Soluble Enzymes from Pea Mitochondria

A method of obtaining an extract of soluble enzymes from peaseedling mitochondria is described. Evidence is presented thatthe mitochondrial extract contains the following enzymes: Diphosphopyridinenucleotide (DPN) and triphosphopy-ridine nucleotide (TPN) specificwocitric dehydrogenases, alcohol dehydrogenase, formic dehydrogenase,aldehyde dehydrogenase, glutamic dehydrogenase, malic enzyme,lactic dehydrogenase, fumarase, aconitase, DPN and TPN cytochrome-creductases, adenylate kinase, phosphopyridine nucleotide transhydrogenaseand oxaloacetic carboxylase. The relative activities of theseenzymes have been quantitatively determined and the resultsdiscussed.     

Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Kinetic studies and fluorescence titration.

Mechanisms involved in the action of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1) have been investigated by means of kinetic studies and fluorescence titration. Our results, as well as those from previous investigations, suggest that the allosteric MWC model (Monod, J., Wyman, J., and Changeux, J. P. (1965), J. Mol. Biol. 12, 88-118) may be used as a first step for the explanation of the properties of the transhydrogenase. The basic reaction of the enzyme is the oxidation of reduced triphosphopyridine nucleotide (TPNH) by diphosphopyridine nucleotide (DPN+). In terms of the model, the functional R state is favored by TPNH, whereas the product triphosphopyridine nucleotide (TPN+) behaves as an allosteric inhibitor, and is therefore assumed to favor the nonfunctional T state. To a slight extent, the T state is also favored by inorganic phosphate. On the other hand, adenosine 2'-monophosphate and several other 2'-phosphate nucleotides function as activators, and hence are presumed to shift the allosteric equilibrium toward the R state. The studies in this paper suggest a specific regulatory site for the transhydrogenase.     

Acyl coenzyme A inhibition of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase: a comparison of the TPN and DPN linked reactions

The inhibitory effects of ATP, coenzyme A, and acetyl, malonyl, and oleyl derivatives of coenzyme A on the TPN and DPN dependent activities of Leuconostoc glucose-6-phosphate dehydrogenase are compared. At pH 7.8, 24°, saturating levels of DPN or TPN, and inhibitor concentrations of 2–4 mM only ATP has an appreciable effect on the TPN dependent reaction, but all were potent inhibitors of the DPN dependent reaction. Oleyl coenzyme A was the most effective (K_i ～ 0.15 mM against glucose-6-phosphate) while acetyl coenzyme A was least effective (K_i ～ 1.0 mM). A possible regulatory role of this inhibition in fatty acid synthesis is suggested.     

The Histochemical Localization of Triphosphopyridine Nucleotide Diaphorase

A histochemical method is described for the localization of triphosphopyridine nucleotide diaphorase using a recently synthesized tetrazolium salt (Nitro-BT). By virtue of the favorable histochemical properties of this reagent, it has been possible to demonstrate that whereas DPN diaphorase is usually restricted to the mitochondria, the TPN diaphorase activity of corresponding cells was distributed throughout the cytoplasm in granules too fine to be considered mitochondria. Furthermore, although the diaphorase alone is responsible for the passage of electrons from TPNH to the tetrazole, it has been found that sites of activity of different TPN-linked dehydrogenases can be visualized in tissue sections, and characteristic loci for each enzyme may be observed. For example, whereas TPN diaphorase and isocitric dehydrogenase have an extensive distribution in the kidney cortex, 6-phosphogluconic dehydrogenase is limited to the cells of the macula densa.     

Pyridine nucleotide metabolism in mammalian cells in culture

The biosynthesis of pyridine nucleotides has been examined in a number of mammalian cell lines in culture. In all lines examined, nicotinamide is incorporated by a biochemical pathway distinct from the Preiss-Handler pathway for nicotinic acid. In at least the human cell line D98/AH2, there is no detectable endogenous synthesis of the pyridine ring from tryptophan. Although most cell lines examined (hamster BHK 21/13, mouse L929 and human D98/AH2) use either nicotinic acid or nicotinamide as a precursor for DPN and TPN, two mouse cell lines, 3T3-4E and LM CIID, are unable to utilize nicotinic acid as a source of the pyridine ring. If nicotinic acid is present in the medium, substantial amounts of intracellular desamido DPN accumulate suggesting that the last step (desamido DPN→DPN) is limiting in the Preiss-Handler pathway. With nicotinamide, the only compound which accumulates in substantial amounts apart from DPN and TPN is nicotinamide ribose; there is no detectable NMN. The results of pulse-labeling experiments suggest that nicotinamide ribose may be an intermediate in the nicotinamide pathway. Following growth of D98/AH2 cells in high concentrations of niacin, biosynthesis of DPN from nicotinamide was completely inhibited for at least six hours. The converse experiment revealed no inhibition of niacin incorporation. This observation suggests that a niacin pathway intermediate, which present evidence indicates is desamido-DPN. can inhibit nicotinamide utilization. Newly synthesized DPN turns over with a half-life of two hours in azaserine-treated D98/AH2 cells. In the absence of azaserine, the nicotinamide moiety of newly synthesized DPN is lost from D98/AH2 cells to the medium with a half-life of eight hours. About 80% of the nicotinamide is lost to medium as nicotinamide ribose.     

Uridine diphosphate galactose 4-epimerase. Alkylation of enzyme-bound diphosphopyridine nucleotide by p-(bromoacetamido)phenyl uridyl pyrophosphate, an active-site-directed irreversible inhibitor.

When UDP-galactose 4-epimerase is inactivated by p-(bromoacetamido)phenyl uridyl pyrophosphate (BUP), the diphosphopyridine nucleotide (DPN) associated with this enzyme as a tightly bound coenzyme cannot be reduced by substrates or by UMP-activated reduction by glucose. Upon acid denaturation of the inactivated enzyme, the DPN released corresponded to 15-30% of that released from the native enzyme. When the enzyme is inactivated by [14C]BUP, about 80% of the radioactivity bound at the active site is released from the protein upon acid denaturation. When epimerase-[3H]DPN is inactivated with [14C]BUP, the 3H and 14C released from the protein upon denaturation of the complex cochromatograph on DEAE-Sephadex. Experiments with [nicotinamide-4-3H]DPN and [adenine-2,8-3H]DPN show that it is the adenine ring that is alkylated. The data suggest that the adenine ring of DPN in epimerase-DPN may be oriented near the glycosyl-binding subsite of this enzyme. Since the nicotinamide ring must also be near this site, it appears that the DPN may not be in an extended conformation when it is bound at the active site of UDP-galactose 4-epimerase from Escherichia coli.     

Prime Ricerche sulla Fisionomia Enzimatica Caratteristica della Fotosintesi

Summary

The rate of oxidation of glucose-6-phosphate, ribose-5-phosphate, fructose-1,6- phosphate, iso-citrate and malate in extracts from green and etiolated pea leaves was determined, using the triphenyltetrazolium technique.

Glucose-6-phosphate, ribose-5-phosphate, and fructose-1,6-phosphate, in the presence of added TPN, where oxidized at a rate about twice higher in the extracts from green than in the extracts from etiolated leaves. Iso-citrate, in the presence of TPN, fructose-1,6-phosphate, in the presence of DPN, and malate, in the presence of either TPN or DPN, were oxidized at about the same rate in the two types of extracts.

These data seem to indicate a preferential synthesis of enzymes involved in the metabolic cycle of phosphorylated sugars during the transition of the leaf from the etiolated to the photosynthetising physiognomy. They seem also favourable to the view assigning to this metabolic system a primary importance in the anabolic pathway of photosynthesis.     

Factors influencing the histochemical demonstration of coenzyme-dependent dehydrogenases and diaphorases

Procedures for the histochemical demonstration of DPN and TPN diaphorases have been presented by other workers. These techniques rely on the coenzyme-dependent dehydrogenases present in the tissue slice to generate the substrate required by the diaphorases. In vitro studies were carried out on kidney and adrenal tissue of the rat, using NT (neotetrazolium) and INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) with various substrates of DPN-dependent dehydrogenases. The solutions used for study contained alcohol and alcohol dehydrogenase, glutamate and malate, malate, glutamate, beta-hydroxybutyrate, or DPNH. It has been possible to demonstrate (1) that histological distribution of dehydrogenases may differ from that of the flavoprotein oxidizing reduced coenzyme I; (2) characteristic patterns of distribution of particular dehydrogenases in the tissue proper; (3) different levels of dehydrogenase in kidney and adrenal; and (4) differences in dehydrogenase distribution in the kidneys of man and rat. The evidence presented clearly indicates the limitations inherent in the accepted procedures for the demonstration of DPN and TPN diaphorases. The possible application of the tetrazolium salts to the study of particular coenzyme-dependent dehydrogenases and the pitfalls which might occur are also discussed.     

Factors Influencing the Histochemical Demonstration of Coenzyme-Dependent Dehydrogenases and Diaphorases

Procedures for the histochemical demonstration of DPN and TPN diaphorases have been presented by other workers. These techniques rely on the coenzyme-dependent dehydrogenases present in the tissue slice to generate the substrate required by the diaphorases. In vitro studies were carried out on kidney and adrenal tissue of the rat, using NT (neotetrazolium) and INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) with various substrates of DPN-dependent dehydrogenases. The solutions used for study contained alcohol and alcohol dehydrogenase, glutamate and malate, malate, glutamate, β-hydroxybutyrate, or DPNH. It has been possible to demonstrate (1) that histological distribution of dehydrogenases may differ from that of the flavoprotein oxidizing reduced coenzyme I; (2) characteristic patterns of distribution of particular dehydrogenases in the tissue proper; (3) different levels of dehydrogenase in kidney and adrenal; and (4) differences in dehydrogenase distribution in the kidneys of man and rat. The evidence presented clearly indicates the limitations inherent in the accepted procedures for the demonstration of DPN and TPN diaphorases. The possible application of the tetrazolium salts to the study of particular coenzyme-dependent dehydrogenases and the pitfalls which might occur are also discussed.     

17beta-Hydroxy-C19-steroid dehydrogenases from male guinea pig liver. Purification and characterization

One major and six minor 17beta-hydroxy-C19-steroid dehydrogenases were isolated each in a highly pure form from male adult guinea pig liver by a combination of gel filtration and ion exchange chromatography. Molecular weight, amino acid composition, sugar content, number of sulfhydryl groups. NH2-terminal amino acid, isoelectric point, substrate specificity, pH optima, Km values, and inhibitory effect of other steroids were studied. The amino acid composition, Km values, and substrate specificity indicated two separate groups of enzymes. The first group possessed a dual coenzyme requirement and specificity for 5beta-androstanes, whereas the second group showed apparent TPN+ and 5alpha-androstane specificities. Phosphate enhanced the DPN+-related activity of the first group and evoked DPN+-linked activity in the second group of enzymes. A molecular weight of 31,000 to 32,000 with a single chain structure was estimated for four of the enzymes. The other three enzymes consisted of two components of 24,000 and 11,000 daltons.     

Preparation of Diphosphopyridine Nucleotide from Bakers’ Yeast

A method for the preparatoin of diphosphopyridine nucleotide (DPN) from bakers’ yeast is described. This method consists of partial purification of crude extract of yeast by charcoal chromatography according to Pontis and coworkers, ion-exchange chromatography on Dowex-l acetate, and precipitation of DPN as the free acid with ethanol. 0.71~.1.1 g of DPN with a purity of 85~90% was obtained from 5 kg of fresh bakers’ yeast by this method.     

Equilibrium perturbation by isotope substitution.

When malic enzyme is added to a mixture of malate-2-d, TPN, CO2, pyruvate, and TPNH at concentrations calculated to be at equilibrium, the TPNH level first drops and then increases slowly to its original level. This equilibrium perturbation is caused by slower cleavage of C-D than C-H bonds during hydride transfer as malate-2-d and TPNH are partly converted into TPND and malate-2-h in the process of establishing isotopic equilibrium. With malate-2-d, isotope effects for malic enzyme at pH 7.1 and malate dehydrogenase at pH 9.3 of 1.45 and 1.70-2.16 (depending on oxaloacetate level) were determined with this method, while the corresponding isotope effects on V/Kmalate and V for the chemical reactions were 1.5-1.8 and 1.0, and 1.9 and 1.5 for the two enzymes. The advantage of this method is its extreme sensitivity, and the lack of interference from various artifacts. The sensitivity is sufficient to permit determination of 13C and 15N isotope effects in favorable cases, and values of 1.031 for malic enzyme with 13CO2, and 1.047 for glutamate dehydrogenase with 15NH4+ have been determined. In the course of this work it was discovered that the equilibrium constants for oxidation by DPN, and oxidative decarboxylation by TPN are lower for malate-2-d than for malate-2-h by a factor of 0.76-0.82. Changes in Keq upon deuterium substitution, which are predicted by the calculations of Hartshorn and Shiner (1972), should be observed for many other reactions as well.     

Bound TPN as the determinant of polymorphism in methemoglobin reductase

The pyridine nucleotide-dependent, dye-linked methemoglobin reductase from human erythrocytes can be resolved chromatographically or electrophoretically into two principal forms (I and II). Form (II), which migrates more rapidly than (I) toward the positive electrode during electrophoresis at pH 8.3, contains 1 mole of non-covalently bound TPN per mole of protein (MW = 22,000) as judged by spectral characteristics (absorbance maxima at 327 and 340 nm when the protein is treated with cyanide and hydrosulfite, respectively) and by analysis (thin-layer chromatography and coenzymatic activity with isocitrate dehydrogenase) of the nucleotide released by heat denaturation. TPN can also be displaced by treatment of (II) with p-chloromercuriphenyl-sulfonate. Incubation of (I) with TPN at neutral pH results in the formation of (II).     

A METABOLIC PATHWAY OF ISOCITRIC ACID IN COTYLEDONS OF A BEAN, VIGNA SESQUIPEDALIS

1. It has been established that the absence of isocitric dehydrogenaseactivity in cotyledons of Vigna sesquipedalisin the germinationstage is due to the lack of endogenous TPN. It is unlikely thatthe TCA cycle in the cotyledon is operative.
2. The activitiesof enzymes of the glyoxylate cycle, a modifiedTCA cycle, weremeasured through the germination stage. Theiractivities wereconsiderably higher in the cotyledon tissuethan in the hypocotyl.A dominant metabolic pathway of isocitricacid in the cotyledonmay be the glyoxylate cycle.
3. DPN kinase which produces TPNfrom DPN, ATP and Mg⁺⁺ was localizedin the supernatant portionof cell components in the cotyledon.Its activity was low. DPNkinase serves as a control factorfor the cotyledon metabolism.

(Received April 17, 1961; )     

Oxidative enzymes in the pancreatic islets of normal and obese-hyperglycemic mice

Summary Histochemical data are presented concerning distributions of succinic dehydrogenase (SD), lactic dehydrogenase (LD), diphosphopyridine nucleotide diaphorase (DPND), triphosphopyridine nucleotide diaphorase (TPND) and glucose-6-phosphate dehydrogenase (G-6-PD) in the pancreas from the American variety of obese-hyperglycemic mice (AO-mice) and their lean litter mates (AN-mice).A high LD activity was found in the exocrine parenchyma, while the reaction in the islet tissue and the duct epithelium was only weak. A considerable reaction for DPND was noted throughout the pancreas. SD activity was slightly more pronounced in the acinar tissue and duct epithelium as compared to the islet tissue, where only a moderate activity appeared. Strong reactions for TPND and G-6-PD were found in the islet cells and duct epithelium, while the activity in the exocrine parenchyma was less pronounced. The hyperactive islet B cells in the AO-mice showed no obvious differences in enzyme activity and distribution compared to that of the AN-mice. The enzyme pattern of the A cells could not be clearly distinguished from that in the B cells.The results suggest the existence at least in the B cells of the mice islet tissue of an active hexosemonophosphate shunt. The probable significance of the hexosemonophosphate shunt for insulin synthesis is briefly discussed.The following abbreviations are used DPN Diphosphopyridine nucleotide - DPND Diphosphopyridine nucleotide diaphorase - DPNH Diphosphopyridine nucleotide, reduced form - EM Embden-Meyerhof - G-6-PD Glucose-6-phosphate dehydrogenase - HMP Hexose monophosphate - LD Lactic dehydrogenase - MTT 3,5-diphenyl-2-(4,5-dimethyl-thiazol-2-yl) tetrazolium bromide - Nitro-BT 2,2-di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - PVP Polyvinyl pyrrolidone (M. W. 11 000) - SD Succinic dehydrogenase - TPN Triphosphopyridine nucleotide - TPND Triphosphopyridine nucleotide diaphorase - TPNH Triphosphopyridine nucleotide, reduced form     

Comparison of Enzymatic Reduction of Diphosphopyridine Nucleotide with Ammonia and Urea

In investigating the properties of the dehydrogenating system from ammonia to nitrate, it has been observed that DPN is reduced with urea also in the presence of an enzyme from fowl liver. The properties of the enzyme are very closely similar to those of ammonium dehydrogenase. However, some fundamental differences have been demonstrated by comparing these two enzymes, especially concerning reduction of TPN, substrate inhibition, formation of some nitrogenous metabolites and reoxidation of DPNH in the reaction mixture. The reaction was postulated to be a dehydrogenation of urea by an enzyme. This activity has also been demonstrated in silkworm and yeast.     

Endogener Rhythmus und CO2-Stoffwechsel bei Pflanzen mit diurnalem Säurerhythmus

Ohne ZusammenfassungFolgende Abkürzungen wurden verwandt ADP Adenosin-Diphosphat - ATP Adenosin-Triphosphat - CAM Crassulaceen-Säurestoffwechsel (Crassulacean acid metabolism - DPN Diphosphorpyridinnucleotid - DPNH₂ reduziertes Diphosphorpyridinnucleotid - Pi anorganisches Phosphat (inorganic phosphate) - PEP Phosphoenolbrenztraubensäure (Phosphoenolpyruvat) - PGA Phosphoglycerinsäure (Phosphoglyceric acid) - RDP Ribulosediphosphat - TPN Triphosphopyridinnucleotid - TPNH₂ reduziertes Triphosphopyridinnucleotid Mit 20 TextabbildungenHerrn Prof. Dr. Dr. h. c. H. v.Guttenberg zum 80. Geburtstag gewidmet.     

灭鼠安、灭鼠优现场灭效试验、解毒及毒理探讨

灭鼠安、灭鼠优是70年代出现的新单剂量灭鼠剂。国外经试验对多种鼠有杀灭效果,适口性较好,对非杀灭对象比较安全,对抗凝血剂有耐药性的鼠能够杀灭等优点。国内军事医学科学院等单位也有合成,我站在1978-1979年对这两类药进行了现场灭效和解毒试验,并对毒理机制进行了探讨,今报告如下。     

Spectroscopic studies of the interactions of coenzymes and coenzyme fragments with pig heart, oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase

Spectroscopic, ultrafiltration, and kinetic studies have been used to characterize interactions of reduced and oxidized triphosphopyridine nucleotides (TPNH and TPN), 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P), and adenosine 2',5'-bisphosphate [Ado(2',5')P2] with with TPN-specific isocitrate dehydrogenase. Close similarity of the UV difference spectra and of the protein fluorescence changes accompanying the formation of the binary complexes provides evidence for the binding of these nucleotides to the same site on the enzyme. From the pH dependence of the dissociation constants for TPNH binding to TPN-specific isocitrate dehydrogenase in the absence and in the presence of Mn2+, over the pH range 5.8-7.6, it has been demonstrated that the nucleotide binds to the enzyme in its unprotonated, metal-free form. The involvement of positively charged residues, protonated over the pH range studied, has been postulated. One TPNH binding site per enzyme subunit has been measured by fluorescence and difference absorption titrations. A dramatic effect of ionic strength on binding has been demonstrated: about a 1000-fold decrease in the dissociation constant for TPNH has been observed at pH 7.6 upon decreasing ionic strength from 0.336 (Kd = 1.2 +/- 0.2 microM) to 0.036 M (Kd = 0.4 +/- 0.1 nM) in the presence and in the absence of 100 mM Na2SO4, respectively. Weak competition of sulfate ions for the nucleotide binding site has been observed (KI = 57 +/- 3 mM). The binding of TPN in the presence of 100 mM Na2SO4 at pH 7.6 is about 100-fold weaker (Kd = 110 +/- 22 microM) than the binding of the reduced coenzyme and is similarly affected by ionic strength. These results demonstrate the importance of electrostatic interactions in the binding of the coenzyme to TPN-specific isocitrate dehydrogenase. The large enhancement of protein fluorescence caused by binding of TPN and Rib-P2-Ado-P (delta Fmax = 50%) and of Ado(2',5')P2 (delta Fmax = 41%) has been ascribed to a local conformational change of the enzyme. An apparent stoichiometry of 0.5 nucleotide binding site per peptide chain was determined for TPN, Rib-P2-Ado-P, and Ado(2',5')P2 from fluorescence titrations, in contrast to one binding site per enzyme subunit determined from UV difference spectral titration and ultrafiltration experiments. Thus, the binding of one molecule of the nucleotide per dimeric enzyme molecule is responsible for the total increase in protein fluorescence, while binding to the second subunit does not cause further change.(ABSTRACT TRUNCATED AT 400 WORDS)     

Untersuchungen an Aspergillus niger van Tiegh. über Zinkaufnahme und Aktivitätsveränderungen einiger Enzyme bei Zinkmangel

Ohne ZusammenfassungVerwendete Abkürzungen Acetyl-CoA Acetyl Coenzym A - AMP Adenosinmonophosphat - cpm counts per minute - Dithizon Diphenylthiocarbazon - DPN Diphosphopyridinnucleotid - EDTA Aethylendiamintetraacetat - FDP Fructose-1,6-diphosphat - F6P Fructose-6-phosphat - G6P Glucose-6-phosphat - G6PD Glucose-6-phosphat-Dehydrogenase - KDPG 2-Keto-3-desoxy-6-phosphogluconat - M Mangelmycel - MTG Myceltrockengewicht - N Normalmycel - p.a. pro analysi - 6PG 6-Phosphogluconat - 6PGD 6-Phosphogluconat-Dehydrogenase - Trispuffer Trishydroxymethylaminomethanpuffer - TPN Triphosphopyridinnucleotid Diese Untersuchungen wurden durch Mittel aus den Arbeitsbeschaffungs-krediten des Bundes ermöglicht, wofür auch an dieser Stelle bestens gedankt sei.