End group analysis of the cytosolic and mitochondrial fumarases from rat liver

Some molecular properties of the cytosolic and mitochondrial fumarases were compared. The carboxyl(C)-terminal amino acid of both the cytosolic and mitochondrial fumarases of rat liver cell was identified as leucine by using carboxypeptidase (CPase) A. As the amino(N)-terminal amino acid of both the cytosolic and mitochondrial fumarases could not be identified by the dansyl chloride method or by the cyanate method, the N-termini of these two fumarases seems to be masked. Both fumarases, after S-carboxymethylation, were completely digested with pronase E and CPase A and B, and the amino acids with blocked amino group were analyzed by high voltage paper electrophoresis and amino acid analysis after acid hydrolysis of these amino acid derivatives. The N-termini of the mitochondrial and cytosolic fumarases were identified as pyroglutamic acid and N-acetylalanine, respectively. To compare the primary structures of the two fumarases in detail, each fumarase was digested with an arginine-specific protease or cleaved with cyanogen bromide. The electrophoretic profiles of the digests of these fumarases were indistinguishable from each other.     

Purification and properties of phosphorylase from baker's yeast

A rapid, reliable method for purification of phosphorylase, yielding 200-400 mg pure phosphorylase from 8 kg of pressed baker's yeast, is described. The enzyme is free of phosphorylase kinase activity but contains traces of phosphorylase phosphatase activity. Phosphorylase constitutes 0.5-0.8% of soluble protein in various strains of yeast assayed immunochemically. The subunit molecular weight (Mr) of yeast phosphorylase is around 100,000. The enzyme is composed of two subunits in various ratios, differing slightly in molecular weight and N-terminal sequence. Both are active. Only the enzyme species containing the larger subunit can form tetramers and higher oligomers. The activated enzyme is dimeric. Correlated with specific activity (1 to 110 U/mg), phosphorylase contained between less than 0.1 to 0.74 covalently bound phosphate per subunit. Inactive forms of phosphorylase could be activated by phosphorylase kinase and [gamma-32P]ATP with concomitant phosphorylation of a single threonine residue in the aminoterminal region of the large subunit. The small subunit was not labeled. The incorporated phosphate could be removed by yeast phosphorylase phosphatase, resulting in loss of activity of phosphorylase, which could be restored by ATP and phosphorylase kinase.     

Purification and properties of three endopeptidases from baker's yeast

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45,000 and 54,000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7-3.2 and casein at pH 2.4-2.8 and 5.5-6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4-6 M urea. Urea also stimulated the enzyme activity by 30-50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X-Tyr and X-Phe type. A proteinase B (PRB) preparation of approximately Mr 33,000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at -20 degrees C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72,000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15-Tyr16 peptide bond of the oxidized beta-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at -20 degrees C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of saccharopine dehydrogenase from baker's yeast.

Saccharopine dehydrogenase (N6-(glutar-2-yl)-L-ly-sine:NAD oxidoreductase (L-lysine-forming)) from baker's yeast was purified to homogenicity. The overall purification was about 1,200-fold over the crude extract with a yield of about 24%. The purified enzyme had a sedimentation coefficient (S20,w) of 3.0 S. The molecular weight determinations by sedimentation equilibrium, Sephadex G-100 gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a value of about 39,000 and, therefore, saccharopine dehydrogenase is a single polypeptide chain enzyme. A Stokes radius of 27 A and a diffusion constant of 7.9 X 10(-7) cm2 s-1 were obtained from Sephadex gel filtration chromatography. The enzyme had a high isoelectric pH of 10.1. The NH2-terminal sequence was Ala-Ala----. The enzyme possessed 3 cysteine residues/molecule; no disulfide bond was present. Incubation of saccharopine dehydrogenase with p-chloromercuribenzoate or iodoacetate resulted in complete loss of enzyme activity. Whereas the coenzyme and substrates were ineffective in protecting from inactivation by p-chloromercuribenzoate, iodoacetate inhibition was protected by excess coenzyme.     

Purification of transketolase from baker's yeast by an immunoadsorbent

Baker's yeast transketolase has been purified by immunoaffinity chromatography on specific TK antibodies covalently linked to CNBr-agarose. Affinity chromatography allows a 480-fold purification of TK from yeast extracts and about 80% recovery of the original activity. The isolated enzyme has a specific activity of 12-60 U/mg and during polyacrylamide gel electrophoresis performed at pH 8.9 migrates as two protein bands possessing a transketolase activity which corresponds to two isoforms of the enzyme.     

Purification and properties of glyoxylate reductase I from baker's yeast.

The purification and properties of NADPH-linked glyoxylate reductase [EC 1. 1. 1. 79] from baker's yeast were studied. Two active fractions (peak I and peak II) were isolated by DEAE-cellulose column chromatography. The peak I fraction was purified to homogeneity by the criteria of disc gel electrophoresis and tentatively designated glyoxylate reductase I. Its molecular weight was calculated to be 31,000 from gel filtration measurements. The enzyme reduced glyoxylate 7 times faster than hydroxypyruvate and was specific for NADPH. The enzyme showed optimum activity between pH 5.5 and 7.2. The Michaelis constants for glyoxylate and NADPH were found to be 13 mM and 4 microM, respectively. The enzymic activity was not significantly affected by anions, except for nitrate and iodide, which were inhibitory.     

Arginyl-tRNA synthetase from baker's yeast. Purification and some properties.

Arginyl-tRNA synthetase from baker's yeast (Saccharomyces cerevisiae, strain 836) was obtained pure by a large-scale preparative method, which involves four chromatographic columns and one preparative polyacrylamide gel electrophoretic step. The enzyme has a high specific activity (9000 U/mg) and consists of a single polypeptide chain of molecular weight approximately 73000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Amino acid analysis of the enzyme permitted calculation of the absorption coefficient of arginyl-tRNA synthetase (A(1 mg/ml 280 nm)=1.26). Concerning kinetic parameters of the enzyme we found the following Km values: 0.28 muM, 300 muM, 1.5 muM for tRNA(Arg III), ATP and arginine in the aminoacylation reaction, and 1400 muM, 2.5 muM, and 50 muM for ATP, arginine and PP(i) in the ATP-PP(i) exchange reaction. Arginyl-tRNA synthetase required tRNA(Arg III) to catalyse the ATP-PP(i) exchange reaction.     

Purification from baker's yeast of an activator of DNA photolyase.

The activity of purified DNA photolyase from Baker's yeast is enhanced by a compound (Activator (III)) obtained from yeast by chloroform extraction ion exchange chromatography and gel filtration. Thin layer chromatography and spectral data indicate that the compound is homogeneous. Activator III emits at 350 and 440 nm when excited at 290 nm, and emits at 440 nm when excited at 358 nm. After acid hydrolysis, emission at 440 nm is produced only by excitation at 358 nm, indicating that activator (III) contains two separate chromophoric moieties. The chromophore excited by 358 nm light has a pK of 9-11, while the other chromophore has a pK of 4-5, and possibly of 9-11. The enhancement of photolytic activity by activator (III) at a concentration equimolar with that of the enzyme and the similarity of the fluorescent spectra of the activator with that of heat-denatured photolyase, suggests that the activator may be the chromophore associated with the enzyme.     

AMP deaminase from baker's yeast. Purification and some regulatory properties.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.     

Purification and properties of tryptophan synthase from baker's yeast (Saccharomyces cerevisiae).

Tryptophan synthase was purified from baker's yeast. The purified enzyme exhibited one band on polyacrylamide-gel electrophoresis, had no detectable N-terminal amino acid and C-terminal alanine. The amino acid composition was close to that predicted by recent studies on the DNA sequence of the structural gene for the enzyme. Kinetic parameters for the following three activities were measured: indole-serine condensation, indolylglycerol phosphate lyase and the overall reaction of serine with 1-(indol-3-yl)glycerol 3-phosphate. The Km for indole was much lower than suggested by previous investigations, and the value of 11 microM was measured by a fluorimetric assay.     

Purification and properties of a major cytochrome b peptide from baker's yeast.

A major cytochrome b peptide was purified from yeast mitochondria by a procedure involving solubilization in deoxycholic and cholic acids, ammonium sulfate fractionation, proteolytic digestion, and sucrose gradient centrifugation in the presence of Tween 80. The homogeneity of the purified protein was established by the criteria that the product was spectrally pure and yielded a single band on both sodium dodecyl sulfate polyacrylamide gel electrophoresis, and by gel isoelectric focusing. The purified cytochrome b polypeptide had absorption maxima at 562, 532, and 430 nm in the reduced form and at 525 to 570 nm and 419 nm in the oxidized form. The reduced minus oxidized difference spectra revealed absorption bands at 562, 532, and 430 nm at room temperature and 559, 529, and 429 nm at 77 K, respectively. The heme group was identified as protoheme by formation of the reduced pyridine hemochromogen. Treatment of the reduced form with carbon monoxide affected the absorption spectrum, indicating that the isolated hemoprotein was modified compared to native cytochrome b. The apparent molecular weight of the preparation was 28,000 based on sodium dodecyl sulfate polyacrylamide-gel electrophoresis and 28,800 based on sucrose gradient centrifugation. The isolated cytochrome b polypeptide showed a strong tendency to aggregate.