Arthur Kornberg

Why purify enzymes

Publisher Summary This chapter explains how an enzyme catalyzes the conversion of one substance to another, and then enzyme purified away from the other enzymes and substances that make up a crude cell extract. It describes the classical approach to enzymology. This approach tracks the molecular basis of cellular function—alcoholic fermentation in yeast, glycolysis in muscle, luminescence in a fly, or the replication of DNA—by first observing the phenomenon in a cell-free system. The responsible enzyme is then isolated by fractionation of the cell extract and is purified to homogeneity. The structure and the regulatory and catalytic functions of the enzyme are then studied. The reverse approach is the neoclassical approach, which first obtains a structure and then looks for its function. The protein is preferably small and stable and is amplified by cloning or is commercially available.

[76] Adenosine phosphokinase:

Publisher Summary This chapter discusses the determination of adenosine phosphokinase. In the process of assay, the phosphorylation of the nucleoside is initiated with small quantities of ATP which are immediately regenerated by the action of pyruvate phosphokinase (added in large excess) on phosphopyruvate. In the presence of myokinase, this leads to the accumulation of the nucleoside mono-, di-, and triphosphates. Thus, for every mole of ATP present 2 moles of TPN are reduced, and for every mole of ADP present 1 mole of TPN is reduced. The extent of TPN reduction can be measured at 340 mμ; the molecular extinction coefficient for TPNH of 6.22 X 10 6 sq. cm./mole is employed. In the purification procedure, Twenty-five grams of dried lager beer yeast, which yielded the most active autolyzate, was suspended in 75 ml. of 0.1 M sodium bicarbonate and incubated for 6 hours at 34°. The mixture was centrifuged and yielded approximately 40 ml. of clear yellow autolyzate.

[124] Isolation of diphosphopyridine nucleotide and triphosphopyridine nucleotide

Publisher Summary Diphosphopyridine nucleotide (DPN) is extracted from yeast with hot water. After removal of other substances from the extract by basic lead acetate, DPN is precipitated as a silver salt. This is decomposed with hydrogen sulfide, and the DPN is obtained as the free acid by precipitation with acetone. Further purification is achieved by anion exchange chromatography. The purity is determined by measurement of the absorption at 340 mμ of the enzymatically reduced product. The extinction coefficient of reduced DPN at 340 mμ is 6.22 × l0 6 cm. per mole. Reduction is achieved by the use of ethanol and yeast alcohol dehydrogenase. Triphosphopyridine nucleotide (TPN) can be isolated horse erythrocytes and liver of hog, including fractional precipitation of mercury, lead, and barium salts, as well as fractional extraction with methanol. TPN has also been obtained by charcoal adsorption and purification by charcoal chromatography. The concentration of TPN is determined by measurement of the absorption at 340 mμ of the enzymatically reduced product.

[116] DPN pyrophosphorylase: NMN+ATP⇋DPN+PP

This chapter discusses the determination of DPN pyrophosphorylase. The assay method is based on the initial rate of formation of DPN starting with a large excess of ATP and NMN. DPN is measured spectrophotometrically after its total reduction by the alcohol dehydrogenase system. The extinction coefficient of 6.22 X 10 6 cm./mole at 340 m/μ is used. One unit of enzyme activity is defined as the amount causing the synthesis of 1 μM. of DPN per hour, and specific activity as units per milligram of protein. Proportionality to enzyme concentration was observed in this test with crude as well as with purified preparations when 1 unit or less was present in the test system. Protein concentration is determined by a nephelometric method with the Beckman spectrophotometer at 340 mμ. The enzyme is purified from both liver and yeast, but most extensively from the latter. Only the preparation of the liver enzyme is described because the starting material is more uniform and more readily obtained and the most purified fraction, although only 5% as pure (on a protein basis) as the purified yeast enzyme, is more stable and is adequate for equilibrium studies.

[117] FAD pyrophosphorylase: FMN+ATP⇋FAD PP

Publisher Summary This chapter discusses the determination of FAD pyrophosphorylase. The assay method is based on the initial rate of formation of FAD, starting with ATP and FMN. The FAD is determined by the method of Warburg and Christian that involves the measurement of O 2 consumption as a function of FAD concentration in the oxidation of DL-alanine by D-amino acid oxidase. ATP served not only as a substrate but also as a competitive inhibitor of FAD hydrolysis by the nucleotide pyrophosphatase present in the various enzyme fractions. One unit of enzyme is defined as that amount causing the synthesis of 1 millimicromole of FAD per hour and specific activity as units per milligram of protein. Protein concentration was determined by a nephelometric method with the Beckman spectrophotometer at 340 mμ. In the course of purification procedure, one hundred grams of dried beer yeast is autolyzed with 300 ml. of 0.1 M sodium bicarbonate (saturated with a mixture of 95% N 2 and 5% CO 2 ) for 24 hours at 23°. All subsequent operations, including storage of solutions, are carried out at 3°, unless otherwise specified. Since the most purified enzyme fractions contained high concentration of nucleotide pyrophosphatase and inorganic pyrophosphatase, specific inhibitors are employed to avoid their interference in balance studies of FAD synthesis and pyrophosphorolysis.

The private life of DNA polymerase I.

Publisher Summary This chapter describes DNA polymerase and discusses the purification of the miniscule activity responsible for DNA synthesis. In the initial search for DNA synthesis in cell-free extracts, DNA was included in the reaction mixture so that it could serve as templates for replication. Several fractions prepared from the crude extract were needed for incorporation of thymidine. Of these, two supplied the nucleases and kinases that generated the missing dNTPs and another the polymerase. From cell extracts prepared by sonic disruption, nucleic acids had to be removed and this was accomplished by a refined precipitation with streptomycin sulfate followed by digestion with DNase. One of the inferences drawn from the replication of a single-stranded, circular template was that DNA polymerase could start a new chain. All three polymerases, although differing significantly in structure, proved to be virtually identical in their mechanisms of DNA synthesis, proofreading, and use of the same building blocks.

[65] Nicotinamide riboside phosphorylase:

Publisher Summary This chapter discusses the nicotinamide riboside phosphorylase. Nicotinamide riboside but not nicotinamide yields a fluorescent condensation product with acetone; the cleavage of nicotinamide riboside is measured by this fluorometric method. The incubation mixture (0.50 ml.) contained 0.05 ml. of NR, 0.10 ml. of phosphate buffer, enzyme (0.5 to 0.7 unit), and water. After 10 minutes at 38°, the reaction was stopped b y a twentyfold dilution with ice-cold water and an aliquot of 0.20 ml. is immediately assayed fluorometrically. One unit of enzyme is defined as that amount which causes the cleavage of 1 micromole of N R in 1 hour. Specific activity is expressed as units per milligram of protein. For the purification procedure, ten grams of beef liver acetone powder is extracted with 100 ml. of 0.1 M Na 2 HPO 4 for 10 minutes with gentle shaking. This and subsequent operations are carried out at 2°. The purified preparation did not attack NMN , DPN , TPN, or methylnicotinamide, nor are there any inhibitory effects of these compounds at equimolar concentration on the rate of NR splitting. The purified enzyme fractions are active in the phosphorolysis of inosine, and, in addition, inosine markedly inhibited NR phosphorolysis.

[131] Preparation of coenzyme A

Publisher Summary In the preparation of coenzyme A, it is extracted from yeast with hot water, chromatographed on charcoal, and precipitated with acetone. The crude preparation is purified by ion exchange chromatography, concentrated on charcoal, and precipitated with acetone, giving a product of 50 to 65 % purity in a yield of 50 to 55 %. It is pointed out that CoA samples of lesser purity prepared from the fractions just preceding the peak fractions from the Dowex column (i.e., the material with a CoA/adenine ratio of less than 0.6) do contain significant amounts of ATP. No reductive steps are employed in the chromatographic procedure, the CoA isolated present mainly as the mixed disulfide derivatives of other sulfhydryl compounds. Paper chromatography of the isolated material in an 80% phenol-20% water solvent shows that the CoA moves to a spot identical with a ninhydrin-reactive material. After reduction of the CoA with hydrogen sulfide it moves with an Rf of 0.51 ; the amino compound has an Rt of 0.46, which is identical with that found for glutathione. These observations suggest that the CoA isolated may be present largely as the mixed disulfide derivative of glutathione.

Chapter 1 Why Purify Enzymes

Publisher Summary The chapter discusses the importance of purifying enzymes. Purifying an enzyme is rewarding all the way, from first starting to free it from the mob of proteins in a broken cell to having it finally in splendid isolation. To attain the goal of a pure protein, the cardinal rule is that the ratio of enzyme activity to the total protein is increased to the limit. Units of activity and amounts of protein must be strictly accounted for, in each manipulation and at every stage. With the purified enzyme, it is easy to learn about its catalytic activities and its responsiveness to regulatory molecules that raise or lower activity. Beyond the catalytic and regulatory aspects, enzymes have a social face that dictates crucial interactions with other enzymes, nucleic acids, and membrane surfaces. To gain a perspective on the enzymes contributions to the cellular economy, it is also important to identify the factors that induce or repress the genes responsible for producing the enzyme. Tracking a metabolic or biosynthetic enzyme uncovers marvelous intricacies by which a bacterial cell gears enzyme production precisely to its fluctuating needs.