Cozymase - Assay of Diphosphopyridine Nucleotide Preparations

Gutcho, Sidney; Stewart, Earl D.

doi:10.1021/ac60024a016

Cited by 50 publications

(10 citation statements)

References 12 publications

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“…Materials.--The DPNH used in these studies was prepared chemically with sodium hydrosulfite (14) from DPN of about 80 per cent purity obtained from the Sigma Chemical Company. However, the DPNH was made up in 0.l m K2HPO4-KH2PO4, pH 7.4, instead of NaHCOa-Na2CO3.…”

Section: Methodsmentioning

confidence: 99%

Intracellular Localization of Enzymes in Spleen

Hj¹

1957

The Journal of Cell Biology

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In 1949, Hogeboom (1) reported that in rat liver homogenates which were prepared in 0.88 M sucrose and separated into four fractions by differential centrifugation, 32 and 58 per cent of the total D P N H 1 cytochrome c reductase activity of the original homogenates were recovered in the mitochondria and microsomes, respectively. He also found that, based on activity per mg. of nitrogen, the enzyme was concentrated 3.3 times in the microsome fraction and only 1.3 times in the mitochondria with respect to the homogenate. In 1951, as part of a study of the D P N H cytochrome c reductase of rabbit liver and heart mitochondria, Eichel (2) observed a 1.6-and 2.9-fold concentration of the enzyme in mitochondrial fractions prepared from isotonic sucrose homogenates of liver and heart, respectively. Cytochrome c oxidase assays on these (and other) fractions and the original homogenates confirmed the mitochondrial nature of the isolated fractions. Subsequently, Brody et al. (3) studied the distribution of D P N H cytochrome c reductase in various fractions obtained from homogenates of rat brain cerebral cortex. In contrast to the results with rat liver, they found a 2.8-fold concentration of the enzyme in the mitochondria with respect to the original homogenate, while the specific activity of the microsomal fraction was slightly less than that of the original homogenate. No data were given for the recovery of total enzyme activity in each of the particulate fractions. Strittmatter and Ball (4) and de Duve el al. (5) have also studied the distribution of the reductase in rat liver fractions. Their results are in essential agreement with those reported previously.

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Section: Methodsmentioning

confidence: 99%

Intracellular Localization of Enzymes in Spleen

Hj¹

1957

The Journal of Cell Biology

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“…Diphosphopyridine nucleotide (DPN) was prepared by the method of Kornberg & Pricer (1953). The DPN preparation was found to be of 70% purity, when assayed by the methods of Gutcho & Stewart (1948) and Racker (1950). Chromatographic analysis (method of Burton & Pietro, 1954) showed that there were onlytraces of triphosphopyridine nucleotide (TPN) present.…”

Section: Methodsmentioning

confidence: 99%

“…Reduced DPN was about 90 % as efficient in converting as DPN when added to the microsome-supernatant preparation plus schradan. In another series of experiments reduced DPN (Gutcho & Stewart, 1948) was added to the liver microsome-supernatant preparation. The oxidation of DPNH at 37°was followed by the decreased absorption at 340 m in a Unicam spectrophotometer (SP.…”

Section: -mentioning

confidence: 99%

The conversion of schradan (OMPA) and parathion into inhibitors of cholinesterase by mammalian liver

Davison

1955

Biochemical Journal

123

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“…Reduced diphosphopyridine nucleotide (DPNH2)t was prepared according to Gutcho and Stewart (1948), and obtained as a dry powder according to Ohlmeyer (1938). Enzymatic activity was measured by a spectrophotometric procedure described by Kornberg and Pricer (1951 Herzog and Slansky (1911).…”

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confidence: 99%

“…sucrose (final concentration: 32%) was added to the supernatant fluid which was used as the source of the enzyme.The mammalian enzyme was prepared by the method of Kornberg and Pricer (1951). Reduced diphosphopyridine nucleotide (DPNH2)t was prepared according to Gutcho and Stewart (1948), and obtained as a dry powder according to Ohlmeyer (1938). Enzymatic activity was measured by a spectrophotometric procedure described by Kornberg and Pricer (1951).…”

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confidence: 99%

KINETICS OF LACTIC DEHYDROGENASES OF SCHISTOSOMA MANSONI AND OF RABBIT MUSCLE*

Mansour¹,

Bueding²

1953

British Journal of Pharmacology and Chemotherapy

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It has been shown that in Schistosoma mansoni glycolysis proceeds at an exceedingly rapid rate (Bueding, 1950) and that this process is more critical for the survival of the parasite than is respiration (Bueding and Peters, 1951 ;Bueding, Peters, Koletsky, and Moore, 1953). Although glycolytic enzymes might catalyse the same reactions in the host as those in the parasite their nature might be different. Such differences would be of importance for the development of chemotherapeutic agents. They would afford opportunities to select specific inhibitors against enzymes of the parasite which would be without effect on those of the host. In an attempt to investigate such a possibility the kinetics of lactic dehydrogenase of Schistosoma mansoni were compared with those of lactic dehydrogenase of rabbit muscle. METHODS Adult worms were dissected from the mesenteric and portal veins of mice six to eight weeks after these animals had been infected with cercariae of Schistosoma mansoni. All subsequent operations were carried out at temperatures varying between 0 and 20 C.The worms were cut with fine scissors in distilled water (1 ml. of water was used per 100 pairs of worms). The process of cutting required two minutes.The suspension containing the worm fragments was stirred for five minutes in a beaker surrounded with cracked ice. This extract was then centrifuged for five minutes at 4,000 r.p.m., and 0.1 ml. of phosphate gel (dry weight per ml.: 1.4 mg.) was added per ml. of supernatant fluid. After stirring, this mixture was centrifuged for 10 minutes at 4,000 r.p.m. Most containing glycylglycine buffer (0.5 M; pH 7.5) and potassium chloride (0.5 M) equivalent to one-half of the original extract. Sucrose was added to this eluate (final concentration: 32%) because this ensured the stability of the enzyme when stored in the frozen state. In a number of experiments a homogenate was used. This was prepared by homogenizing the worms in glycylglycine buffer (0.01 M; pH 7.5). After centrifugation for 10 minutes at 4,000 r.p.m. sucrose (final concentration: 32%) was added to the supernatant fluid which was used as the source of the enzyme.The mammalian enzyme was prepared by the method of Kornberg and Pricer (1951). Reduced diphosphopyridine nucleotide (DPNH2)t was prepared according to Gutcho and Stewart (1948), and obtained as a dry powder according to Ohlmeyer (1938). Enzymatic activity was measured by a spectrophotometric procedure described by Kornberg and Pricer (1951). This method is based on the change in the optical density at a wavelength of 340 m/A as a result of the oxidation or reduction of diphosphopyridine nucleotide (DPN)t during the following reaction:Pyruvate + DPNH2 'lactate + DPN All measurements were made in a Beckman spectrophotometer in an air-conditioned room at 26°C. Under optimal conditions the activity of the enzyme was proportional to its concentration. The latter was adjusted so that under optimal conditions 0.013 to 0.016 /'M of DPN was reduced or oxidized in one minute. The total volume of the ...

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Cozymase - Assay of Diphosphopyridine Nucleotide Preparations

Cited by 50 publications

References 12 publications

Intracellular Localization of Enzymes in Spleen

Intracellular Localization of Enzymes in Spleen

The conversion of schradan (OMPA) and parathion into inhibitors of cholinesterase by mammalian liver

KINETICS OF LACTIC DEHYDROGENASES OF SCHISTOSOMA MANSONI AND OF RABBIT MUSCLE*

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