Aromatische Disulfide, Dibenzyldisulfid und Diphenyldiselenid dissoziieren bei Bestrahlung niit kurzwelligem UV im Hochvakuum in Radikale des einbindigen Schwefels (R-S.) und Selens ( C~H S -S~. ) . Sie sind bei 77" K stabil. Beim Erwarmen rekombinieren sie zum Disulfid oder bilden Mercaptan durch Disproportionierung bzw. Dehydrierung uberschussigen Disulfids. Elektronendonatoren oder ein Phenylkern in p-Stellung stabilisieren die Arylschwefel-Radikale und verschieben ihre Lichtabsorption nach groBeren Wellenlangen. p Xehylschwefel-Radikal entsteht auch durch Photolyse von p-Xenylmercdptan. Die ESR-Spektren der Radikale zeigen einen anisotropen, meist rrahezu axialsymmetrischen g-Faktor.
Glutamate dehydrogenase from Cundidu utilis undergoes a reversible conformational transition between an active and an inactive state at low pH and low temperature. This conformational transition can also be followed by fluorescence measurements.The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 "C and a A H value of 148 kcalimol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 "C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Cundidu utilis enzyme is not caused by change in the quaternary structure.The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candidu utilis enzyme.The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s -l . Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition.The sedimentation coefficient ( s !~,~) and the molecular weight were found to be 11.0 S and 276000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47000In contrast to glutamate dehydrogenases in animals, those from other organisms are specific for either NAD or NADP and appear not to be regulated by purine nucleotides in physiological concentrations [3]. From the comparison of the primary structure between liver enzymes and the NADP-dependent enzyme from Neurosporu crussu it is suggested that the conformations should be similar, if not identical, in some parts 141 and that they have a common evolutionary This paper is dedicated to Hans Herloff Inhoffen on the occasion This work has been described in a preliminary report [I]. For of his 70th birthday. the previous paper in this series, see [2].origin [5]. The homologous parts may be responsible for the catalytic process, whereas differences occur in those which are involved in the regulation and the coenzyme specificity. If the non-animal glutamate dehydrogenases, in general, are not regu...
The investigation of the association-dissociation equilibrium of beef liver glutamate dehydrogenase by light-scattering measurements over a wide range of protein concentration (0.02 to 50 mg/ml) has shown that this equilibrium can be described as an open equilibrium (Mt + Mh + Mi, h , i and h 2 I ~ M = oligomer) with identical equilibrium constants for all steps and without any limit. The dependence of the apparent weight-average molecular weight on the protein concentration reaches a maximum a t about 9 mg/ml. The decrease of the molecular weight at higher protein concentrations must be attributed to effects of nonideality (as expressed in relation to a first approximation by the second virial coefficient). At 20 "C and in M/15 sodium phosphate buffer the equilibrium constant was found to be 9.0 x lo5 M-l (corresponding to a free energy change of 7.8 kcal/mol for each consecutive step in the association reaction) and the second virial coefficient to be 9nmol x 1 x g2. The calculation of the dependence of the reduced viscosity on the protein concentration based on these results conforms with the experimental data. However, it was found that the length of the oligomer obtained from light-scattering measurements was dependent on the wavelength. The reason for this phenomenon is not quite obvious.Low p H and low ionic strength promote the association of the oligomers. It was found that the sedimentation coefficient a t pH 6.2 and an ionic strength of 0.04 is 31 S (34 S in the presence of 4.4 mM NAD+) compared to 27 S a t an ionic strength of 0.115. The pH dependence of the sedimentation coefficient a t low ionic strength indicates the participation of a group in the association with a pK near 7, possibly an imidazole group. At high ionic strength the sedimentation coefficient is almost pH independent,.From the results it may be concluded that, in the interaction between the oligomers at low ionic strength, ionic effects are involved that are shielded a t higher ionic strength.GTP and NADH cause the dissociation into the oligomer characterized by an s & w value of 13.0 0.3. The dissociation is complete in the presence of 20 mM GTP and NADH. The result that the sio,w values of the oligomer are the same in the absence and in the presence of effectors, GTP/NADH, conforms to our X-ray small-angle measurements.
ation by splitting offaldeh5de even when, as in the case of the formaldehyde compound, the b. p. of the hemiacetal ( 3~) is higher than that of the acctal (2a). With amines, hydrazines, and thiols, the corresponding types ofmixed acetal are formed, viz. (4)-(7), e . g . from (Zo): RO-RN-CH-NR'?(4) R =-CH,, R I N = pipertdine, b p. 61-62 "CjIO mni RO-RN-CHI N R -NRI ( 5 ) R=-CH,, h.p. 46"C/t2mni RO-RN-CHz SR' (6) R = CH?, R'= C4H9, b.p. 74 'C/IOnim o r from (3151 RO-RN -C H R S R (7) K CH,, R' -C T H~, b.p. 101 'C/23 iniii The acetals can be split by acyl chloridcs, just as Bu/inw and Haitke [3] found for aminals, e.g.: + Ph-CO CI + Ph -CO-NR-OR -1-RO CO -CI i RO-CO-NR-OR ( 2 a) --f RO RN C H z -C I Received, M a y Ihth, 1963 [Z 5081339 IE] German version: Angew. Chem. 75, 640 (1963).
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