An Assessment of Some of the Methods Available for the Determination of Molecular Weights of Proteins as Applied to Aspartate Aminotransferase from Pig Heart

Banks, Barbara E. C.; Doonan, Shawn; Flögel, Mirna; Porter, Patricia B.; Vernon, C. A.; Walker, John M.; Crouch, Thomas H.; Halsey, John F.; Chiancone, Emilia; Fasella, P.

doi:10.1111/j.1432-1033.1976.tb11135.x

Cited by 8 publications

(4 citation statements)

References 22 publications

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“…where (dp/dC2)" is the change in solution density with respect to calmodulin concentration under isopotential conditions (Banks et al, 1976), is the rotation rate in radiants/second, R is the gas constant, and T is the absolute temperature. Both in the presence and absence of Ca2+, the molecular weight of calmodulin was 18 700.…”

Section: Resultsmentioning

confidence: 99%

“…No time-dependent change in the concentration pattern along the cell was detected after 12 h, which was taken to indicate equilibrium. The buoyancy term (dp/dC2)" (Banks et al, 1976) was determined by a magnetic densimeter (Kupke & Crouch, 1978). Two samples containing approximately 2 mL of 8 mg/mL calmodulin were dialyzed against the buffers used for the ultracentrifugation experiments.…”

Section: Circular Dichroism and Uv-difference Spectroscopicmentioning

confidence: 99%

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Positive cooperative binding of calcium to bovine brain calmodulin

Crouch¹,

Klee²

1980

Biochemistry

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Equilibrium dialysis measurements of the binding of Ca2+ to calmodulin have confirmed the existence of four high affinity Ca2+-binding sites (Kd between 3 X 10(-6) and 2 X 10(-5) M). In the presence of 3 mM Mg2+, the dissociation constants for Ca2+ are increased two- to fourfold (Kd between 5 X 10(-6) and 4 X 10(-5) M). Positive cooperativity of Ca2+ binding was observed at low Ca2+ concentrations with Hill coefficients of 1.33 and 1.22 in the absence and presence of 3 mM Mg2+, respectively. The positive cooperativity is compatible with the steepness of the Ca2+ dependence of the conformational transition associated with the binding of 2 mol of Ca2+/mol of calmodulin. This conformational change, which affects the environment of the aromatic residues of calmodulin as measured by UV absorption and near-UV circular dichroism spectroscopy, is not the result of a monomer-dimer equilibrium mediated by Ca2+. Binding of Ca2+ to calmodulin is believed to occur by a sequential mechanism generating at least four different conformers of the protein and its free and liganded states. Even though the major conformational change is almost complete upon binding of 2 mol of Ca2+/mol of calmodulin, the activation of cyclic nucleotide phosphodiesterase measured in the presence of limiting concentrations of calmodulin suggests that a calmodulin Ca3-42+ complex is required for interaction of calmodulin with the enzyme. As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex (Kd = 1-3 X 10(-9) M), the Ca2+ dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold of Ca2+ concentration for control of enzyme activity.

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Section: Resultsmentioning

confidence: 99%

Section: Circular Dichroism and Uv-difference Spectroscopicmentioning

confidence: 99%

Positive cooperative binding of calcium to bovine brain calmodulin

Crouch¹,

Klee²

1980

Biochemistry

Self Cite

351

160

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“…50-720-3285, Roche Diagnostics no. 10105554001, from pig heart type I GOT1/cAST, MW 46.6 kDa, suspension in 3.2 M ammonium sulfate solution, 4460 U mL –1 ), NaHCO 3 , NaH 2 PO 4 ·H 2 O, Na 2 HPO 4 , HCl, NaCl, CaCl 2 ·2H 2 O, Mg(NO 3 ) 2 , and NaOH were from Fisher. Multiwalled carbon nanotubes (CNTs, ∼95% nominal purity) were purchased from Nanolab (Brighton, MA).…”

Section: Methodsmentioning

confidence: 99%

Electrochemical Coupled-Enzyme Assays at Carbon Nanotubes

2014

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The recently developed internally calibrated electrochemical continuous enzyme assay (ICECEA) has proved to work well for single-enzyme systems. In the present work, its relevance to more challenging coupled-enzyme measurements was investigated by using a model enzyme pair comprising aspartate transaminase (AST) and malic dehydrogenase. The ICECEA was performed at an electrode modified with carbon nanotubes (CNTs), which were dispersed in a polysaccharide chitosan that acted as an adhesive. The 7 min assay required a 100 μL sample and relied on an AST-free calibration. It had a limit of detection equal to 5.0 pM AST (0.10 U L(-1)) with no need for the incubation period. Its linear range extended up to 3500 pM (70 U L(-1)). Perhaps the most promising was the fact that the assay and its calibration could be performed in the same solution even though the composition of the assay solution for the coupled-enzyme assays is typically more complex than that for the single-enzyme assays. This and the fast electrode kinetics of the signal transducing reaction of nicotinamide adenine dinucleotide at CNTs accounted for the low limit of detection. The unique shape of the ICECEA amperogram allowed for the selective determination of AST in the complex matrix of serum samples containing redox active potentially interfering species. Given these advantages, the prospects for the ICECEA in the development of other coupled-enzyme assays were also discussed.

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“…The molecular weight of enzymically active molecules was estimated with the parallel column using the following markers : cytosolic aspartate aminotransferase of pig heart purified in this laboratory (molecular weight, 92000) [22,23], bovine serum albumin (molecular weight, 68 000), egg albumin (molecular weight, 43 000) and soybean trypsin inhibitor (molecular weight, 21500). The inner volume of the column was determined from the difference in elution volume between blue dextran and dinitrophenyl-Lalanine.…”

Section: Separation Of Enzymically Active Molecules From the Renaturementioning

confidence: 99%

Latent Active Site in Rat-Kidney gamma-Glutamyl Transpeptidase. The Refolding Process of the Large Subunit and Characterization of the Renatured Enzyme

1980

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Rat kidney y-glutamyl transpeptidase (EC 2.3.2.2) is composed of two non-identical subunits (the large and small subunits). In the native state of this enzyme, the active site resides on the small subunit. The present investigation has been performed to confirm and extend in further detail the previously described finding that the large subunit isolated from the native dimeric enzyme is also catalytically active [S. Horiuchi, M. Inoue and Y. Morino (1978) Biochem. Biophys. Res. Commun. 80,873 -8781. A pure preparation of y-glutamyl transpeptidase which had been completely inactivated by affinity labeling with 6-diazo-5-oxo-~-norleucine was dissociated into the small subunit and the large subunit under denaturating conditions. Upon renaturation, only the large subunit restored the enzymic activity.Sephadex G-200 column chromatography of the renatured preparation of the large subunit revealed the presence both of enzymically inactive aggregate forms and the enzymically active species. The enzymically active molecules of the renatured large subunit behaved as particles having a molecular weight of 48000 2 2000. This indicates that the enzymically active species of the large subunit has a monomeric structure.Under the present experimental conditions, the restoration of enzymic activity of the large subunit was optimal at a protein concentration of approximately 150 pg/ml and seemed to require the presence of glutathione or its S-methyl derivative as a substrate ligand.The restoration of the enzymic activity of the large subunit was accompanied by a conformational change as judged by both circular dichroic and immunochemical properties.Some kinetic properties of the reaction with y-glutamyl-p-nitroanilide and the affinity label, 6-diazo-5-oxo-~-norleucine were essentially identical between the renatured large subunit and the native oligomeric enzyme. y-Glutamyl transpeptidase catalyzes the transfer reaction of a y-glutamyl moiety of glutathione or S-substituted glutathione derivatives to acceptors such as amino acids and peptides [1,2]. Many proposals have been made on the possible participation of this enzyme in physiological functions, including ammoniagenesis in metabolic acidosis [3,4], mercapturate biosynthesis [5,6], amino acid and peptide transport [7 -91 and so forth. Rat kidney y-glutamyl transpeptidase consists of two non-identical subunits (the large and small subunit) [lo-121. The small subunit has been shown to have an active site which is labeled by 6-diazo-5-oxo-~-norleucine, an affinity label for this enzyme [13,14], suggesting that the small subunit is a catalytic component of the enzyme. However, little has been known about the functional Enzyme. y-Glutamyl transpeptidase (EC 2.3.2.2) aspect of the large subunit except that this subunit may be mainly involved in anchoring this membranous enzyme into the renal brush border membrane [ l l , 12,151.When we studied subunit-subunit interaction of y-glutamyl transpeptidase to gain an insight into the possible functional role of the large subunit, a re...

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An Assessment of Some of the Methods Available for the Determination of Molecular Weights of Proteins as Applied to Aspartate Aminotransferase from Pig Heart

Cited by 8 publications

References 22 publications

Positive cooperative binding of calcium to bovine brain calmodulin

Positive cooperative binding of calcium to bovine brain calmodulin

Electrochemical Coupled-Enzyme Assays at Carbon Nanotubes

Latent Active Site in Rat-Kidney gamma-Glutamyl Transpeptidase. The Refolding Process of the Large Subunit and Characterization of the Renatured Enzyme

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