Dissoziation der Rinderleber-Katalase in ihre Untereinheiten

Sund, Horst; Weber, Katharina; Mölbert, Elisabeth

doi:10.1111/j.1432-1033.1967.tb00088.x

Cited by 124 publications

(44 citation statements)

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“…24, and unpublished data). Itappears that the size of an enzyme and the complexity of its function are generally not correlated; e.g., lysozyme has a molecular weight of 14,500 (25) and 3-galactosidase is 540,000 (26,27). It may well be that, at least to a certain degree, the size of an enzyme molecule is the consequence of evolutionary chance events that do not follow selective pressure.…”

Section: Number Of Chains In Core Complexmentioning

confidence: 99%

Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

Vogel

Hoehn

Henning

1972

Proc. Natl. Acad. Sci. U.S.A.

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The pyruvate dehydrogenase core complex from E. coli K-12, Since the pioneering experiments of Gunsalus and Hager (1) and of Koike, Reed, and Carroll (2), a-ketoacid dehydrogenase complexes from various sources have been extensively studied. Several years ago, we began to study the Escherichia coli pyruvate dehydrogenase complex from a genetic point of view (3), because it appeared that the molar ratio of the constituent polypeptide chains was far from unity. We therefore began to study the regulation of the synthesis of the enzyme complex, and it became clear during these studies (4-6) that one would be hampered in further such experiments without a precise knowledge about the composition of this multienzyme complex; thus, we turned our attention toward an elucidation of its structure (7,8,4 MATERIALS AND METHODSCells and Reagents. Pyruvate dehydrogenase complex was prepared from K-12 strain YMel and from the regulatory mutants K1-1 LR8-13 and K1-1 LR8-16, which synthesize the enzyme complex constitutively (6). Growth conditions, purification procedures, enzymc assays, and sources of reagents were described (8).Determination of Flavin-Adenine Dinucleotide (FAD). Our instrumentation was checked with FAD purchased from Boehringer iIannheim GmbH. The expected extinction coefficient (450 nm) of 1.13 cm2/mmol (9) was found after the substance was dried for 3 days under reduced pressure at 600 over P205. All spectra were measured in 0.05 M potassium phosphate, pH 7.5, with a Zeiss PMQJI spectrophotometer. Reduction and reoxidation of enzyme-bound FAD in the presence of 6.5 M urea was performed exactly as described by Massey, Hofmann, and Palmer (10). All protein concentrations for these spectral analyses were determined by the biuret reaction (11); the relation of dry weight to biuret assay has been reported$.Determination of Polypeptide Chain Ratio. Pure polypeptide chains were isolated by preparative dodecylsulfate-polyacrylamide gel electrophoresis (8). They were subjected to analytical electrophoresis under conditions essentially the same as those of Weber and Osborn (12). The gels contained 10% acrylamide, 0.135% methylene bisacrylamide, 0.05% sodium dodecylsulfate, and 0.05 M phosphate, pH 7.0. The gel columns were 60-mm high and had a diameter of 5 mm. Electrophoresis was for about 2.5 hr at 6 mA per column. The gels were stained for 2 hr with Commassie Brilliant Blue (12), and were immediately destained electrophoretically. The gels were then kept for 8 days in 7.5% acetic acid-5% methanol to remove all background stain. Stain intensity was measured with a Joyce-Loebl microdensitometer equipped with a red filter. Each column was measured twice; after the first tracing the column was rotated around its longitudinal axis by 900 and a second tracing was taken. The difference between the two measurements did not exceed 5% per band. Each point recorded in Fig. 1 is an average value of these two measurements. The areas below the tracings were cut out and weighed with a microbalance. (The density of the r...

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Section: Number Of Chains In Core Complexmentioning

confidence: 99%

Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

Vogel

Hoehn

Henning

1972

Proc. Natl. Acad. Sci. U.S.A.

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“…The concentration of the modified protein was determined by Kjeldahl analysis or by integrating the area under the concentration gradient of thc sedimentation pattern in the analytical ultracentrifuge. Sedimentation velocity measurements and the calculation of the sedimentation coefficients were performed as previously described [17].…”

Section: Chemical Modificationmentioning

confidence: 99%

Studies of Glutamate Dehydrogenase: Chemical Modification and Quantitative Determination of Tryptophan Residues

Witzemann

Koberstein

Sund

et al. 1974

European Journal of Biochemistry

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The effect of the modification of beef liver glutamate dehydrogenase with 2-hydroxy-5-nitrobenzyl bromide was studied with respect to the association-dissociation equilibrium as well as the enzymatic and regulatory properties. Upon incorporation of 2.1 molecules of the reagent per polypeptide chain, the association of the enzyme is strongly reduced, whereas the enzymatic activity is only slightly decreased (SOo/, maximum velocity). The K , values and the regulation by ADP and GTP, respectively, remain unaltered. Evidence is presented that only tryptophan residues are modified. Magnetic circular dichroism measurements of the modified enzyme suggest partial disubstitntion of tryptophan residues. From the reduced incorporation of 2-hydroxy-5-nitrobenzyl groups a t high enzyme concentration it is concludcd that tryptophan is located a t the association areas of the enzyme.Quantitative determination of the tryptophan content of glutamate dehydrogenase with 2-hydroxy-5-nitrobenzyl bromide, magnetic circular dichroism and peptide analysis yields four tryptophan residues per polypeptide chain contrary to three residues previously suggested from structural analysis. The fourth tryptophan, not present in the reported sequence, is recovered in a chymotryptie peptide Qlu-Trp.The temperature dependence of the linear association of glutamate dehydrogenase to higher aggregates indicatcs hydrophobic interactions between the oligomers [2,3]. From the influence of toluene and benzene on this association equilibrium, further evidence for hydrophobic interactions or sandwich complexes involving aromatic amino acids is presented [4,51. These results prompted us to investigate the role of tryptophan residues in the association process IS]. In this paper we present the results of the chemical modification of native glutamate dehydrogenase with 2-hydroxy-5-nitrobenzyl hromide (BzlOHN0,Br) which primarily reacts with tryptophan residues [ 7 ] . The influence of this modification on the association behavior and on the enzymatic properties of glutamate dehydrogenase is described.I n the course of this work, we obtained results indicating a total of four tryptophan residues per polypeptide chain instead of three found in the sequence analysis [S, 91. Earlier amino-acid analyses do not provide an unequivocal number for tryptophan residues, since values between 3 and 5.5 residues per polypeptide chain have been reported [lo -131.To clarify this point, we carried out quantitative tryptophan determinations using three independent methods : magnetic circular dichroism [14], sequence analysis and modification with BzlOHN0,Br under denaturing conditions [15].

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“…The GluDH concentration was determined using an absorbance factor Azso = 0.97 [cm' mg-' ] . Sedimentation velocity and diffusion measurements were made with the Spinco model E analytical ultracentrifuge (Beckman Instruments, Miinchen, Germany) and the calculation of the hydrodynamic properties were performed as previously described [7]. For the experiments the enzyme solutions were dialyzed for 36 hr at 2" to 4' against 0.067 M sodium phosphate buffer, pH 7.6, or citrate buffer (cl = 0.04), pH 6.0 or 6.9 and the ionic strength was increased as noted by adding sodium chloride.…”

Section: Methodsmentioning

confidence: 99%