Storage of yeast alcohol dehydrogenase in crystal suspension causes a decrease of the enzymatic activity which is strongly related to the number of free -SH groups. With a rate slower than the rate of inactivation the enzyme molecule (molecular weight reinvestigated from hydrodynamic measurements t o be 141 000) dissociates into four polypeptide chains (molecular weight 35000) which are enzymatically inactive. By sulfitolysis in 4 M urea and amperometric titration it was shown that free -SH groups are oxidized mainly to disulfide groups during inactivation.The formation of disulfide bonds does not occur between --SH groups of different polypeptide chains. It is assumed that the inactivation process from the fully active enzyme molecule to the inactive polypeptide chains is not an all-or-none process but passes through three stages : 141 000 (fully active) --f 141 000 (partially active) +-141 000 (inactive) + 4 x 35000 (inactive). The more inactive the alcohol dehydrogenase, the more sensitive it is t o heat denaturation and peptidase attack.By treatment with mercaptoethanol or Cleland's reagent the disulfide bonds could be reduced and the enzymatic activity was recovered partially. The undissociated inactive molecules could be fully reactivated. With one of the investigated enzyme preparations which contains a peptidase it was also possible t o reactivate and reassociate alcohol dehydrogenase from the polypeptide chains.Storage of yeast alcohol dehydrogenase causes a decrease of the enzymatic activity which is strongly related to the number of free -SH groups [2]. I n addition a slower sedimenting component with a sedimentation coefficient of 2.6 S compared to a sedimentation coefficient of 7.7 S of the native enzyme molecule [3], appears during this inactivation [4]. The partial dissociation shows that not all enzyme molecules are equally involved in the aging process. These results raise two questions. (a) Is the process of inactivation an all-or-none process producing completely inactive enzyme molecules from fully active ones without intermediates having lower specific activities, and (b) what has happened t o the missing -SH groups ' 1 To answer these questions we investigated firstly the content of -SH and disulfide groups and the enzymatic activity of differently aged preparations of alcohol dehydrogenase, secondly the molecular weight of the enzyme and the dissociation product, and thirdly the reactivation of aged, inactivated enzyme preparations.

The binding of NADH and NADPH to beef liver glutamate dehydrogenase has been investigated using fluorescence titration and the preparative as well as the analytical ultracentrifuge over a wide range of protein and coenzyme concentrations. The glutamate dehydrogenase oligomer (molecular weight 336 000, six polypeptide chains) binds independent of enzyme concentration a total of twelve molecules of each reduced coenzyme, NADH and NADPH. The first six binding sites, the "active sites", bind NADH and NADPH in the same manner, whereas the weaker binding to the other six sites, the "nonactive sites", is remarkably different for both coenzymes : probably due to the steric hindrance or electrostatic repulsion the affinity of the enzyme for NADPH is about 10 times lower than that for NADH. This result explains the different kinetic behaviour of the two coenzymes. From the results obtained at different protein concentrations it is concluded that the coenzyme binding sites and the association sites are located at different parts of the enzyme surface.A procedure is described for three-parameter fitting of titration curves using a programmable desk calculator with on-line x-y recorder.Glutamate dehydrogenase from beef liver catalyzes the reversible oxidation of glutamate to 2-0x0-glutarate with NAD as well as NADP as coenzyme. Some experimental data indicate that a t high coenzyme concentrations NADf acts as an activator and NADH as an inhibitor of the enzymatic reaction, whereas in the presence of NADP+ or NADPH in general a normal Lineweaver-Burk plot is observed [5]. This difference in kinetic behaviour and the rate of dissociation induced by the coenzyme in the presence of GTP were explained by assuming two distinct binding sites per polypeptide chain for NAD but only one such site for NADP [5,6]. However other kinetic studies [7,8] show no difference in principle between the two oxidized coenzymes. The oligomer is composed of six polypeptide chains with identical sequence [9] and from a chemical point of view it seems reasonable to assume an identical reaction mechanism for both coenzymes [lo], even if quantitative differences between corresponding kinetic parameters exist. This paper is dedicated to Hugo Theorell on the occasion of his 70th birthday. This work has been described partly in preliminary reports [1,2]. For the previous paper in this series see [3].Abbreviations. E, enzyme; R, reduced coenzyme (NADH,Enzyme. Glutamate dehydrogenase or L-glutamate:NAD(P) oxidoreductase (deaminating) (EC 1.4.1.3).Quantitative analyses of the binding of NADH to the enzyme also yielded controversial results. Some reports in the literature indicate that the coenzyme binding capacity of the enzyme depends on the state of association [ll--131, other results are contrary to that finding, indicating only one [15,16] or slightly more than one binding site per polypeptide chain and negative or positive cooperativity between these sites [l, 17,181. Circular dichroism studies give direct evidence for the existence of a second coenzy...

Surprisingly, the template effect of a hydrocarbon has been detected for the first time: the yield in the one‐pot synthesis of the tetracationic macrocycle 1c is raised from 3–5% to 16% when excess phenanthrene is present. The new phane‐like tetracations 1a‐c and 2a‐c act as π‐acceptor host compounds and take up arenes as π‐donors in their cavities in a sandwich‐like manner.

Der Templateffekt eines Kohlenwasserstoffs konnte überraschend erstmals nachgewiesen werden: Bei der Eintopfsynthese des tetrakationischen Makrocyclus 1c steigt die Ausbeute von 3–5% auf 16%, wenn Phenanthren im Überschuß zugegen ist. Als neue π‐Acceptor‐Wirtverbindungen nehmen die phanartigen Tetrakationen 1a–1c und 2a–2c Arene als π‐Donoren sandwichartig in ihren Hohlraum auf. magnified image

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