James B. Sumner

d-Glyceraldehyde 3-phosphate dehydrogenase; a comparison with liver aldehyde dehydrogenase

Abstract Using d -glyceraldehyde 3-phosphate, d -glyceraldehyde, and acetaldehyde as substrates, crude and crystalline preparations of d -glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle have been investigated with respect to requirement for phosphate and arsenate (in acetaldehyde oxidation), pH optimum, rates of reactions, Michaelis constants, and inhibition by tetraethylthiuram disulfide (antabuse). Our crude preparation of d -glyceraldehyde 3-phosphate dehydrogenase has a much higher affinity for low concentrations of acetaldehyde than does the crystalline enzyme. Also, the rate of acetaldehyde oxidation as compared to glyceraldehyde oxidation is considerably higher with crude than with crystalline d -glyceraldehyde 3-phosphate dehydrogenase. However, most of our data suggest that d -glyceraldehyde 3-phosphate dehydrogenase may be the only DPN-linked aldehyde dehydrogenase in rabbit muscle. d -Glyceraldehyde 3-phosphate dehydrogenase, especially our crude preparation, has many properties in common with Rackers (10) preparation of liver aldehyde dehydrogeanse. However, Rackers (10) liver aldehyde dehydrogenase, which was somewhat purified, has not been shown to oxidize d -glyceraldehyde 3-phosphate. Investigations by various authors also indicate some other differences between aldehyde dehydrogenase from liver and d -glyceraldehyde 3-phosphate dehydrogenase from muscle. A further comparison of the two enzymes is warranted. d -Glyceraldehyde 3-phosphate dehydrogenase is strongly inhibited by tetraethylthiuram disulfide. The drug appears to compete with the substrate ( d -glyceraldehyde 3-phosphate, d -glyceraldehyde, acetaldehyde) for the enzyme. The apparent dissociation constant for the enzyme-inhibitor complex is 5 × 10−6, M.

[137] Liver catalase

Publisher Summary This chapter discusses the determination of liver catalase. Next to urease, beef liver catalase is possibly the easiest enzyme to obtain in crystalline condition. The reasons for this are the unusual stability of this enzyme, its insolubility in water at its isoelectric point, and its relatively high concentration in beef liver. Since the preparation of crystalline catalase from beef liver by Sumner and Dounce crystalline catalases have been obtained from a number of other sources. These are: lamb liver, horse liver, beef erythrocytes, horse kidney and human liver, guinea pig liver, Micrococcus lysodeikticus, and pig liver. Catalase can be purified by being adsorbed on tricalcium phosphate in a Tswett column at pH 5.7 and later by eluting with phosphate buffer of pH 8.0. However, since most preparations of tricalcium phosphate are rather impermeable to water, it is probably better to adsorb on Celite. The chapter also describes a method for the determination of catalase activity which in all probability is the best yet devised for solutions of pure or partially purified catalase.

General Properties of Enzymes

This chapter focuses on enzymes and their general properties. An enzyme, or ferment, is a definite chemical substance of organic nature, thermolabile and elaborated by plants, animals and microorganisms, and capable of increasing the velocity of a chemical reaction without being used up in the process, or becoming a part of the product formed. Many enzymes are simple proteins, while others, especially those concerned with oxidation, are conjugated proteins, where the apoenzyme or protein part is either loosely or firmly bound to some such prosthetic group as hematin, flavin adenine dinucleotide, riboflavin phosphate, cocarboxylase, and pyridoxal phosphate. Enzymes can be classified into four major groups on the basis of the reactions that they catalyze: (1) enzymes that catalyze the addition or removal of water, (2) enzymes that catalyze the transfer of electrons, (3) enzymes that transfer a radical from one molecule to another, and (4) enzymes that split or form a C—C bond without group transfer.

Sulfhydryl Compounds and Crystalline Urease

Perlzweig 1 states that both crystalline urease and extracts of jack bean meal give a positive sulfhydryl test with nitroprusside and that the more active preparations give stronger tests than the weaker ones. He estimates one of his samples of jack bean meal to contain the equivalent of 70 mg. of glutathione per 100 gm. and believes the substance in the meal to be glutathione, rather than cysteine, since the material is negative to the Sullivan reaction until after hydrolysis. He suggests that urease activity is due in part to the sulfhydryl compound and that it may be possible to separate a sulfhydryl activator from the urease proper. We have independently observed that urease, prepared from jack bean meal by the Sumner procedure and twice recrystallized from 30% alcohol, produces a moderate amount of red color when treated with nitroprusside, ammonium sulfate and ammonia, and had ascribed this reaction to the presence of sulfhydryl groups attached to the urease molecule. It was recently shown that no co-enzyme for urease is present in the soy bean, or the jack bean. 2 However, in view of the statements of Perlzweig we have undertaken experiments to ascertain the connection between crystalline urease and the sulfhydryl groups. Perlzweig refers to studies of Waldschmidt-Leitz and his students upon the significance of sulfhydryl compounds in enzymatic reactions. 3 Attention should be called to the fact that the claim made by Waldschmidt-Leitz that liver arginase is wholly inactive in the absence of sulfhydryl compounds, while it is active in the presence of hydrogen sulfide, cysteine, or reduced glutathione has been vigorously denied by Edlbacher, Kraus, and Walter, 4 as well as by Klein and Ziese, 5 who claim that arginase is never activated by cysteine or by glutathione, but instead is inhibited.

The nature of insoluble urease

Soluble urease is prepared by the authors from the jack bean by the use of two principles: (1) repeated precipitation from slightly acid 30 per cent alcohol by cooling and centrifuging; (2) removal of the two globulins, concanavalin A and B, by allowing them to crystallize out from the solution of urease in dilute aqueous neutral phosphate solution. Urease prepared in this manner has an activity of nearly 30,000 units per gram of dry material. A unit is defined by the authors as the amount of urease capable of producing 1 mg. of ammonia nitrogen from a urea phosphate solution in 5 minutes at 20° C. Contrary to the previous belief of the authors, it is not possible to obtain urease entirely free from carbohydrate by the above mentioned procedure. A large amount of a pentose gum can be washed out during the precipitation by cooling, but small amounts of the gum appear to be firmly bound to some of the protein present. It is possible also that soluble urease is contaminated with traces of the third jack bean globulin, canavalin, although canavalin is insoluble in acid 30 per cent alcohol, the solvent used for extracting urease. The authors have been able to free urease from the last traces of pentose gum, and also from any canavalin that might be present, by means of a third new principle, the conversion of urease into an insoluble, though still active form. This is effected by adding small amounts of sodium chloride to1 neutral 30 per cent alcohol urease, and allowing the solution to stand in a cool place for one or two days. The alcohol and sodium chloride are not added until after the crystallizable globulins have been removed and the material has been filtered.

A study of the kinetics of aconitase inhibition and activation

Abstract 1. 1. The inhibition of aconitase activity by chelating agents has been studied over a wide range of substrate concentrations. The chelating agents, o -phenanthroline, pyrophosphate, 8-hydroxyquinoline, and α,α′-dipyridyl inhibit aconitase activity, whereas EDTA has a slight activating effect. Excess substrate will not reverse the inhibition. 2. 2. Activation experiments indicate that ferrous ions and substrate may combine independently with the enzyme. 3. 3. Aconitase can utilize a ferrous-aconitate chelate as substrate and shows greater activity in the presence of this chelate than in the presence of substrate alone. A ferrous-isocitrate chelate can also be utilized but the enzyme has an equal or higher affinity for free isocitrate than the isocitrate-iron chelate.

Parallel Adsorption of Crystalline Pepsin and Peptic Activity Upon Casein and Ovalbumin.

Waldschmidt-Leitz and Kofrányi 1 found that by repeatedly shaking a solution of crystalline pepsin with small quantities of suspension of cantaloupe seed globulin they could adsorb the peptic activity while the crystalline pepsin-protein was left in solution. They accordingly claim that Northrops 2 crystalline pepsin is simply a case of an enzyme adsorbed upon a protein. Their evidence for the non-adsorption of the pepsin-protein was obtained by determining the weight of dry matter left after centrifuging off the suspended material and by blank determinations made with cantaloupe globulin alone. It is unfortunate that these authors did not employ a specific test for the protein of crystalline pepsin, for their centrifuged solutions may have contained not only crystalline pepsin, but also cantaloupe globulin in quantity not indicated by blank determinations, as well as digestion products. By the employment of a test which is specific for crystalline pepsin-protein, provided other proteins are not present in the solution, I have been able to demonstrate that when the peptic activity of a solution of crystalline pepsin is completely adsorbed upon casein or coagulated ovalbumin the pepsin-protein is also completely adsorbed. Furthermore, I have found that the rates of adsorption of peptic activity and of pepsin-protein are the same. These findings conclusively disprove the work of Waldschmidt-Leitz and Kofrányi unless one is willing to make the highly improbable assumption that crystallized cantaloupe seed globulin possesses some peculiar action upon pepsin which is not exerted by casein or by denatured ovalbumin. Casein and coagulated ovalbumin were chosen as adsorbents because of their insolubility at pH 4.8. Edestin could not be used since it was too soluble below pH 5 and it was not desirable to subject pepsin to solutions less acid than this. Coagulated edestin formed large lumps which could not be pipetted in suspension. The casein was prepared by washing 10 gm. of Mercks casein repeatedly with dilute acetate buffer of pH 4.8. The casein was then mixed with 90 cc. of water and 10 cc. of 0.5 M acetate buffer of pH 4.8. The ovalbumin was prepared by heating 1000 mg. of recrystallized, dialyzed ovalbumin in 90 cc. of water for 15 minutes, cooling and adding 10 cc. of acetate buffer. Both proteins were suspended by shaking just before pipetting. The casein was lumpy and difficult to pipette.

The role of the reducing agent in the activation of aconitase

Abstract When ferrous ions are added to citrate, there is an increased absorbance at 240 mμ. Paralleling this increase in optical density, ferrous ions are oxidized to ferric ions at pH 4.6. The same results were obtained at pH 7.4. The presence of a reducing agent will inhibit this non-enzymic reaction at pH 4.6 and pH 7.4. The application of these data to aconitase activation suggests that the primary function of the reducing agent is to maintain iron in the divalent state.

The Copper Enzymes

This chapter focuses on enzymes that contain copper such as tyrosinase, laccase, and ascorbic acid oxidase. Tyrosinase is found in plants, molds, crustacea, and molluscs. It occurs in bacteria. The mealworm, Tenebrio mollitor, is a good animal source. Potatoes, dahlia bulbs, wheat bran, and certain mushrooms are good vegetable sources. Tyrosinase and other similar enzymes are present in higher animals and human skin. It is an aerobic oxidase and in the presence of gaseous oxygen, it catalyzes the oxidation of tyrosine and various phenols. Laccase is a polyphenol oxidase and it is found in the latex of the lac tree. It is stated to occur in many plants and fungi. It is present in potatoes, sugar beets, apples, cabbages, and various mushrooms. In the presence of oxygen, laccase oxidizes ortho and para dihydric phenols. Laccase does not act upon monohydric phenols and resorcinol. It oxidizes ascorbic acid and p-phenylenediamine and is not poisoned by carbon monoxide.

Dehydrogenases Containing Coenzymes I and II

This chapter focuses on dehydrogenases containing coenzymes I and II. Dehydrogenases are enzymes that start the series of reactions that lead ultimately to the formation of water. The dehydrogenases act specifically upon certain substrates to remove hydrogen and thereby bring about oxidation. Some of the dehydrogenases can transfer hydrogen directly to gaseous oxygen. Coenzymes I and II are known by a variety of names. The term coenzyme I is synonymous with cozymase, codehydrogenase I (Co I), and diphosphopyridine nucleotide (DPN). Coenzyme II is synonymous with codehydrogenase II (Co II) and triphosphopyridine nucleotide (TPN). Coenzyme I is almost universally present in living things. Coenzyme I functions as the coenzyme for many dehydrogenases. When such a dehydrogenase catalyzes the oxidation of its substrate, coenzyme is reduced by the hydrogen from the substrate. Coenzyme II acts in enzymic reactions by taking up two atoms of hydrogen. It is the pyridine residue that gives the compound this property.