Browsing by Author "EYZAGUIRRE, J"

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    AFFINITY LABELING OF RABBIT MUSCLE PYRUVATE-KINASE WITH DIALDEHYDE-ADP
    (1982) HINRICHS, MV; EYZAGUIRRE, J
    Periodate-oxidized ADP (dialdehyde-ADP) inactivates rabbit muscle pyruvate kinase and combines irreversibly to the enzyme. This inactivation is first-order with respect to dialdehyde-ADP and follows saturation kinetics, indicating that the enzyme first forms a reversible complex with the inactivator. Low Mg2+ concentrations stimulate the rate of inactivation, while higher concentrations have a protective effect. ADP and ATP, especially in the presence of Mg2+, protect very strongly against inactivation, while phosphoenolpyruvate and pyruvate are less effective. Dialdehyde-ADP is not a substrate, but acts as competitive inhibitor of ADP, with a KI of 4.5 mM. The analog has somewhat lower affinity to the enzyme than Mg-ADP, which has a Kd of 1.2 mM. Based on kinetic data, 1 molecule of reagent must combine per enzyme active site in order to inactivate the enzyme. Incorporation of [14C]dialdehyde-ADP into the enzyme and treatment of the data by the Tsou plot shows that 6-7 residues/subunit react with the modifier, 2 of them being essential for activity. Dialdehyde-ADP behaves as an affinity label of rabbit muscle pyruvate kinase, the inactivator binds probably to lysine residues at or near the active site, forming morpholine-like structures; and the enzyme possesses 2 modifiable groups essential for activity, the reaction of one of them being sufficient to cause total loss of activity.
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    BETA-GLUCOSIDASE FROM PENICILLIUM-PURPUROGENUM - PURIFICATION AND PROPERTIES
    (1992) HIDALGO, M; STEINER, J; EYZAGUIRRE, J
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    CHEMICAL MODIFICATION OF ARGINYL RESIDUES IN RABBIT MUSCLE PYRUVATE-KINASE
    (1981) CARDEMIL, E; EYZAGUIRRE, J
    Rabbit muscle pyruvate kinase was inactivated with the arginine-specific reagent 2,3-butanedione, in borate buffer, following psuedo-first-order kinetics. Graphical representation of the kobs as a function of inactivator concentration follows a straight line which is compatible with the following mechanism: .**GRAPHIC**. Values for k1 = 4.6 M-1 min-1 and k1 = 0.0022 min-1 can be estimated. The order of the reaction was near 1, suggesting that the modification of a single amino acid residue of the enzyme is responsible for its inactivation. Almost full catalytic activity was recovered when excess butanedione and borate buffer were removed by Sephadex G-25 gel filtration, which suggests that only arginyl residues were modified. The inactivation was partially prevented by phosphoenolypyruvate in the presence of K+ and Mg2+, but not by the competitive inhibitors lactate and bicarbonate, or ADP, with or without added metal ions. Mg2+ alone increased the rate of inactivation. Quantification of residual arginine residues after chemical modification, using amino acid analysis, gives no precise correlation between the number of residues modified and the residual activity; however, .apprx. 4 residues react per enzyme subunit, showing the presence of reactive, nonessential arginines. One essential arginyl residue is present per subunit in rabbit muscle pyruvate kinase, and this residue is probably located near the binding site of the phosphate group of phosphoenolpyruvate.
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    CHEMICAL MODIFICATION OF ESSENTIAL ARGININES IN PIG-LIVER PHOSPHOMEVALONATE KINASE
    (1982) VERGARA, M; ALVEAR, M; CARDEMIL, E; JABALQUINTO, AM; EYZAGUIRRE, J
    Phosphomevalonate [MVAP] kinase, an enzyme of the polyisoprenoid biosynthesis pathway, catalyzes the transfer of phosphate from ATP to MVAP, with the formation of pyrophosphomevalonate and ADP. The pig liver enzyme, a monomer of MW 22,000, possesses 1 cysteinyl residue, which is essential for catalysis. By means of chemical modification of a partially purified preparation, the participation of arginine residues in the enzyme active site was studied. Butanedione and phenylglyoxal were chosen as group-specific reagents. The kinetics of inactivation by both reagents is rather complex, suggesting that several arginine residues, directly or indirectly related to the active site, are being modified. Both substrates, MVAP and Mg-ATP, protect against inactivation but to a different extent, depending on the modifier used. With butanedione, almost total protection is achieved with Mg-ATP. Better protection with MVAP is observed for the modification with phenylglyoxal, but with this reagent, Mg-ATP protects strongly only at very high concentrations. A reversible inactivation mechanism is followed with butanedione, while for phenylglyoxal this mechanism appears to be irreversible, in agreement with findings by other authors. MVAP kinase probably presents 2 or more arginyl residues in or near its active site, one of them being involved in the binding of Mg-ATP, and at least another located in the neighborhood of the MVAP binding site. A more precise determination of the number of essential residues requires its measurement by chemical methods, utilizing a homogeneous enzyme preparation.
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    CONDITIONS FOR SELECTIVE DEGRADATION OF LIGNIN BY THE FUNGUS GANODERMA-AUSTRALIS
    (1992) RIOS, S; EYZAGUIRRE, J
    The white-rot fungus Ganoderma australis selectively degrades lignin in the ecosystem "palo podrido". Using conditions that simulate those of "palo podrido' in the laboratory, it was found that low nitrogen content and low O2 tension stimulate the production of manganese peroxidase and lignin degradation, and depress cellulose degradation and cellulase production. The inverse is found at high nitrogen concentration and high O2 tension. This agrees with previous results indicating that low O2 tension and low nitrogen stimulate selective lignin degradation by this fungus.
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    CULTURE CONDITIONS FOR ENHANCED CELLULASE PRODUCTION BY A NATIVE STRAIN OF PENICILLIUM-PURPUROGENUM
    (1994) STEINER, J; SOCHA, C; EYZAGUIRRE, J
    A cellulolytic wild-type strain of Penicillium purpurogenum was isolated from a soil sample in southern Chile. It grew best at 28-degrees-C from an inoculum of 4 x 10(7) spores/100 ml medium. Highest endoglucanase activity was with Sigmacell as carbon source and corn steep liquor as nitrogen source. Wheat bran enhanced the production of endoglucanase and beta-glucosidase. The enzymes in the crude supernatants were stable up to 50-degrees-C and between pH 4.4 and 5.6 for 48 h.
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    EVIDENCE OF ESSENTIAL ARGINYL RESIDUES IN RABBIT MUSCLE PYRUVATE-KINASE
    (1979) CARDEMIL, E; EYZAGUIRRE, J
    Rabbit muscle pyruvate kinase [EC 2.7.1.40] is inactivated by 2,3-butanedione in borate buffer. The inactivation follows pseudo-1st-order kinetics with a calculated 2nd-order rate constant of 4.6/M per min. The modification can be reversed with almost total recovery of activity by elimination of the butanedione and borate buffer, suggesting that only arginyl groups are modified. This result agrees with the loss of arginine detected by amino acid analysis of the modified enzyme. Using the kinetic data, it was estimated that the reaction of a single butanedione molecule/subunit of the enzyme is enough to completely inactivate the protein. The inactivation is partially prevented by phosphoenolpyruvate in the presence of K+ and Mg2+, but not by the competitive inhibitors lactate and bicarbonate. These findings point to an essential arginyl residue being located near the phosphate binding site of phosphoenolpyruvate.
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    ISOLATION AND SEQUENCE DETERMINATION OF AN ACTIVE-SITE PEPTIDE OF RABBIT MUSCLE PYRUVATE-KINASE
    (1987) BEZARES, G; EYZAGUIRRE, J; HINRICHS, MV; HEINRIKSON, RL; REARDON, I; KEMP, RG; LATSHAW, SP; BAZAES, S
    Rabbit muscle pyruvate kinase was inactivated by 2'',3''-dialdehyde ADP with the incorporation of one molecule of reagent per enzyme subunit. The inactivated protein was digested with trypsin after reduction and carboxymethylation. The labeled peptide was isolated by gel filtration and further purified by HPLC. The peptide was sequenced both by liquid-phase and gas-phase automatic Edman degradation. A 34-residue peptide was obtained. This peptide labeled with trinitrobenzenesulfonate, isolated and sequenced by Johnson et al. (Biochem. Biophys. Res. Commun. (1979) 90, 525-530) from bovine muscle pyruvate kinase. Available evidence suggests that dialdehyde ADP labels the enzyme at the same lysine in position 25 of the peptide, as found by Johnson et al. The high homology between the isolated peptide and regions of other pyruvate kinases from low to high eukaryotes supports the idea that this peptide is related to the enzyme active site.
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    MULTITEST-II - A PROGRAM FOR THE GENERATION, CORRECTION, AND ANALYSIS OF MULTIPLE-CHOICE TESTS
    (1990) LIRA, P; BRONFMAN, M; EYZAGUIRRE, J
    Multitest II is a powerful program for the generation, correction, and analysis of multiple choice examinations. The program is written in Pascal and has been implemented for the IBM-PC and compatible microcomputers, and for the VAX minicomputer. The program allows the random generation of tests from a master test, and supports several types of multiple choice tests. The tests are graded by means of a user-definable numerical grading system. Certain statistical analyses can also be performed on the tests results.
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    PENICILLIUM-PURPUROGENUM PRODUCES SEVERAL XYLANASES - PURIFICATION AND PROPERTIES OF 2 OF THE ENZYMES
    (1995) BELANCIC, A; SCARPA, J; PEIRANO, A; DIAZ, R; STEINER, J; EYZAGUIRRE, J
    The fungus Penicillium purpurogenum produces several extracellular xylanases. The two major forms (xylanases A and B) have been purified and characterized. After ammonium sulfate precipitation and chromatography in Bio-Gel P 100, xylanase A was further purified by means of DEAE-cellulose, hydroxylapatite and CM-Sephadex, and xylanase B by DEAE-cellulose and CM-Sephadex. Both xylanases showed apparent homogeneity in SDS-polyacrylamide gel electrophoresis. Xylanase A (33 kDa) has an isoelectric point of 8.6, while xylanase B (23 kDa) is isoelectric at pH 5.9. Antisera against both enzymes do not cross-react. The amino terminal sequences of xylanases A and B show no homology. The results obtained suggest that the enzymes are produced by separate genes and they may perform different functions in xylan degradation.
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    PHOTOAFFINITY-LABELING OF PYRUVATE-KINASE FROM RABBIT MUSCLE
    (1988) BAZAES, S; BOSCH, M; SCHAFER, HJ; EYZAGUIRRE, J
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    PIG-LIVER PHOSPHOMEVALONATE KINASE .1. PURIFICATION AND PROPERTIES
    (1980) BAZAES, S; BEYTIA, E; JABALQUINTO, AM; SOLISDEOVANDO, F; GOMEZ, I; EYZAGUIRRE, J
    Pig liver phosphomevalonate kinase (EC 2.7.4.2) was purified to homogeneity as shown by polyacrylamide gel electrophoresis. The MW estimates range from 21,000 to 22,500. Each molecule is composed of 1 polypeptide chain. The presence of SH-containing reagents is essential for preservation of enzyme activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM were obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, 1 cysteine residue/molecule of enzyme, and the absence of methionine. The enzyme apparently plays no regulatory function is cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.
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    PIG-LIVER PHOSPHOMEVALONATE KINASE .2. PARTICIPATION OF CYSTEINYL AND LYSYL GROUPS IN CATALYSIS
    (1980) BAZAES, S; BEYTIA, E; JABALQUINTO, AM; SOLISDEOVANDO, F; GOMEZ, I; EYZAGUIRRE, J
    Phosphomevalonate kinase from pig liver is inactivated by 5,5''-dithiobis(2-nitrobenzoate) and pyridoxal 5''-phosphate. The substrate phosphomevalonate protects the enzyme against inactivation by these reagents. Inactivation by 5,5''-dithiobis(2-nitrobenzoate) is complete and may be reverted by 2-mercaptoethanol or dithiothreitol. Experiments carried out with partially inactivated enzyme show no change in the kcat or in the apparent Km for the substrates, as compared with the native enzyme, indicating the existence of 2 populations of molecules, one intact and the other totally inactive. 5,5''-Dithiobis(2-nitrobenzoate) apparently reacts with the only cysteinyl residue of the enzyme and this residue is located in or near the active site. Inhibition by pyridoxal 5''-phosphate can be reverted, either by dialysis or by the addition of lysine, but not if the partially inactivated enzyme is treated previously with NaBH4, in agreement with the formation of a Schiff base between pyridoxal 5''-phosphate and an amino group of the enzyme. This is further supported by the appearance of an absorption band with a maximum at 325 nm in the enzyme treated with pyridoxal 5''-phosphate and NaBH4. Pyridoxal and pyridoxamine 5''-phosphate are weaker inhibitors than pyridoxal 5''-phosphate, suggesting a specific effect due to the phosphate and aldehyde groups. The enzyme is not completely inactivated by pyridoxal 5''-phosphate, even at a molar ratio of 350, or by a 2nd inactivation treatment after reduction with NaBH4. The partially modified enzyme shows a lower Km for phosphomevalonate than the native enzyme, suggesting that the reactive group is located near the binding site of phosphomevalonate. The lower Km may reflect an effect of the positive charge of the pyridoxal 5''-phosphate ring N, enhancing the binding of phosphomevalonate. Values of 8.15 at 24.degree. C and 7.95 at 31.degree. C were determined for the pK of the reactive group. A .DELTA.Hi of 11.8 kcal/mol was estimated, in agreement with the values expected for an amino group. One amino group/active site is involved in the enzyme inactivation as shown by kinetic data. Quantification of the number of moles of pyridoxal 5''-phosphate bound/mole of enzyme is not conclusive but supports this assertion. This group may correspond to an .epsilon.-amino group of lysine.
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    POSSIBLE INVOLVEMENT OF CYSTEINYL, LYSYL AND ARGINYL GROUPS IN THE ACTIVE-CENTER OF PIG-LIVER PHOSPHOMEVALONATE KINASE
    (1981) BAZAES, S; BEYTIA, E; JABALQUINTO, AM; DEOVANDO, FS; GOMEZ, I; EYZAGUIRRE, J
    Pig liver phosphomevalonate kinase has a MW of about 22,000, its structure is monomeric and it shows only 1 cysteine residue/molecule. The presence of cysteinyl and lysyl residues at the active site was studied by chemical modification. Preliminary work was also performed on the involvement of arginyl residues. Modification of cysteinyl residues was attempted using 5,5''dithiobis (2-notrobenzoate) (DTNB). Pyridoxal phosphate (PLP) was utilized as reagent for lysyl residues, and arginyl residues were modified with butanedione. Inactivation of the enzyme by DTNB is complete but may be reversed by 2-mercaptoethanol or dithiothreitol. Kinetic analysis shows that 1 mol of reactive SH group/mol of enzyme is responsible for catalytic activity. The substrate phosphomevalonate, but not Mg-ATP, protects against inactivation. Since no change in Km or kcat is observed on partial inactivation, this is interpreted as indication that the modified molecule is totally inactive. PLP inactivates the enzyme, but even at high concentrations total inactivation was not observed. Addition of lysine or dialysis reactivates the modified enzyme, but this does not occur after the addition of sodium borohydride. Pyridoxal and pyridoxamine 5''-phosphate are weaker inhibitors than PLP. As with DTNB, phosphomevalonate but not MgADP protects against inactivation. The partially modified enzyme shows a lower Km for phosphomevalonate than the native enzyme. Kinetic data suggest that 1 PLP molecule reacts/enzyme active center, while spectrophotometric titration shows a lower value. pK values 7.2 (at 24.degree. C) and 7.5 (at 31.degree. C) are obtained for the modified group, in agreement with values described for reactive lysines. Inactivation by butanedione can be protected by phosphomevalonate, but only to a smaller extent by Mg-ATP. The cysteinyl residue of the enzyme plays an essential role in the catalytic mechanism, while the lysine residue modified may not be essential for activity. Both residues, and possibly an arginyl group, may be located at or near the phosphomevalonate binding site of the enzyme.
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    PURIFICATION AND CHARACTERIZATION OF AVIAN LIVER MEVALONATE-5-PYROPHOSPHATE DECARBOXYLASE
    (1982) ALVEAR, M; JABALQUINTO, AM; EYZAGUIRRE, J; CARDEMIL, E
    Mevalonate-5-pyrophosphate decarboxylase was purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of MW 85,400 .+-. 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0-6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column choromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM were obtained for mevalonate-5-pyrophosphate and ATP, respectively.
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    PYRUVATE-KINASE - STUDIES ON AFFINITY LABELING AND ACTIVE-SITE STRUCTURE USING THE RABBIT MUSCLE ENZYME
    (1985) EYZAGUIRRE, J; BAZAES, S; BEZARES, G; HINRICHS, MV; HEINRIKSON, RL; REARDON, I
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    YEAST PYRUVATE-KINASE - ESSENTIAL LYSINE RESIDUES IN THE ACTIVE-SITE
    (1988) IMARAI, M; HINRICHSEN, P; BAZAES, S; WILKENS, M; EYZAGUIRRE, J
    Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by trinitrobenzenesulfonate and by P1, P2-bis (5'' pyridoxal) diphosphate. Labeled peptides were isolated and their amino acid composition was determined. The results suggest that yeast pyruvate kinase has an essential lysine residue, and that this residue is in a location equivalent to an essential lysine described in the muscle enzyme. Protection experiments indicate that this lysine is located at the nucleotide binding site.