Kirromycin activates functions of elongation factor Tu (EF-Tu) that normally require the presence of specific effectors [Wolf et al. (1974) Proc. Nut1 Acud. Sci. U.S.A. 71, 4910-49141. As a result, the EF-Tu GTPase activity is uncoupled from aminoacyl-tRNA and ribosomes. For a better understanding of the action of the antibiotic, we have studied its effect on the interaction between EF-Tu and guanine nucleotides and compared this with the action of the physiological effectors aminoacyltRNA and elongation factor Ts (EF-Ts). Kirromycin affects both association (k; and dissociation (k' 1) rates of EF-Tu and GTP, but in opposite ways, the k: being strongly increased, whereas the kL is even more strongly decreased. This causes a lowering of the apparent K ' of this complex by two orders of magnitude, i.e. approaching that of EF-Tu . GDP. By contrast, the k : 1 and k l 1 are both increased to the same extent and consequently the apparent K ' of this complex is unchanged in the presence of the antibiotic. Thus the action of kirromycin resembles that of EF-Ts opening the EF-Tu site for GDP, thereby increasing the rate of the EF-Tu.GDP/GDP exchange, but in contrast to EF-Ts kirromycin binds preferentially to EF-Tu . GTP. Like aminoacyl-tRNA, the antibiotic induces a specific conformation that locks GTP in its site on EF-Tu. The regeneration of EF-Tu . GTP from EF-Tu .GDP does not limit the rate of GTP hydrolysis induced by kirromycin, even when aminoacyl-t RNA or ribosomes' stimulate this reaction. By contrast, the turnover activity occurring in the absence of the antibiotic and depending on aminoacyl-tRNA plus ribosomes is limited by the dissociation rate of the EF-Tu.GDP complex. Our results indicate that kirromycin induces a conformation of EF-Tu that shares features with the conformations evoked sequentially by EF-Ts, aminoacyl-tRNA and ribosomes during the elongation cycle.In each round of polypeptide chain elongation, the elongation factor Tu (EF-Tu) forms a ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) that interacts with mRNA .ribosomes, resulting in the binding of aa-tRNA to the ribosome, hydrolysis of GTP and release of EF-Tu.GDP (for a review, see [ I -31). The regeneration of the ternary complex from EF-Tu .GDP is accelerated by elongation factor Ts (EF-Ts). We have shown that kirromycin, an inhibitor of protein biosynthesis and the first antibiotic having EF-Tu as its target, binds to EF-Tu in a one-to-one ratio and that the EF-Tu . kirromycin complex thus formed can still react sequentially with GTP and aa-tRNA leading to the formation of a quaternary complex [4-81. However, after the enzymatic binding of this complex to the ribosome.mRNA and associated GTP hydrolysis, the modifications introduced by kirromycin in the interactions between EF-Tu, guanine nucleotides and aa-tRNA inhibit the dissociAbbreviations. EF-Tu, elongation factor Tu; EF-Ts, elongation factor Ts; k;, , apparent association rate constant; kl, , apparent dissociation rate constant; aa-tRNA, aminoacyl transfer RNA. ation of EF-Tu f...

complexes trans,mer-[(PR3)2(CO)3M]2(«-L) with the symmetrically bridging ligands w-L = pyrazine (pz), 4,4'bipyridine (bp) and 3,6-bis(4-pyridyl)-1,2,4,5-tetrazine (4,4'-bptz) were studied by cyclic voltammetry and by IR, UV/vis/near-IR, and EPR spectroscopy. Oxidation of the electron-rich and dissociatively labile (Mo > W) systems yields M(I) species, including stable (Kc > 106 *) mixed-valent d5/d6 cations {[(PPrsMCO^Mhlpz)}"1". These mixed-valent complex ions exhibit complete delocalization on the vibrational time scale and show similar spectroscopic features as the structurally related Creutz-Taube ion {[(HgNisRuht/i-pz)}5"1". Reversible twoelectron oxidation processes and thus no mixed-valent states were observed for the bp-and 4,4'-bptz-bridged ditungsten systems. Whereas the oxidation of (PCy3)2(CO)3W(?y2-H2) is irreversible with the assumed loss of a proton, the solvates (PR3)2(CO)3M(THF) are reversibly oxidized and reduced, the latter process requiring rather negative potentials. In nonpolar solvents the neutral dinuclear complexes display very intense and strikingly narrow charge transfer bands in the near-infrared region, suggesting very little geometrical change between ground and MLCT excited states. One-electron reduction of the dinuclear complexes produces EPR-detectable radical anion complexes which show the loss of one PR3 ligand per metal center, i.e. the preference for a 16 + <5 rather than an 18 + valence electron configuration. The tungsten-centered oxidation of complexes (PR3)2(CO)3W(L) is facilitated in the order L = mpz+, pz, H2, 4,4'-bptz, bp, and THF, illustrating quantitatively the well-balanced donor and a* acceptor characteristics of the H2 ligand. From the results of this study it appears that the (PR3)2-(CO)3M fragments are suited to bind H2 because of a very finely tuned combination of -acceptor and fairly strong but not excessive -donor characteristics, in addition to the proper amount of steric shielding.

SUMMARY1. Sarcoplasmic reticulum vesicles and mitochondria were prepared from red and white skeletal muscles of the rabbit. The preparations were characterized in terms of their specific activities of citrate synthase, basal (Mg2+-dependent) and Ca2+-dependent ATPase (the latter two in the presence of NaN3 and ouabain), and their specific carbonic anhydrase activities were determined.2. Skeletal muscle mitochondria had high specific activities of citrate synthase (700-1200 mu. mg protein-1) and low carbonic anhydrase activities (0 1-0 4 u. ml mg protein-1). The latter are likely to be due to a contamination of the preparations with sarcoplasmic reticulum (s.r.)3. Preparations of s.r. vesicles showed negligible activities of citrate synthase and the expected differing patterns of basal and Ca2+-dependent ATPase in red and white muscles. Specific carbonic anhydrase activities in s.r. from both muscle types were high (2-4 u. ml mg protein-'). The highest carbonic anhydrase activity, 11 u. ml mg protein-1, was found in s.r. from rabbit m. masseter. The inhibition constant of s.r. carbonic anhydrase towards acetazolamide was 4-6 x 10-8 M and similar but not identical to that of cytosolic carbonic anhydrase II.4. It appears possible that the carbonic anhydrase II-like enzyme previously found by us in muscle homogenates (Siffert & Gros, 1982) originates from the s.r.5. Histochemical studies using the dansylsuphonamide method described previously (Dermietzel, Leibstein, Siffert, Zamboglou & Gros, 1985) showed an intracellular pattern of carbonic anhydrase staining compatible with the presence of the enzyme in s.r.: spots homogeneously distributed across the fibre crosssections in transversely sectioned fibres and thin, longitudinally oriented, bands in longitudinally sectioned fibres.6. It is estimated that s.r. carbonic anhydrase accelerates C02 hydration within the s.r. approximately 1000-fold. Thus, CO2 and HCO3-react fast enough to provide a rapid source and sink for protons leaving and entering the s.r. in exchange for Ca2+.

No abstract

A series of GTP and GDP analogues modified in the terminal phosphate has been synthesized and their activities were investigated in elongation factor G dependent reactions. All of the analogues, with the exception of guanosine 5'-O-(3-thiotriphosphate), were not hydrolyzed by EF-G and ribosomes, but were competitive inhibitors of the ribosome-dependent EF-G GTPase. The most active inhibitors were P3-fluoro P1-5'-guanosine triphosphate and P3-methyl P1-5'-guanosine triphosphate with a Ki of 1.0 X 10(-6) and 2.5 X 10(-6) M, respectively. The activity of the GTP alkyl ester derivatives decreased with increasing number of carbon atoms in the side chain. GTP analogues were much more effective inhibitors than the corresponding GDP derivatives. This points out the necessity of the presence of at least three negative charges in the phosphate chain of the nucleotide for an effective interaction with the active site of the ribosomal EF-G GTPase. Guanosine 5'-O-(3-thiotriphosphate), which was hydrolyzed at one-third the rate of GTP, was able to support poly(U)-directed poly(phenylalanine) polymerization. Possible mechanisms of ribosome-EF-G GTP hydrolysis that arise from our results are discussed. Activity of the nucleotide analogues in EF-G-ribosome complex formation compared well with their ability to inhibit ribosome-dependent EF-G GTPase, P3-fluoro P1-5'-guanosine triphosphate and P3-methyl P1-5'-guanosine triphosphate being again the most effective ones. The stabilizing action of fusidic acid on the EF-G-ribosome complex formation induced by the various nucleotides could not be correlated to any of the structural modifications of the substrate. Guanylyl methylene diphosphonate was displaced more readily than GDP from the EF-G-ribosome complex by GTP analogues insensitive to fusidic acid.

Microsomal membranes from bovine heart homogenates were subfractionated by density gradient centrifugation. Fractions with high levels of a sarcolemmal (SL) marker are enriched in specific carbonic anhydrase (CA) activity up to ninefold compared with the microsomes. Fractions with high levels of a sarcoplasmic reticulum marker and a mitochondrial marker, respectively, exhibit specific CA activities that are similar to the one found in the microsomes. Determination of cytosolic markers reveals that the CA activity in the SL fraction is not due to contamination by cytosolic CA, and it is shown by Triton X-114 phase separation that the CA activity is due to an integral membrane protein. In cryosections from rabbit heart the SL region of cardiomyocytes is stained by the fluorescent CA inhibitor dansylsulfonamide. Intracellular staining occurs also, with a pattern suggesting the presence of CA associated with intracellular membranes. Although it cannot be excluded that there is a contribution by endothelial membranes, it appears likely that most CA of the heart is bound to the SL. The possible involvement of the enzyme in extracellular proton buffering is discussed.

We have studied the distribution of carbonic anhydrases (CA) in several skeletal muscles of the hindlimb of rabbits and rats and in cardiac muscle of the rabbit. To remove erythrocyte CA, hindlimbs and hearts were thoroughly perfused with dextran solution, and the effectiveness of the perfusion was in most cases assessed by determining the contamination of the muscles with radioisotopes that had been used to label the erythrocytes before the perfusion was started. We observed three forms of CA: (1) cytosolic (sulphonamide-resistant) CA III; (2) a cytosolic sulphonamide-sensitive CA, probably isoenzyme II; (3) a membrane-bound form that was extracted from the particulate fraction using Triton X-100. These CA isoforms were distributed as follows. (1) CA III is located in the cytoplasm of slow, oxidative skeletal muscles and is absent from or low in fast skeletal and cardiac muscle; this holds for rabbits and rats and is identical with the pattern previously described for several other species. (2) The cytosolic sulphonamide-sensitive CA is present in fast rabbit muscles and absent from slow muscles of this species. In contrast, all skeletal muscles of the rat studied here lack, or possess only very low, activity of this isoenzyme. (3) The membrane-bound form of CA is present in all rabbit muscles studied; its activity appears somewhat higher in fast than in slow skeletal muscles. (4) Cardiac muscle constitutes an exception among all striated muscles of the rabbit as it possesses no form of cytosolic CA but a high activity of the membrane-bound form.

Transfer of a 36-kilobase piece of human DNA containing the beta-interferon (IFN-beta) gene into mouse Ltk-cells leads to transient expression of human interferon even without an exogenous inducer. A low level of human interferon expression is also found in most stable clones containing the transferred DNA. With double-stranded RNA or Newcastle disease virus (NDV) as inducer, human interferon expression is greatly increased. The induced transcript is identical to normal human IFN-beta mRNA. Neighbouring genes contained on the transferred DNA are co-induced but are not essential for the production of human interferon in mouse L cells.