In a previous paper, we described the kinetic characteristics of the inhibition exerted by the protease inhibitors tosylphenylalanyl and tosyllysyl chloromethanes on superoxide production by human polymorphonuclear leukocytes when stimulated by phorbol esters [E. C. Conseiller & F. Lederer (1989) Eur. J. Biochem. 183, 107-1141. The results suggested the existence of a specific target which was affinity labeled by the inhibitors. The target appeared to be neither a protease, nor intracellular enzymes which can be inhibited in vitro by the chloromethanes (protein kinase C, hexokinase and enzymes of the hexose monophosphate shunt). In the present work, using the cell-free reconstitution assay for superoxide production, we substantiate the hypothesis that the chloromethanes, target is on the plasma membrane. We have radiolabeled the membranes of cells inactivated before or after phorbol ester stimulation, using either [3H]KBH4 reduction after reaction with unlabeled inactivator, or tritiated tosylphenylalanyl chloromethane. In all cases, besides a certain background of non-specific labeling, a radioactive band of M , 15 000 can be observed upon SDSjPAGE of radiolabeled membranes. We suggest that it is the chemical modification of this protein which is responsible for inactivation of superoxide production. Its identity and its role in the oxidative burst remain to be determined.NADPH oxidase, an enzymatic complex present in blood phagocytes, produces superoxide anion when the cells are stimulated by soluble or particulate stimuli. This oxygen derivative then undergoes a series of enzymatic and non-enzymatic reactions producing a variety of oxidizing agents which play an essential role in host defense mechanisms against infectious diseases [I -41. Various studies have demonstrated a requirement for at least two redox components, a flavoprotein [5 -71 and a low-potential cytochrome b termed cytochrome b245 or b55t,, which is partially or totally absent from patients with X-linked chronic granulomatous disease (CGD) [8 -101. In activated cells, the superoxide anion-producing activity is found attached to the plasma membrane [I1 -131.Proteins other than the redox components are required for appearance of the enzymatic activity, which is dormant in resting cells. Studies initiated by Bromberg and Pick [14] and Curnutte [I51 have shown it is possible to induce the oxidase activity in a cell-free system by combining the cytosolic and particulate fractions of resting cells in the presence of SDS or arachidonate. Studies with such a system led to the implication Abbreviations. CGD, chronic granulomatous disease; Dip-F, diisopropyl fluorophosphate; HBSS; Hank's balanced salt solution; Me'SO; dimethylsulfoxide; PhMeS02F, phenylmethanesulfonyl fluoride; PMA, phorbol12-myristate 13-acetate; PMN, polymorphonuclear leukocytes ; TosLysCH2C1,N"-p-tosyl-L-lysyl chloromethane; TosPheCH2C1, N-p-tosyl-1,-phenylalanyl chloromethane.Enzymes. NADPH oxidase (EC 1.6.99.6); superoxide dismutase (EC 1.25.1.1) ~-of several cytosolic factors in...

Die Hydrolyse von verdünnten salpetersauren Wismutnitratlösungen (10−2 bis 10−3 m) wurde im pH‐Bereich von 1,04–1,88 zwischen 21,0° C und 80,0° C untersucht. Es entstehen Hydrolyseprodukte der Zusammensetzung [Bi6Ox(OH)12–2x] (OH)3(NO3)3 · n H2O, die bei 25,0° C und bei pH = 1,50 mit einer Halbwertszeit von etwa 70 Minuten zerfallen. Der Zerfall ist formal 2. Ordnung; Zerfallskonstante bei 25,0° C: k = 0,32 ± 0,021 · Mol−1 · Min−1, Aktivierungsenergie des Zerfalls: ΔHA = 1,4 ± 0,5 kcal/Mol. Bei pH ≥ 1,6 wird der Zerfall der Hydrolyseprodukte durch die Bildung kristalliner Niederschläge überlagert, für die die Zusammensetzung [Bi6O4(OH)4](OH)(NO3)5 · 0,5 H2O (± 0,5 H2O) wahrscheinlich gemacht werden konnte. Dieses basische Wismutnitrat kristallisiert monoklin in der Raumgruppe P 21/c−C 2h5 mit 8 Formeleinheiten in der Elementarzelle; Gitterdimensionen: a = 17,58 ± 0,04 Å, b = 9,15 ± 0,03 Å, c = 27,20 ± 0,05 Å, β = 83,2 ± 0,3°. – Die Löslichkeiten wurden bei 25,0° C als Funktion der Acidität der Lösung bestimmt. Zwischen den in Lösung vorliegenden Polykationen [Bi6Ox(OH)12−2x]6+ einerseits und den Hydrolyseprodukten und den thermodynamisch stabilen kristallinen Niederschlägen andererseits bestehen enge Zusammenhänge.

It is shown that AcSDKP a new regulator of the hematopoietic system can he generated from thymosin β4 by a one‐step enzymatic cleavage in vitro and in vivo. AcSDKP and Tβ4 were both detected in bone marrow cells (BMC'). Incubation of [3H]Tβ4 with either intact or lysed BMC led to the formation of [3H]AcSDKP whereas the labelled tetrapeptide was not degraded under these conditions. Model enzymatic degradation of Tβ4 carried out with bacterial enzymes suggests that a mammalian endoproteinase Asp‐N might be involved in the formation of AcSDKP through the specific cleavage of the 4Pro‐5 Asp peptide bond of T β4.

S u m m a r yT h e s y n t h e s i s of the t r i c h l o r o m e t h y l a n a l o g u e of be n z y I ox y c ar b o n y 1 -t h r e o n i n e 1' hi s precursor was isomer. They were separated by HPLC using a chiral stationnary phase. They have a molar specific activity of 3190 GBq/mmol, 86 Ci/mmol.i s d e s c r i bed .reduced by tritium gas to give [methyl-3 H3]-threonine and its all0Key words : selective CrO3 oxidation, 3II-labelling, trichloromethylation, trichlorinated 2-threonine. I n t r o d u c t i o nBiological investigations with peptides and proteins frequently require radio-labelled compounds with high specific activities.IIuman insulin differs from porcin insulin by only one aminoacid content : threonine replaces alanine at the C-terminal position of the a-chain. We develop an original procedure for labelling the human insulin, which consists of substituing the alanine of the porcine insulin with labelled threonine by the aid of carboxypeptidase.Catalytic reduction of halogenated or unsaturated precursor by tritium gas is t h e most convenient way of labelling organic compounds. So we focused our attention on the synthesis of the trichlorinated analogue 1 of Z-threonine. The benzyloxycarbonyl group was chosen as the masking agent of the amino function because of its lability under reductive conditions : the labelling step could directly lead to the free amino-acid. A high specific activity could be expected from the tritiation of the poly-halogenated group.

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