EZ-FIT: A practical curve-fitting microcomputer program for the analysis of enzyme kinetic data on IBM-PC compatible computers

Perrella, Frank W.

doi:10.1016/0003-2697(88)90042-5

Cited by 275 publications

(139 citation statements)

References 30 publications

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“…Analyses of Data-Kinetic data were analyzed with the EZ-FIT enzyme kinetic model-fitting program (62). Statistical analyses were performed with SigmaPlot software.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

Park

O'Brien

Carman

2003

Journal of Biological Chemistry

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The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C. We examined the hypothesis that Ser 36 , Ser 330 , Ser 354 , and Ser 454 , contained in a protein kinase C sequence motif in CTP synthetase, were target sites for the kinase. Synthetic peptides containing a phosphorylation motif at these serine residues served as substrates for protein kinase C in vitro. Ser 3 Ala (S36A, S330A, S354A, and S454A) mutations in CTP synthetase were constructed by site-directed mutagenesis and expressed normally in a ura7 ura8 double mutant that lacks CTP synthetase activity. The CTP synthetase activity in extracts from cells bearing the S36A, S354A, and S454A mutant enzymes was reduced when compared with cells bearing the wild type enzyme. Kinetic analysis of purified mutant enzymes showed that the S36A and S354A mutations caused a decrease in the V max of the reaction. This regulation could be attributed in part by the effects phosphorylation has on the nucleotide-dependent oligomerization of CTP synthetase. In contrast, CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of purified enzyme showed that the S330A mutation caused an elevation in the V max of the reaction. In vitro data indicated that phosphorylation of CTP synthetase at Ser 330 affected the phosphorylation of the enzyme at another site. The phosphorylation of CTP synthetase at Ser 36 , Ser 330 , Ser 354 , and Ser 454 residues was physiologically relevant. Cells bearing the S36A, S354A, and S454A mutations had reduced CTP levels, whereas cells with the S330A mutation had elevated CTP levels. The alterations in CTP levels correlated with the regulatory effects CTP has on the pathways responsible for the synthesis of the membrane phospholipid phosphatidylcholine.In the yeast Saccharomyces cerevisiae CTP synthetase catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP (1, 2). GTP stimulates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (2-5). URA7 (6) and URA8 (7) are duplicate genes that code for CTP synthetase in S. cerevisiae. The yeast CTP synthetase enzymes (6, 7) contain a conserved glutamine amide transfer domain (see Fig. 1) common to CTP synthetases from other organisms (8 -16). The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme (17) and is responsible for the majority of the CTP synthesized in vivo (7). Neither the URA7 nor the URA8 gene is essential as long as cells possess one functional CTP synthetase gene (6, 7). CTP synthetase is an indispensable enzyme because its reaction product CTP is essential for the synthesis of nucleic acids and membrane phospholipids (18). The importance of understanding the regulation of CTP synthetase is emphasized by the fact that unregulated levels of its activity is a common property of various cancers in humans (19 -26).The yeast CTP synthetase is regulated by genetic and biochemical m...

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“…Analyses of Data-Kinetic data were analyzed with the EZ-FIT enzyme kinetic model-fitting program (62). Statistical analyses were performed with SigmaPlot software.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

Park

O'Brien

Carman

2003

Journal of Biological Chemistry

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“…The measurements were performed in the absence and presence of V 10 , while maintaining the concentrations of other ions (Na + , K + and Mg 2+ ) constant. The experimental data were fitted to the Michaelis-Menten equation by nonlinear regression analysis using EZ-FIT program (Perrella 1988). Maximum enzymatic velocity (V max ) and Michaelis constant (K m ) values were derived by nonlinear regression fitting (hyperbola function) of the experimental data as well as from a Lineweaver-Burk plot and expressed in μmol Pi/h/mg protein and mmol/l of ATP.…”

Section: Kinetic Analysismentioning

confidence: 99%

Influence of decavanadate on rat synaptic plasma membrane ATPases activity

Krstić¹,

Čolović²,

Bošnjaković-Pavlović³

et al. 2009

gpb

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Abstract.The in vitro influence of decameric vanadate species on Na + /K + -ATPase, plasma membrane Ca 2+ -ATPase (PMCA)-calcium pump and ecto-ATPase activity, using rat synaptic plasma membrane (SPM) as model system was investigated, whereas the commercial porcine cerebral cortex Na + /K + -ATPase served as a reference. The thermal behaviour of the synthesized decavanadate (V 10 ) has been studied by differential scanning calorimetry and thermogravimetric analysis, while the type of polyvanadate anion was identified using the IR spectroscopy. The concentration-dependent responses to V 10 of all enzymes were obtained. The half-maximum inhibitory concentration (IC 50 ) of the enzyme activity was achieved at (4.74 ± 1.15) × 10 -7 mol/l for SPM Na + /K + -ATPase, (1.30 ± 0.10) × 10 -6 mol/l for commercial Na + /K + -ATPase and (3.13 ± 1.70) × 10 -8 mol/l for Ca 2+ -ATPase, while ecto-ATPase is significantly less sensitive toward V 10 (IC 50 = (1.05 ± 0.10) × 10 -4 mol/l) than investigated P-type ATPases. Kinetic analysis showed that V 10 inhibited Na + /K + -ATPase by reducing the maximum enzymatic velocity and apparent affinity for ATP (increasing K m value), implying a mixed mode of interaction between V 10 and P-type ATPases.

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“…Glc released was measured by the Glc oxidase method as described previously (Hrmova et al, 1998b). Kinetic data were processed by a proportional weighted fit using a nonlinear regression analysis program based on Michaelis-Menten model equations (Perella, 1988 …”

Section: Steady State Kinetic Analysesmentioning

confidence: 99%

“…Residual enzyme activity was monitored spectrophotometrically at 410 nm as specified above. The K i constant was determined by a proportional weighted fit using a nonlinear regression analysis program (Perella, 1988) as described previously (Hrmova et al, 1998b). …”

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confidence: 99%

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

et al. 2002

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Family 3 ␤ -D -glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of ␤ -D -glucosyl residues from nonreducing termini of a range of ␤ -D -glucans and ␤ -D -oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley ␤ -D -glucan glucohydrolase in complex with nonhydrolyzable S -glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite ؊ 1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite ؉ 1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite ؉ 1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent ␤ -D -glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development. INTRODUCTION␤ -D -Glucan glucohydrolases have been purified and characterized from barley seedlings, maize coleoptiles, soybean cultures, Acacia cells, nasturtium cells, and cultured tobacco cells (Cline and Albersheim, 1981;Nari et al., 1982;Lienart et al., 1986;Labrador and Nevins, 1989;Hrmova et al., 1996;Kotake et al., 1997;Crombie et al., 1998;Kim et al., 2000;Koizumi et al., 2000). They can hydrolyze glycosidic linkages in several ␤ -D -glucans, in ␤ -D -oligoglucosides containing (1 → 2)-, (1 → 3)-, (1 → 4)-, or (1 → 6)-linkages, in aryl ␤ -D -glucosides such as 4 Ј -nitrophenyl ␤ -D -glucopyranoside (4NPGlc), and in some ␤ -D -oligoxyloglucosides (Crombie et al., 1998;Hrmova and Fincher, 1998;Kim et al., 2000). The barley ␤ -D -glucan glucohydrolases also hydrolyze cyanogenic ␤ -D -glucosides, albeit with low activity (M. Hrmova and G.B. Fincher, unpublished data). Single Glc molecules are released from the nonreducing termini of these substrates, with retention of the anomeric configuration (Hrmova et al., 1996). Their broad substrate specificity makes it difficult to assign these higher plant ␤ -D -glucan glucohydrolases to current Enzyme Commission classes; therefore, they have been described variously as ␤ -D -glucan glucohydrolases, (1 → 3)-␤ -D -glucan exohydrolases, and ␤ -D -glucosidases. Nevertheless, they can be classified according to the structural criteria of Henrissat (1991) and fall into the family 3 group of glycoside hydrolases (http://afmb.cnrsmrs.fr/CAZY/).The distribution of the broad-specificity ␤ -D -glucan glucohydrolases in various tissues of monocotyledons and dicotyledons, together with the presence of expressed sequence tags in gymnosperm sequence databases (e.g., Pinus taeda ), suggests that they may play a fundamental role in plant growth and development...

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EZ-FIT: A practical curve-fitting microcomputer program for the analysis of enzyme kinetic data on IBM-PC compatible computers

Cited by 275 publications

References 30 publications

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

Influence of decavanadate on rat synaptic plasma membrane ATPases activity

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

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