold for epoxy resin embedding allows enhanced microscopic analysis of microcores extracted from woody plants

A novel mold was devised to embed microcores extracted from stems of trees in epoxy resin, which has been widely used for optical and electron microscopic analysis of xylem formation. The embedding mold of a tight cylindrical shaped tube was designed to avoid displacement of microcores from the right position during the process of resin embedding. Microcores of a ring-porous hardwood species, Quercus crispula, with higher wood density and much larger differentiating vessel elements laid down on the boundary between the current xylem and the previous one, which generally cause difficulty in thin sectioning and breaks in sections, respectively, were embedded in the cylindrical molds full of epoxy resin. Locations of the three principal planes of wood anatomy could be determined in cylindrical resin-embedded microcores as follows: the transverse plane could be found on their side of cylinder, the radial one was vertical to the transverse, and the tangential ones were their circular ends of cylinder. The present embedding mold, therefore, can provide all three principal sections for microscopic wood anatomy from the side or ends of the same cylindrical microcore in principle. To confirm the usefulness of the resin-embedded microcores, we examined the differentiation of vessel elements during the period of earlywood formation on their transverse sections under microscopes, consequently could observe cell division in the cambial zone and sequential stages of vessel element differentiation, including cell expansion and deposition of the secondary cell wall. The present embedding mold for epoxy resin is simple but highly useful and innovative for a wide range of applications of microcores in microscopy for studies on tree-ring formation.     

Ultrastructure of the contractile apparatus of rat skeletal muscle embedded in an aqueous medium

The method of tissue embedding in melamine resin was applied to rat skeletal muscle. This method does not require tissue dehydration with organic solvents; only aqueous solutions are used. Electron micrographs of muscles embedded in melamine differ from those embedded in the conventional epoxy resin. In melamine-embedded muscles the actin and myosin filaments appear larger in diameter and subunits can be recognized in cross-sectioned myosin filaments. Within the Z-line, the characteristic patterns described for muscles embedded in epoxy resin are not visible; the spaces between the actin filaments are filled with electron-dense material. This suggests that the Z-line is more compact than could be concluded from epoxy resin-embedded muscle specimens. The M-line appears to be different from what is observed in epoxy-embedded muscle. The membranes appear as several clearly delineated layers. Dehydration rather than the action of the organic solvents per se is the main reason for the differences in the structure of the contractile apparatus between melamine- and epoxy-embedded muscles.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

Spectrofluorometric assay of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity

A simple, rapid, and sensitive spectrofluorometric assay for 15-hydroxyprostaglandin dehydrogenase activity was developed in which the rate of production of NADH was monitored. The cytosolic fraction prepared from human placental tissue was employed as the enzyme source. The assay was conducted at pH 9.5 since 15-ketoprostaglandin Δ¹³-reductase and NADH oxidase activities were inhibited at this pH, thereby minimizing the interference of the reactions catalyzed by these enzymes in the assay of prostaglandin dehydrogenase activity.     

Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria

Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.     

The activity of 3 alpha- and 3 beta-hydroxysteroid dehydrogenases and 5 alpha-reductase, together with androgen levels in male rat pituitary during sexual maturation

In all subcellular pituitary fractions, 3 alpha-hydroxysteroid dehydrogenase (3 alpha-ol dehydrogenase) activity is high (1 to 3 pmol/mg/h) with NADH or NADPH as cofactor, and 3 beta-hydroxysteroid dehydrogenase (3 beta-ol dehydrogenase) activity much lower. The highest activity of the latter (0.15 pmol/mg/h) is detected in cytosol with NADH as cofactor. During sexual maturation, cytosolic (NADH-dependent) 3 alpha- and 3 beta-ol dehydrogenase activities remain constant, whereas the 5 alpha-reductase activity is maximum at 37 days. The levels of different pituitary androgens were evaluated by radioimmunoassay. At 28 days, testosterone level is 4 ng/g of tissue, then after 42 days the level remains between 4.5 and 6 ng/g at a level higher than the DHT level. In all cases during the maturation of the rat, the different 5 alpha-reduced androgens are in the same ratio: DHT greater than 3 alpha-diol greater than 3 beta-diol, and the sum of these three 5 alpha-reduced androgens decreases between the 28th and the 90th day.     

Interaction of rhodanese with mitochondrial NADH dehydrogenase

NADH dehydrogenase is an iron-sulfur flavoprotein which is isolated and purified from Complex I (mitochondrial NADH: ubiquinone oxidoreductase) by resolution with NaClO4. The activity of the enzyme (followed as NADH: 2-methylnaphthoquinone oxidoreductase) increases linearly with protein concentration (in the range between 0.2 and 1.0 mg/ml) and decreases with aging upon incubation on ice. In the present work a good correlation was found between enzymic activity and labile sulfide content, at least within the limits of sensitivity of the assays employed. Rhodanese (thiosulfate: cyanide sulfurtransferase (EC 2.8.1.1) purified from bovine liver mitochondria was shown to restore, in the presence of thiosulfate, the activity of the partly inactivated NADH dehydrogenase. Concomitantly, sulfur was transferred from thiosulfate to the flavoprotein and incorporated as acid-labile sulfide. Rhodanese-mediated sulfide transfer was directly demonstrated when the reactivation of NADH dehydrogenase was performed in the presence of radioactive thiosulfate (labeled in the outer sulfur) and the 35S-loaded flavoprotein was re-isolated by gel filtration chromatography. The results indicated that the [35S]sulfide was inserted in NADH dehydrogenase and appeared to constitute the structural basis for the increase in enzymic activity.     

An improved ultrastructural double-staining method for rat growth hormone and its mRNA using LR White resin: a technical note

An improved new method for the simultaneous visualization of mRNA and encoded protein in LR White resin-embedded specimens is described. This pre-embedding electron microscopical in situ hybridization (procedure) localized rat growth hormone mRNA specifically as high electron-density products on the polysomes of the rough endoplasmic reticulum. A subsequent post-embedding immunoreaction, using protein A colloidal gold particles, identified growth hormone as gold particles both in the cisternae of the rough endoplasmic reticulum and on the secretory granules. In our previous report, we used Epon resin for tissue embedment, which required an etching process using hydrogen peroxide or sodium periodate for immunoreactivity retrieval. In general, osmification and embedment in Epon resin are reported to decrease the immunoreactivity of the targeted protein, and the etching process using hydrogen peroxide or sodium periodate results in deosmification and shades off the signals of mRNA. To resolve these problems, we have recently used LR White resin for tissue embedment. In LR White resin-embedded tissues, retrieval of immunoreactivity using hydrogen peroxide or sodium periodate is not required, and, therefore, the gradation of the signals of mRNA can be avoided. © 1998 Chapman & Hall     

NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain

Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity. In comparison, membranes from E. coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity. Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol. Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH. Inside-out membrane vesicles from E. coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate. Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity. The data provide a strong indication that the E. coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.     

Purification and characterization of two types of NADH-quinone reductase from Thermus thermophilus HB-8

Two types of the NADH-quinone reductase were isolated from Thermus thermophilus HB-8 membranes, by use of the nonionic detergent, dodecyl beta-maltoside, and NAD-agarose affinity, DEAE-cellulose, hydroxyapatite, and Superose 6 column chromatography. One of these (NADH dehydrogenase 1) is a complex composed of 10 unlike polypeptides, and the other (NADH dehydrogenase 2) exhibits a single band (Mr 53,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase 1 was about 14 times higher than that of the dodecyl beta-maltoside extract and partially rotenone sensitive. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase 2 was about 30-fold as high as that of the dodecyl beta-maltoside extract and rotenone insensitive. The purified NADH dehydrogenase 1 contained noncovalently bound FMN, non-heme iron, and acid-labile sulfide. The ratio of FMN to non-heme iron to acid-labile sulfide was 1:11-12:7-9. The high content of iron and labile sulfide is suggestive of the presence of several iron-sulfur clusters. The purified NADH dehydrogenase 2 contained noncovalently bound FAD and no non-heme iron or acid-labile sulfide. The activities of both NADH dehydrogenases were stable at temperatures of greater than or equal to 80 degrees C. The occurrence of two distinct types of NADH dehydrogenase as a common feature in the membranes of various aerobic bacteria is discussed.     

Biochemical Basis of Obligate Autotrophy in Nitrosomonas europaea

The specific activities of isocitric dehydrogenase, alpha-ketoglutaric dehydrogenase, succinic dehydrogenase, malic dehydrogenase, and reduced nicotinamide adenine dinucleotide (NADH) oxidase were determined in extracts of Nitrosomonas europaea and compared with the corresponding values for Anacystis nidulans and autotrophically grown Hydrogenomonas eutropha. In common with other obligate autotrophs and in contrast to facultative autotrophs, Nitrosomonas extracts lacked alpha-ketoglutaric dehydrogenase and KCN-sensitive NADH oxidase activity and had low succinic dehydrogenase activity. The Nitrosomonas NADH oxidase appeared to be of the peroxidase type.     

Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris)

The present study compares the exogenous NAD(P)H oxidation and the membrane potential ([delta][psi]) generated in mitochondria isolated from different tissues of an important agricultural crop, sugar beet (Beta vulgaris}. We observed that mitochondria from taproots, cold-stored taproots, and in vitro-grown tissue cultures contain a functional NADH dehydrogenase, whereas only those isolated from tissue cultures displayed a functional NAD(P)H dehydrogenase. It is interesting that the NADH-dependent [delta][psi] of mitochondria from cold-stored taproots and from tissue cultures was not affected by free Ca2+ ions, whereas free Ca2+ was required for the mitochondrial NADPH oxidation by in vitro-grown cells and cytosolic NADH oxidation by mitochondria from fresh taproots. A tentative model accounting for the different response to Ca2+ ions of the NADH dehydrogenase in mitochondria from cold-stored taproots and tissue cultures of B. vulgaris is discussed.     

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.     

Purification and properties of a peptic heme peptide from cytochrome c1.

Highly purified mouse liver plasma membranes have been used to define the properties of an NADH dehydrogenase activity associated with plasma membrane. The NADH indophenol reductase activity is two-fold stimulated at 5 × 10^?8 M glucagon and the stimulation is inhibited by atebrin. Corresponding activity in endoplasmic reticulum is not stimulated by glucagon. The NADH indophenol reductase is 90% inhibited by insulin at 7 × 10^?11M and shows a return to the original activity at higher insulin concentrations. NADH dehydrogenase activity in endoplasmic reticulum is inhibited up to 50% by insulin at a similar concentration. Triiodothyronine at 10^?7M also inhibits the plasma membrane dehydrogenase whereas thyroxine has little effect. The response of this dehydrogenase to hormones suggests a role in regulation of cellular function.     

Influence of ammonium and nitrate nutrition on the pyridine and adenine nucleotides of soybean and sunflower

Total pyridine nucleotide concentration of root tissue for young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var. Mammoth Russian) plants is the same with either ammonium or nitrate, but nitrate results in an increased proportion of total oxidized plus reduced NADP (NADP[H]) seemingly at the expense of NAD. The activity of NADH- and NADPH-dependent forms of glutamic acid dehydrogenase is correlated with the ratio of total oxidized plus reduced NAD to NADP(H). The low NAD: NADH ratio maintained in nitrate roots despite active NADH utilization via nitrate reductase and glutamic acid dehydrogenase may be the result of nitrate-stimulated glycolysis. Nitrate roots also maintain a high level of NADPH, presumably by the stimulatory effect of nitrate utilization on glucose-6-phosphate dehydrogenase activity. In the presence of nitrate rather than ammonium, the highly active nitrate-reducing leaves of soybean show a greater proportion of total pyridine nucleotide in the form of NADP(H) than do the inactive leaves of sunflower.     

Association of MT-ND5 gene variation with mitochondrial respiratory control ratio and NADH dehydrogenase activity in Tibet chicken embryos

NADH dehydrogenase (complex I) couples the oxidation of NADH for the reduction of ubiquinone with the generation of a proton gradient that can be used for the synthesis of ATP. We have found a missense mutation in the MT-ND5 subunit of NADH dehydrogenase in the Tibet chicken breed. In the present study, the mitochondrial respiratory control ratio (RCR) and NADH dehydrogenase activity in Tibet chicken embryonic brain with different genotypes were measured. Significant differences between animals carrying mitochondria with the EF493865.1:m.1627A vs. EF493865.1:m.1627C alleles were observed for RCR and enzyme activity.     

Relation of NADH/NAD to contraction in vascular smooth muscle

The relationship of NADH/NAD to O2 consumption with respect to the different phases of contraction in vascular smooth muscle in response to a maximal depolarizing concentration of KCl was investigated. The NADH bound to cellular proteins could be distinguished from free NADH in whole tissue homogenates. Evidence suggested that the NADH was bound to pyruvate dehydrogenase and perhaps to other dehydrogenases since binding paralleled the changes in the activity of pyruvate dehydrogenase with contraction. The measured changes in NADH were attributed to that within the mitochondrial compartment since the contribution of reducing equivalents within the cytoplasmic compartment was negligible. During the phase of contraction in which force was initially being generated and at which O2 consumption was the highest, there was a net increase in NADH/NAD. After stable isometric force was maintained, at which time O2 consumption had returned to slightly above the basal pre-contraction level, there was a net decrease in NADH/NAD. Previous evidence indicates the phosphorylation potential (ATP/ADP) may decrease during this phase of contraction. It is concluded that contraction of vascular smooth muscle is accompanied by a changing pool of reducing equivalents. Factors which govern O2 consumption may change during the different phases of muscle contraction.     

NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane studied using inside-out submitochondrial particles

Submitochondrial particles (SMP) were isolated from potato ( Solanum tuberosum L. cv. Bintje) tubers. The SMP were 91% inside-out and they were able to form a membrane potential, as monitored by oxonol VI, with succinate, NADH and NADPH. The pH dependence and kinetics of NADH and NADPH oxidation by these SMP was studied using three different electron acceptors – O₂, duroquinone and ferricyanide. In addition, the SMP were solubilized, fractionated by non-denaturing polyacrylamide gel electrophoresis, and the gels were stained for NAD(P)H dehydrogenase activity and specificity at different pH using Nitro Blue Tetrazolium. From the results we conclude that there are at least two distinct NAD(P)H dehydrogenases on the inner surface of the inner membrane: (1) Complex 1 which oxidizes NADH and deamino-NADH in a rotenone-sensitive manner, (O₂ as acceptor) with optimum activity at pH 8 and a very low K_m(NADH) of 3 μ M . It also oxidizes NADPH and deamino-NADPH in a rotenone-sensitive manner, but with a pH optimum at pH 5.8 and a very high K_m(NADPH) of more than 1 m M . This complex is found as a broad, diffuse band at the top of the gels. (2) A second dehydrogenase which oxidizes NADH in a rotenone-insensitive manner with optimum activity at pH 6.2 and a higher K_m(NADH) of 14 μ M . It also oxidizes NADPH in a rotenone-insensitive manner with an activity optimum at pH 6.8 and low K_m(NADPH) of 25 μ M . This dehydrogenase does not oxidize deamino-NAD(P)H. One of the sharp bands around the middle of the native gels may be caused by this dehydrogenase indicating that it has a relatively low molecular mass compared to Complex I. Several other NAD(P)H dehydrogenase bands were observed on the gels which we cannot yet assign.