In the first 15-30 sec after addition of I0 mM NH,+ to Escherichia coli cells, one observes a 20-fold increase in the concentration of glutamine, an approximate one-third reduction in the stationary concentration of glutamate, an approximate goo/, decrease in the concentration of ATP and a decrease in the activity of glutamine synthetase to a few percent of the initial value. I n the subsequent 30 sec, glutamine synthetase remains inactive, glutamine concentration decreases, glutamate concentration increases 2 to 3-fold, and the ATP concentration rises slowly. The above-mentioned changes after addition of NH,+, together with previous observations on properties of purified glutamine synthetase and adenylyl transferase, make the following sequence of events probably: NH,+ enters the cells and is quickly incorporated into glutamate yielding glutamine, with a concomitant utilization of ATP. The accumulated glutamine stimulates the adenylyl transferase resulting in an inactivation of glutamine synthetase. Thus, S5 sec after addition of NH,+, further synthesis of glutamine is prevented. Consequently, glutamate concentration increases, glutamine concentration decreases, and ATP concentration increases. On the basis of these findings and conclusions, the following biological functions of NH,+ inactivation of glutamine synthetase are discussed : (a) prevention of a too high level of glutamine and (b) prevention of a sustained decrease in the ATP concentration which would be harmful to the cell.Within a few seconds following addition of NH,+ to Escherichia coli cells which were grown in an NH,+-free medium, Mecke and Holzer [l] observed an inactivation of glutamine synthetase to a level less than 10°/, of the initial activity. Mecke et al. [2,3] showed that the inactivation of glutamine synthetase was caused by a second enzyme and that this enzyme was stimulated in vitro by glutamine. An ATPdependent adenylylation of the glutamine synthetase was established as the mechanism of the inactivation reaction [4,5] .However, glutamine did not cause inactivation of the glutamine synthetase in intact E . coli cells as it did in cell-free systems. As an explanation for this apparent discrepancy it was postulated [2,3, S5] that NH,+ in the cells would be quickly synthesized to glutamine from glutamate and ATP. The accumulated glutamine then causes the inactivation of glutamine synthetase by stimulation of the adenylyltransferase. I n the present report it will be shown that in the first few seconds after addition of NII,+, the observed changes in the concentration of glutamate, glutamine and ATP, as well as the activity level of glutamine synthetase, are in agreement with this concept. With respect to the Enzymes. Glutamine synthetase or L-glutamate : ammonia ligase (ADP) (EC 6.3.1.2); adenylyltransferase or ATP : glutamine synthetase adenylyltransferase (EC 2.7.7.-).biological function of the glutamine-stimulated inactivation of glutamine synthetase, the following two points will be discussed: (a) an uneconomical accumulation of...

Individual subunits of yeast transaldolase were studied by the method of matrix-bound derivatives. Native dimeric transaldolase was bound to Sepharose activated with low amounts of cyanogen bromide to give matrix-bound transaldolase in which the majority of molecules were bound covalently via only one subunit. Exhaustive washing with 6 M guanidinium chloride and renaturation in triethanolamine-EDTA buffer resulted in a derivative (boundsubunit transaldolase) which retained 65O/, of the original protein and 63O/, of the original activity. This active form of transaldolase is more sensitive to 2 M urea than bound-dimeric transaldolase. Bound-subunit transaldolase is able to associate with ('nascent'' soluble transaldolase subunits generated in situ by diluting a small aliquot of guanidinium-chloridedenatured transaldolase into a much larger volume of buffer. The ability of bound-subunit derivative to pick up soluble "nascent" transaldolase subunits is highly specific and reaches a plateau level close to the original protein and activity content of bound transaldolase. The product of such treatment (bound-renatured transaldolase) has properties virtually indistinguishable from those of bound transaldolase. These results indicate that individual subunits of transaldolase are catalytically active.Bound-subunit transaldolase has K, values similar to those of the bound-dimeric derivatives and can be inactivated with sodium borohydride in the presence of the substrate fructose 6-phosphate. However, the pH profile of the subunit form is substantially altered suggesting a shift in the pKa of an essential protonated group from 9.2 to 8.6.Transaldolase from Candida utilis has been shown to be composed of two similar or identical subunits [1,2]. Substantial work has been done on the mechanism of action of this enzyme (for review see MATERIALS AND METHODS MaterialsTransaldolase (type 111) was purified and crystallized from Candida utilis by the procedure of Tchola and Horecker [9] with the following modifications. The acid ammonium sulfate fractionation was omitted and a preparative DEAE-cellulose (Whatman DE-52) chromatographic step performed in order to separate the three isoenzymes of transaldolase. 10 acetone fractions (32000 units, 10 g protein) were dialyzed overnight a t 4 "C against 5 1 of 0.05 M triethanolamine buffer pH 7.0. Distilled Eur. J. Biochem. 40 (1973)

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Optically active D-arylglycines, which are of interest for preparation of semisynthetic penicillins and cephalosporins, were isolated from the racemic mixtures of their derivatives using immobilized proteolytic enzyme subtilisin (EC No. 3.4.4. 16). The performance of these reactions in two-phase systems, consisting of water and an immiscible organic solvent, improved the yield, purity, and economics of the process by increasing the substrate solubility and reducing the rate of nonenzymatic hydrolysis. The proportion of the organic phase can be as much as 75% of the overall volume without seriously impairing the enzymatic activity. The optically pure D-and L-arylglycines were liberated from their D- and L-derivatives by acid hydrolysis. The substituent influence of the various arylglycine derivatives on the rate of the enzymatic cleavage reaction was investigated.