The partial specific volume of various proteins and its dependence on concentration and temperature of the solution

Pilz, Ingrid; Czerwenka, G.

doi:10.1002/macp.1973.021700115

Cited by 29 publications

(2 citation statements)

References 4 publications

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“…The partial specific volume used was calculated from the amino acid composition according to the revisions of Perkins (1986); the value found was 0.734 mL/g. This was corrected to 5 °C with the factor 3.9 X 10"4 mL g"1 deg"1 (Pilz & Czerwenka, 1973). The buffer density was calculated to be 1.0044 g/mL at 21 °C and 1.0066 at 5 °C.…”

Section: Methodsmentioning

confidence: 99%

Expression, purification, and characterization of human factor XIII in Saccharomyces cerevisiae

Bishop¹,

Teller²,

Smith³

et al. 1990

Biochemistry

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Factor XIII is the terminal enzyme of the clotting cascade. A cDNA sequence encoding human placental factor XIII was expressed in Saccharomyces cerevisiae with the yeast ADH2-4c promoter. Expression levels were a strong function of the noncoding flanking DNA content of the construction. When the terminal 3'-flanking noncoding DNA was removed, expression increased approximately 50-fold. The protein was produced in quantity by high-yield fermentation and purified to homogeneity. The recombinant protein was cleaved by thrombin at the same activation site as purified human placental FXIII and exhibited 100% enzymatic activity. At high thrombin concentrations rFXIIIa was cleaved into inactive 54- and 25-kDa polypeptides. The identity of these cleavage sites and the blocked N-terminus to that of the human protein was revealed by amino acid microsequencing. A time course of thrombin activation was performed and the relative distribution of the thrombin-cleaved subunits to the uncleaved zymogen subunits determined; the results were consistent with the half of the sites catalytic model for transglutaminase activity proposed by Chung et al. (Chung, S. I., Lewis, M. S., & Folk, J. E. (1974) J. Biol. Chem. 249, 940-950, 1974) and Hornyak et al. (Hornyak, T. J., Bishop, P. D., & Shafer, J. A. (1989) Biochemistry 28, 7326-7332). Equilibrium and velocity sedimentation analysis indicated that rFXIII exists as a 166-kDa nondissociating dimer that behaves as a compact particle of 8.02 S. Thus, all of the properties of rFXIII thus far examined are consistent with those reported for human platelet and placental FXIII.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Expression, purification, and characterization of human factor XIII in Saccharomyces cerevisiae

Bishop¹,

Teller²,

Smith³

et al. 1990

Biochemistry

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“…In order to find the correct value of the molecular parameters from these experiments, we corrected the value of sm to the conditions of the experiment. Of course, v2 must also be corrected to the experimental conditions for which we used dv2/dT = 3.9 X 10"4 mL g~* deg-1 (Pilz & Czerwenka, 1973). Then,n, vc, and Mc could be calculated from eq 10 under the experimental conditions.…”

Section: Discussionmentioning

confidence: 99%

Self-association and active enzyme forms of Naja naja naja and Crotalus atrox phospholipase A2 studied by analytical ultracentrifugation

Bukowski¹,

Teller²

1986

Biochemistry

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The dimerization of phospholipase A2 (PLPA2) from Naja naja naja (Pakistani cobra) and Crotalus atrox (Western Diamondback rattlesnake) has been studied from pH 2.5 to 11 at 20 degrees C in 1 mM CaCl2, 0.21 M ionic strength. For the C. atrox enzyme, it was found necessary to use a combination of sedimentation equilibrium and fluorescence yield data to analyze the association. Sedimentation equilibrium in the analytical ultracentrifuge sufficed for the study of the N. naja PLPA2. In the region of enzymatic activity at pH 8, the dimerization association constants found were k2 = 2.8 X 10(6) L/mol and k2 = 6.9 X 10(4) L/mol for the C. atrox and N. naja enzymes, respectively. Analytical linked functions are presented which describe the data. Because the associations are linked to Ca2+ as well as the hydrogen ion, no attempt was made to interpret the ionization of residues in terms of the molecular structure. Active-enzyme sedimentation velocity experiments have been used to study the relation between enzymatic activity and association for both the C. atrox and N. naja enzymes. The substrate 1,2-dibutyryl-sn-glycero-3-phosphocholine (diC4PC) did not dissociate the C. atrox PLPA2. The substrate 1,2-dihexanoyl-sn-glycero-3-phosphocholine (diC6PC) at 7.5 mM dissociated the C. atrox PLPA2 when monitored either as the active enzyme or as the Sr2+-inhibited enzyme. At low enzyme concentrations, 40 mM diC4PC had no effect on N. naja PLPA2 dimerization. However, the sedimentation coefficients observed at enzyme concentrations above 0.2 mg/mL in active-enzyme sedimentation velocity experiments were larger than the values predicted from the thermodynamic studies. Sedimentation coefficients observed for the N. naja PLPA2 acting on diC6PC were larger than those of the monomeric protein, which was the form layered on this substrate. The dissociation of the C. atrox PLPA2 effected by diC6PC was analyzed by the thermodynamics of association and the kinetic Michaelis constant. The analysis suffices to account for the observed sedimentation coefficient. The sedimentation behavior of the N. naja PLPA2 acting on diC6PC substrate was analyzed in terms of a protein-lipid complex. With this model, 68 +/- 16 phospholipid molecules per protein monomer were determined. It is proposed that this enzyme has two micelle nucleation sites per monomer. These putative sites promote micelle formation of the substrate on the enzyme below the critical micelle concentration of the lipid alone.

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Molecular thermodynamic properties of protein solutions from partial specific volumes

1995

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The partial specific volume of various proteins and its dependence on concentration and temperature of the solution

Cited by 29 publications

References 4 publications

Expression, purification, and characterization of human factor XIII in Saccharomyces cerevisiae

Expression, purification, and characterization of human factor XIII in Saccharomyces cerevisiae

Self-association and active enzyme forms of Naja naja naja and Crotalus atrox phospholipase A2 studied by analytical ultracentrifugation

Molecular thermodynamic properties of protein solutions from partial specific volumes

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