scholarly journals Purification and properties of the nicotinamide–adenine dinucleotide phosphate-dependent isocitrate dehydrogenase from pig liver cytoplasm

1970 ◽  
Vol 118 (2) ◽  
pp. 253-258 ◽  
Author(s):  
J. A. Illingworth ◽  
K. F. Tipton
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Soluble Fraction ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Turnover Number ◽  
Pig Liver ◽  
Extinction Coefficients ◽  
Purification And Properties

The NADP-dependent isocitrate dehydrogenase from pig liver soluble fraction was purified over 500-fold with an overall yield of 25%. The purified enzyme, which is homogeneous by all the usual criteria, has a molecular weight of about 75000 and is composed of two identical subunits. This has been demonstrated by ultracentrifugation, fluorescence titration and peptide `fingerprinting'. The maximal turnover number, extinction coefficients at 280nm and 260nm and amino acid analysis are described.

scholarly journals The purification and properties of butyryl-coenzyme A dehydrogenase from Peptostreptococcus elsdenii

1971 ◽  
Vol 125 (3) ◽  
pp. 879-887 ◽  
Author(s):  
Paul C. Engel ◽  
V. Massey
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Simple Procedure ◽  
Potassium Bromide ◽  
Prosthetic Group ◽  
Pig Liver ◽  
Peak Absorption ◽  
Purification And Properties ◽  
Green Form ◽  
Spectrophotometric Titrations

Butyryl-CoA dehydrogenase prepared by a simple procedure from Peptostreptococcus elsdenii has a molecular weight of approx. 150000. The enzyme has FAD as its prosthetic group. The amino acid analysis is reported. This enzyme, like most of the corresponding mammalian ones, is green. The absorption band at 710nm can be abolished irreversibly by dithionite reduction and air reoxidation; it can be abolished reversibly by phenylmercuric acetate or potassium bromide. The enzyme as isolated appears to be a mixture of a green and a yellow form, both of which are active. This view is supported by the variable ‘greenness’ of different preparations and the biphasic curve obtained in anaerobic spectrophotometric titrations with dithionite. It can be calculated from the titration results that fully green enzyme would have a peak-to-peak absorption ratio (E710/E430) as great as 0.54. The green form is much less rapidly reduced by dithionite than the yellow form, but is nevertheless much more readily reduced by dithionite than the enzyme from pig liver. It is also more readily reoxidized by air and shows less tendency to form a semiquinone. Treatment with sodium borohydride produces an unusual reduced species that is probably the 3,4-dihydroflavin.

scholarly journals Purification and Properties of Staphylocoagulase

1975 ◽  
Author(s):  
A.D. Muller ◽  
B. M. Bas ◽  
H. C. Hemker
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Composition ◽  
Aspartic Acid ◽  
Acid Composition ◽  
Isoelectric Point ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Terminal Amino

Staphylocoagulase, an exoprotein of coagulase positive staphylocoagulase, has been purified to a state in which only trace amounts of contaminating proteins are detectable.Purification was more than 35,000 fold, which is 7 times more than the highest value reported in the literature. The yield was about 15%.Aspartic acid was found as a single N-terminal amino acid in this preparation. The molecular weight is 61,000 and the isoelectric point lies at pH 4.53.The amino acid composition was determined.

Protein

1995 ◽  
Author(s):  
Roland Lüthy ◽  
David Eisenberg
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Isoelectric Point ◽  
Extinction Coefficient ◽  
Titration Curve ◽  
Side Chains ◽  
Pka Values ◽  
Extinction Coefficients ◽  
Molecular Weights

Given a protein sequence, the amino acid composition can be determined by counting the number of residues of each type. Then a molecular weight can be calculated by summing the molecular weights of the individual amino acid residues, taking into account the loss of one H2O molecule per peptide bond. Table 1 lists the molecular weights of the twenty amino acids and water. This approach assumes that the protein has not been covalently modified. Because of extensive glycosylation of some proteins, this approach can significantly underestimate the actual molecular weight. With the pKa values of Table 1, it is possible to calculate the theoretical charge of a protein at a given pH by summing the charges of the amino acid side chains and of the amino terminus and carboxyl terminus. By performing this calculation over a pH range, one obtains a theoretical titration curve and an isoelectric point (the pH at which the protein hasanetchargeof zero). This method assumes that all normally titratable groups are accessible to water, and that all side chains have the intrinsic pKa values listed in Table 1. This assumption is not completely correct, and consequently, the theoretical isoelectric point may differ from the experimentally determined value. Figure 1 shows the calculated titration curve for pancreatic ribonuclease: the calculated isoelectric point is 8.2, whereas the measured value is 9.6 (Lehninger, 1977). The calculation of extinction coefficients (Gill and von Hippel, 1989) is performed in much the same way as that of the isoelectric point Individual residues are treated as if they are free amino acids, and the overall extinction coefficient is calculated as the sum of the extinction coefficients of the residues. The same basic assumption is made: Residues are assumed to be in typical environments and not to show unusual absorption due to their local environments. In the case of the extinction coefficient, however, this assumption seems to be generally acceptable; calculated extinction coefficients are typically within a few percent of the experimentally determined value, and errors of more than 15% are rare (Gill and von Hippel, 1989).

Molecular cloning and characterization of glucose-6-phosphate dehydrogenase from Brugia malayi

Parasitology ◽  
2013 ◽  
Vol 140 (7) ◽  
pp. 897-906 ◽  
Author(s):  
ANITA VERMA ◽  
MANISH K. SUTHAR ◽  
PAWAN K. DOHAREY ◽  
SMITA GUPTA ◽  
SUNITA YADAV ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Brugia Malayi ◽  
Pentose Phosphate ◽  
Subunit Molecular Weight ◽  
Optimum Ph ◽  
Sh Group ◽  
Glucose 6 Phosphate Dehydrogenase

SUMMARYGlucose-6-phosphate dehydrogenase (G6PD), a regulatory enzyme of the pentose phosphate pathway from Brugia malayi, was cloned, expressed and biochemically characterized. The Km values for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) were 0·25 and 0·014 mm respectively. The rBmG6PD exhibited an optimum pH of 8·5 and temperature, 40 °C. Adenosine 5′ [γ-thio] triphosphate (ATP-γ-S), adenosine 5′ [β,γ-imido] triphosphate (ATP-β,γ-NH), adenosine 5′ [β-thio] diphosphate (ADP-β-S), Na+, K+, Li+ and Cu++ ions were found to be strong inhibitors of rBmG6PD. The rBmG6PD, a tetramer with subunit molecular weight of 75 kDa contains 0·02 mol of SH group per mol of monomer. Blocking the SH group with SH-inhibitors, led to activation of rBmG6PD activity by N-ethylmaleimide. CD analysis indicated that rBmG6PD is composed of 37% α-helices and 26% β-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.

Purification and properties of a phenol carboxylic acid acyl esterase from Aspergillus flavus

1971 ◽  
Vol 17 (11) ◽  
pp. 1455-1463 ◽  
Author(s):  
J. J. Child ◽  
T. Oka ◽  
F. J. Simpson ◽  
H. G. Krishnamurty
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Carboxylic Acid ◽  
Aspergillus Flavus ◽  
Protocatechuic Acid ◽  
Hydroxybenzoic Acid ◽  
Purification And Properties ◽  
Wide Range ◽  
Hydrolysis Of ◽  
Active Preparation

Aspergillus flavus produces an extracellular esterase that hydrolyses phenolic carboxylic acid acyl esters. An assay based upon the measurement of the rate of release of phloroglucinol on hydrolysis of the ester of phloroglucinol and protocatechuic acid is described. The most active preparation hydrolyzed 30.8 μmoles of substrate per minute per milligram of protein and was active against a wide range of esters of meta and para hydroxybenzoic acid derivatives.The enzyme was isolated as a homogeneous protein, as judged by ultracentrifugation and by electrophoresis and had an isoelectric point of pH 4.45. The molecular weight was determined as 166 000. The enzyme is a glycoprotein containing 42.8% carbohydrate and the amino acid composition is described.

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