bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in membrane ionic conductance, but not changes in slip, can account for the non‐linear dependence of the electrochemical proton gradient upon the electron‐transport rate in chromatophores
Authors: COTTON Nicholas P. J. , CLARK Adam J. , JACKSON J. Barry
Published: March 2005

Decrease in the rate of cyclic electron transport (JE) measured from the absorbance changes associated with reaction centre bacteriochlorophyll led to a less than proportionate decrease in the membrane potential (ΔΨ) measured by electrochromism. In principle this result can be explained either by a ΔΨ‐dependent slip in the H+/e− coupling ratio (nE) or by a ΔΨ‐dependent change in the membrane ionic conductance. Simultaneous measurement of the membrane ionic current (JDIS) did not reveal any significant changes in the H+/e− ratio (JDIS/JE) and showed that conductance changes (JDIS/ΔΨ) account quantitatively for the curved dependence of ΔΨ on JE.

Simultaneous recordings of JDIS and the extravesicular pH from cresol‐red absorbance changes, suggest that protons are the main current‐carrying species across the chromatophore membrane at high values of ΔΨ in the presence and absence of F0‐ATPase inhibitor. At reduced ΔΨ the flux of other ions outweighs the hydrogen ion current.
Oxido‐reduction of B800–850 and B880 holochromes isolated from three species of photosynthetic bacteria as studied by electron‐paramagnetic resonance and optical spectroscopy
Authors: PICOREL Rafael , LEFEBVRE Suzie , GINGRAS Gabriel
Published: March 2005

Certain redox properties of bacteriochlorophyll a were used to probe the structure of several light‐harvesting pigment‐protein complexes or holochromes. To attribute redox properties unequivocally to a given holochrome, we worked with purified holochromes. We developed purification procedures for the B880 holochromes from Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Ectothiorhodospira sp. and for the B800–850 holochromes from the latter two species. In all these holochromes, bacteriochlorophyll a could be oxidized by ferricyanide as witnessed by the bleaching of their near‐infrared absorption bands. However, only in B880 holochromes was this oxidation reversible. Another important difference between the B800–850 and the B88O holochromes is that oxidation of the latter gives rise to a g= 2.0025 electron paramagnetic resonance (EPR) signal with linewidth varying, according to species, from 0.37mT to 0.48mT. Both the reversible EPR signal and absorption changes titrate with a midpoint redox potential (pH 8.0) of approximately 570 mV. Linewidth narrowing can be interpreted by delocalization of the free electron spin over approximately 12 bacteriochlorophyll molecules. While the B880 holochromes from the three species considered had indistinguishable redox properties, the B800–850 holochromes differed from one another by their circular dichroic spectra and by the relative ease of oxidation of their 800‐nm and 850‐nm bands. This indicates that, contrary to the B880 holochromes, the B800–850 holochromes may not form a homogeneous class.
Histone H1 kinase from mouse plasmacytoma
Authors: QUIRIN‐STRICKER Christiane
Published: March 2005

A cAMP‐independent protein kinase which phosphorylates histone H1 to a high level and which may correspond to the mitotic H1 kinase has been partially purified and characterized from mouse plasmacytoma microsomes [Quirin‐Stricker, C., and Schmitt, M. (1981) Eur. J. Biochem. 118, 165–172]. The present study compares the microsome‐associated and the chromatin‐associated histone H1 kinases isolated from mouse plasmacytoma cells. The results indicate that the two H1 kinases are indistinguishable by several criteria.

The molecular structure of the microsome‐associated histone H1 kinase has been determined (a) by exclusion chromatography on Ultrogel, (b) by electrophoresis in non‐denaturing polyacrylamide gels of graded porosity and (c) by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the H1 kinase activity peak from an AcA‐34 Ultrogel column. All these techniques gave the same result: H1 kinase may exist in a native form as a monomeric enzyme with an apparent relative molecular mass of 90000 ± 8000.
Repression by dexamethasone of epinephrine‐induced modulation of the fatty acyl‐CoA desaturase system in Tetrahymena microsomes
Authors: UMEKI Shigenobu , NOZAWA Yoshinori
Published: March 2005

Epinephrine modulation of the fatty acyl‐CoA desaturase activities in Tetrahymena microsomes, namely the stimulation of Δ9 desaturase activity with a decreased activity of Δ12 desaturase, was markedly inhibited by dexamethasone administered 30min prior to epinephrine addition. This effect by dexamethasone was dose‐dependent. With epinephrine or dexamethasone prior to epinephrine administration, the activities of Δ9‐terminal and Δ12‐terminal components were found to change in parallel with those of Δ9 and Δ12 desaturase activities respectively. Furthermore, addition of dexamethasone as early as 30 min before epinephrine repressed the epinephrine‐mediated stimulation of cyclic AMP production and of adenylate cyclase activity. Therefore, it is suggested that dexamethasone may inhibit the modulation by epinephrine of the terminal component activity, which plays a crucial role in microsomal fatty acyl‐CoA desaturase system, through the reduction of cyclic AMP accumulation by decreased responsiveness to epinephrine of Tetrahymena cells.
Enzymic synthesis of the iron‐sulfur cluster of spinach ferredoxin
Authors: PAGANI Silvia , BONOMI Franco , CERLETTI Paolo
Published: March 2005

A biologically active spinach ferredoxin was reconstituted from the apoprotein by incubation with catalytic amounts of the sulfurtransfcrase rhodanese in the presence of thiosulfate, reduced lipoate and ferric ammonium citrate. Analytical and spectroscopical features of the reconstituted ferredoxin were identical to those of the native one; yield of the reconstitution reaction was 80%. Yields and kinetic parameters of the enzymic and chemical rcconstitution were also compared. The higher efficiency of the enzymic system is ascribed to a productive interaction between rhodanese and apoferredoxin favouring the process of cluster build‐up and insertion. The physiological relevance of this synthetic activity is discussed.
Conformation of physalaemin
Authors: BERNIER Jean‐Luc , HÉNICHART Jean‐Pierre , HELBECQUE Nicole
Published: March 2005

The conformational and spatial configuration of the biologically active undecapeptide physalaemin was studied using 350‐MHz 1H NMR. The NMR analyses suggested the existence of a strong hydrogen bond between the amide proton of the Phe7 and a carbonyl group in the N‐terminal moiety, most likely the Pro4 one. Other bondings were postulated, involving the side‐chain amine of Lys6 and the side‐chain amide of Asn5 and respectively the side‐chain carboxyl of Asp3 and the terminal amide carbonyl of Met‐NH2. Thus unlike its shorter peptidic fragments, physalaemin exhibited a stable molecular structure in solution, giving some insight into the conformation required for interaction at the biological receptor of tachykinins.
Conformational studies on the pancreatic polypeptide hormone family
Authors: GLOVER Ian D. , BARLOW David J. , PITTS James E. , WOOD Stephen P. , TICKLE Ian J. , BLUNDELL Thomas L. , TATEMOTO Kazuhiko , KIMMEL Joe R. , WOLLMER Axel , STRASSBURGER Wolfgang , ZHANG You‐Shang
Published: March 2005

Pancreatic polypeptide has been extracted and sequenced from a wide range of species. The 36‐residue polypeptides have some hormonal characteristics, and show a high degree of sequence homology. Two recently isolated polypeptides, from porcine gut and brain, also show a high degree of sequence homology with the pancreatic polypeptides. It was proposed that these polypeptides were members of a related family.

The X‐ray determined structure of one member of the family, turkey pancreatic polypeptide, is known to high resolution, but there is no structural information for the others. Studies designed to give an insight into the tertiary structure of these related molecules have been carried out, including model building using interactive computer graphics, circular dichroic spectroscopy and secondary structure prediction using a variety of algorithms.

The results indicate that a compact globular conformation, similar to that observed in turkey pancreatic polypeptide may be adopted by all molecules and that this may be more highly conserved than the individual amino acid sequences.
Calmodulin antagonists inhibit activity of myosin light‐chain kinase independent of calmodulin
Authors: ZIMMER Manfred , HOFMANN Franz
Published: March 2005

The calmodulin antagonists W‐7, trifluoperazine and R24571 in vitro inhibited calmodulin‐dependent and independent myosin light chain kinase activity with IC50 values of about 300 μM, 140 μM and 18 μM in the presence of 8 mg/ml myosin light chains. These IC50 values decreased to 15 μM. 6 μM and 2.5 μM when the concentration of myosin light chains was decreased to 0.4 mg/ml in the presence of saturating concentrations of calmodulin. Endogeneous tyrosine fluorescence of myosin light chain measured at 334 nm was quenched concentration dependently by trifluoperazine and R24571. In addition, fluorescence of W‐7 measured at 370 nm was quenched concentration dependently by myosin light chains. The quenching of fluorescence which was independent of calcium, suggested that all three compounds bound to myosin light chain. The IC50 values for trifluoperazine obtained from fluorescence quench curves at different concentrations of myosin light chain were almost identical, with those obtained under similar conditions from inhibition curves of myosin light chain kinase. These results indicate that ‘calmodulin antagonists’ inhibit the activity of myosin light chain kinase independent of calmodulin by binding to myosin light chain. The implication of this finding for the interpretation of results obtained in vivo by the use of ‘calmodulin antagonists’ is discussed.
Phosphorylation of the ribosomal protein S6 during agonist‐induced exocytosis in exocrine glands is catalyzed by calcium‐phospholipid‐dependent protein kinase (protein kinase C)
Authors: PADEL Ulrike , SÖLING Hans‐Dieter
Published: March 2005

The ribosomal protein S6 in exocrine cells is phosphorylated during stimulated during stimulation of exocytosis by cAMP‐dependent or calcium‐dependent agonists. Under both conditions the same tryptic S6 phosphopeptides (termed A, B, and C) were found [Padel, Kruppa, Jahn & Söling (1983) FEBS Lett. 159, 112–118]. Studies have now been made of the phosphorylation pattern of protein S6 from purified guinea pig parotid ribosomes following in vitro phosphorylation with calmodulin‐dependent, phospholipid‐dependent, and cAMP‐dependent protein kinases. Only the phospholipid‐dependent enzyme led to the phosphorylation of peptides A, B, and C, while the cAMP‐dependent enzyme phosphorylated only peptides A and C, and the calmodulin‐dependent enzyme did not phosphorylate any of the phosphopeptides found in S6 from unstimulated or stimulated intact cells. Guinea pig parotid microsomes contain substantial phospholipid‐dependent protein kinase activity. Stimulation of intact parotid glands with tetradecanolyphorbol acetate led to a significant phosphorylation of S6 and a similar tryptic S6 phosphopeptide pattern as seen with carbamoylcholine. It is concluded that activation of phospholipid‐dependent protein kinase is responsible for the phosphorylation of protein S6 during stimulation with calcium‐dependent and cAMP‐dependent secretagogues.
Reversible chemical cross‐linking of the light‐harvesting polypeptides of Rhodopseudomonas viridis
Authors: LUDWIG Franz Rupert , JAY Frances Alexandra
Published: March 2005

The topography of the light‐harvesting polypeptides of Rhodopseudomonas viridis was investigated using cleavable chemical cross‐linkers. To this end a set of succinimidyl esters and surface‐specific sulfosuccinimidyl esters of different span widths were synthesized. The corss‐linking reagents have been characterized using NMR and infrared spectroscopy and thin‐layer chromatography. The cross‐linking reaction was carried out under physiolocigal conditions and the aggregates were analyzed by the methods of one‐ and two‐dimensional polyacrylamide gel electrophoresis and by immunoblot analysis. We found cross‐linkage between B1015‐α and B1015‐α, between B1015‐α and B1015‐β and B1015‐β. Aggregates of higher molecular mass were hetero‐oligomers of B1015‐α and B1015‐β contaning three and four polypeptides, respectively. The results obtained in this work indicate a very tight contact among the light‐harvesting polypeptides. We assume that the light‐harvesting polypeptides are localized alternately as dimers of B1015‐α and B1015‐β around the reaction centre core.
An azide‐insensitive low‐affinity ATPase stimulated by Ca2+ or Mg2+ in basal‐lateral and brush border membranes of kidney cortex
Authors: ILSBROUX Ingrid , VANDUFFEL Luc , TEUCHY Henri , DE CUYPER Marcel
Published: March 2005

Basal‐lateral and brush border membranes from pig kidney cortex were prepared by differential centrifugation followed by free‐flow electrophoresis. In each type of membrane, azide‐insensitive, low‐affinity Ca2+‐ATPase and Mg2+‐ATPase activities are demonstrated. A comparative study for both membranes further reveals the following analogies between these ATPase: (a) they show maximal activity between pH 8 and 8.5; (b) they exhibit Km values for Ca‐ATP or Mg‐ATP in the millimolar range and have a comparable low substrate specificity; (c) they are insensitive to 10 μM of vanadate, N,N′‐dicyclohexylcarbodiimide, diethylstillbestrol, quercetin, harmaline and amiloride. The partial inhibition by 1 mM of the various compounds is rather aspecific. In view of these similarities it is concluded that only one enzyme entity is responsible for the activity which is measured in both membrane types.

The HCO3−‐stimulated Mg2+‐ATPase activity in pig kidney cortex was also studied. This enzyme, however, is clearly of mitochondrial origin since the HCO3−‐stimulation coincides with the distribution profile of succinate dehydrogenase, a mitochondrial marker; and since it is inhibited by azide.
Folding of thermolysin fragments
Authors: VITA Claudio , FONTANA Angelo , CHAIKEN Irwin M.
Published: March 2005

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH‐terminal fragments 121–316, 206–316 and 225(226)–316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two‐fragment complex composed of fragments 5–224(225) and 225(226)–316. In aqueous solution at neutral pH, all the COOH‐terminal fragments attain a native‐like conformation, as judged both by the content of secondary structure deduced from far‐ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH‐terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn · HCl). The phase transition curves were analyzed for Tm (temperature of half‐denaturation) and Gibbs free energies (ΔGD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)–316 ≤ 206–316 < 121–316 < thermolysin S < thermolysin. The ranking of ΔGD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C‐labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206–316 of native thermolysin indicate that the COOH‐terminal fragments adopt native‐like conformations which are in equilibrium with non‐native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)–316, to 206–316, to 121–316. Fragment 225(226)–316, when combined with fragment 5–224(225) in the thermolysin S complex, adopts a more stable native‐like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH‐terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.
Second lytic target of β‐lactam compounds that have a terminal d‐amino acid residue
Authors: TSURUOKA Tsutomu , TAMURA Atsushi , MIYATA Aiko , TAKEI Tamako , INOUYE Shigeharu , MATSUHASHI Michio
Published: March 2005

A new biochemical mechanism of lysing bacterial cells by treatment with certain β‐lactam compounds that possess a terminal d‐amino acid moiety in their side chain was demonstrated. The two functions of the molecule, the β‐lactam and terminal d‐amino acid moiety, are both involved in the activity of lysing gram‐negative bacteria, which is characterized by very rapid lysis of the cells in the first few hours after their contact with the compound. This mechanism was proved by studies on one such compound, named MT‐141, which contains a terminal d‐cysteine moiety with free amino and carboxyl groups in the 7β‐side chain of the 7α‐methoxy‐cephalosporin skeleton. This compound bound to the cell‐wall peptidoglycan of Escherichia coli through the d‐amino group of its terminal d‐amino acid moiety and this seemed to cause rapid cell lysis. Both activities, of binding to peptidoglycan and of causing rapid cell lysis, were inhibited by certain d‐amino acids, but not by l‐amino acids. Mutants were isolated that had simultaneuously gained decreased senstivity to this kind of α‐lactam compound and supersensitivity to globomycin, an inhibitor of formation of lipoproteins which function in linking the peptidoglycan to the outer membrane. These results that binding of the terminal d‐amino acid moiety of the β‐lactam compound to peptidoglycan somehow influences formation of the linkage between the outer membrane and the peptidoglycan and consequently enhances the cell lytic activity of the β‐lactam portion of the molecule.
Scatter analysis of discrete‐sized chromatin fragments favours a cylindrical organization
Authors: LASTERS Ignace , WYNS Lode , MUYLDERMANS Serge , BALDWIN John P. , POLAND George A. , NAVE Colin
Published: March 2005

Fragments of chromatin containing 23±2.5 nucleosomes have been fractionated after light nuclease treatment of chicken erythrocyte nuclei. Low‐angle scattering measures the total z‐average radius of gyration of the already well‐defined particles and the shape of scatter curves can be compared with three‐dimensional analysis as opposed to cross‐section analysis of long chromatin fragments. The data show that the particles are not spherical, have no detectable hole in the center of the structure and are best represented by a solid rod‐like shape such as that generated by a coil of nucleosomes with the centre perhaps filled with linker DNA and histone H1/H5. 23 nucleosome fragments, where the DNA is partially fragmented, have near‐identical scatter curves to the above‐defined intact particles, indicating the primary importance of histone proteins in maintaining the integrity of the chromatin higher‐order structure. Neutron scattering shows the radii of gyration to be contrast‐independent, which fits in with the model calculations for solenoids. Particles with fragmented DNA and the intact particles, therefore, behave as sections of a solenoidal higher‐order structure and possibly are observed as ‘superbeads’ only during the folding and unfolding pathways of nucleosome multimers.
Non‐disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels
Authors: HOLLER Eggehard , FISCHER Heinrich , SIMEK Helmut
Published: March 2005

A non‐disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels. The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography. Cell extracts from a variety of organisms were studied using this method. The activity from Escherichia coli crude extracts migrated in a position corresponding to a higher molecular mass than did purified preparations of DNA polymerase I. DNA polymerases of higher organisms generally migrated in positions corresponding to 400–900 kDa, in some cases, close to 200 kDA.
The amino acid sequences of the copper/zinc superoxide dismutases from swordfish and Photobacter leiognathi confirm the predictions made from the compositions
Authors: CORNISH‐BOWDEN Athel
Published: March 2005

Recent suggestions that the amino acid sequence of the copper/zinc superoxide dismutases of swordfish and Photobacter leiognathi do not support the theory that the bacterium obtained the gene for the enzyme by transfer from its eucaryotic symbiont [Rocha, H. A., Bannister, W. H. and Bannister, J. V. (1984) Eur. J. Biochem. 145, 477–484] are examined. The amount of difference between the sequences is in good agreement with expectation from the amino acid compositions. Moreover, the gene‐transfer hypothesis cannot be discarded without postulating an enormous increase in the rate at which the superoxide dismutase gene has accumulated amino acid substitutions since the divergence of the swordfish and cattle lineages.
Glutamic acid decarboxylase in tubules and glomeruli isolated from rat kidney cortex
Authors: BURGMEIER Nicole , ZAWISLAK Richard , DEFEUDIS Francis V. , BOLLACK Claude , HELWIG Jean‐Jacques
Published: March 2005

4‐Aminobutyric acid (GABA) synthesis was examined in purified glomeruli and tubules of rat kidney cortex that were incubated in the presence of [2,3‐3H2]glutamate. The GABA that was formed was separated from glutamate using anion‐exchange resin, and identified by means of an automatic amino acid analyser. In the renal cortex only the tubules were able to form GABA (35.0 nmol mg‐1 h‐1); the remaining GABA synthesis found in the glomerular preparations can most probably be attributed to a contamination by cortical tubules (9%), as shown by determination of a known tubular marker enzyme (l‐γ‐glutamyltransferase). Hydroxylamine (1 mM) and ethanolamine‐O‐sulfate (10 mM), well‐known inhibitors of cerebral GABA formation and GABA catabolism respectively, inhibited renal tubular GABA formation at 100% and 44% respectively.
Adenosine kinase from bovine adrenal medulla
Authors: ROTLLAN Pedro , MIRAS PORTUGAL Maria Teresa
Published: March 2005

Adenosine kinase from bovine adrenal medulla was purified 1600‐fold by using ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration yielded a relative molecular mass around 42000 and Michaelis constants were 0.2 μM for adenosine and 20 μM for MgATP. The enzyme showed a broad specificity for purine nucleoside triphosphate as phosphate donors. Both free Mg2+ and ATP were inhibitors.

AMP was a competitive inhibitor with regard to adenosine and a non‐competitive inhibitor versus MgATP, while ADP was a uncompetitive inhibitor with regard to adenosine and a non‐competitive inhibitor versus MgATP.

Adenosine kinase was strongly inhibited by the bis(adenylyl) polyphosphates Ap4A and Ap5A. These compounds inhibited the enzyme competitively versus MgATP (Ki= 0.06 μM for Ap4A and 0.4 μM for Ap5A) and uncompetitively with regard to adenosine. The results of the kinetic analysis suggest an ordered bi‐bi mechanism, adenosine being the first substrate. The phosphorylation of adenosine was unaffected in the presence of vanadate ions.
Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense
Authors: REINWALD Erwin
Published: March 2005

The effects of tunicamycin on different aspects of structure and biosynthesis of variant surface glycoprotein from Trypanosoma congolense have been studied.

Deglycosylated variant antigen becomes synthesized in vitro, is transported through the cell, and is deposited on the cell surface in equivalent amounts compared to the glycosylated species.

In contrast to the glycosylated molecule only marginal amounts of high‐molecular‐mass fragments can be removed from the parasitic cell by externally added proteases in the case of tunicamycin‐treated cells.

Most of the material removed by proteases from the cell surface of tunicamycin‐treated cells has a molecular mass lower than 2 kDa.

Many additional proteolytic cleavage sites become accessible after removal of the glycan chains. There is no indication that in the deglycosylated molecule the same prefential protease‐sensitive site exists as is found in the glycosylated species.

These results suggest that glycosylation of variant surface glycoprotein could be important for the survival of the parasite within the host organism.
Biosynthesis of the O9 antigen of Escherichia coli
Authors: JANN Klaus , PILLAT Michael , WEISGERBER Christoph , SHIBAEV Vladimir N. , TORGOV Vladimir I.
Published: March 2005

Synthetic monosaccharide derivatives (α‐glucosyl, β‐glucosyl, α‐mannosyl) and disaccharide derivative (α‐manosyl‐1,2‐α‐glucosyl, α‐mannosyl‐1,3‐α‐glucosyl, α‐mannosyl‐1,4‐α‐glucosyl, α‐mannosyl‐1,6‐α‐glucosyl) of diphosphomoraprenol were used as putative mannose acceptors in the biosynthesis of Escherichia coli O9 antigen. Membranes of E. coli O9 derived from the rfe mutant F 1357 were reconstituted with these compounds and then incubated with different concentrations of GDP‐[14C]mannose. Of the monosaccharide derivatives tested, only α‐glucodiphosphomoraprenol was a mannose acceptor and the only disaccharide derivative which accepted mannose was α‐mannosyl‐1,3‐α‐glucosyldiphosphomoraprenol. The α‐glucosyl derivative accepted only one mannose unit at 4 μM GDP‐[14C]mannose, and above 50 μM GDP‐[14C]mannose about 25% of the product had a minimum size of about 30 mannose units. The α‐mannosyl‐1,3‐α‐glucosyl derivative was only a mannose acceptor at a GDP‐[14C]mannose concentration of 50 μM and higher, and the product had a minimum size of about 30 mannose units. The results are discussed with respect to requirement of mannose acceptors.
Prokaryotic triterpenoids
Authors: RENOUX Jean‐Marie , ROHMER Michel
Published: March 2005

Together with bacteriohopanetetrol, which is identical to bacteriohopanetetrol isolated from Bacillus acidocaldarius, three other bacteriohopane derivatives were isolated from the methylotrophic bacterium Methylobacterium organophilum. Three different kinds of polar moieties were found linked to the C‐35 hydroxyl group of bacteriohopanetetrol: glucosamine linked through a glycosidic bond and two new polyhydroxylated methylcyclopentanoids differing one from each other by the presence of an amino or a guanidino group and linked through an ether bond. The significance of the presence of these compounds in terms of membrane reinforcement and DNA/triterpenoid interactions is discussed.
Evidence that treatment of platelets with phorbol ester causes proteolytic activation of Ca2+‐activated, phospholipid‐dependent protein kinase
Authors: TAPLEY Peter M. , MURRAY Andrew W.
Published: March 2005

Incubation of human platelets with the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) caused the accumulation of a protein kinase in the particulate fraction which was not dependent on Ca2+ and phosphatidylserine (Ptd‐Ser). The Ca2+/Ptd‐Ser‐independent kinase eluted from DEAE‐cellulose at a NaCl concentration of 0.18–0.22 M compared with 0.08–0.16 M for Ca2+/Ptd‐Ser‐dependent protein kinase (C‐kinase). Formation of the Ca2+/Ptd‐Ser‐independent kinase in 12‐O‐tetradecanoylphorbol‐13‐acetate‐treated platelets was blocked by leupeptin, an inhibitor of Ca2+‐dependent neutral proteases. The Ca2+/Ptd‐Ser‐independent kinase and C‐kinase both catalysed the same pattern of phosphorylation of smooth muscle myosin light chains and histone H1 as detected by one‐dimensional or two‐dimensional peptide mapping after tryptic digestion. The phosphorylation sites were different from those obtained with myosin light chain kinase or cAMP‐dependent kinase. The Ca2+/Ptd‐Ser‐independent kinase and C‐kinase had Mr values of about 50000 and 77000 respectively as determined by sucrose‐gradient centrifugation. It was concluded that 12‐O‐tetradecanoylphorbol‐13‐acetate induces the proteolytic cleavage of C‐kinase to a Ca2+/Ptd‐Ser‐independent form.
Modulation of adenylate cyclase of human platelets by phorbol ester
Authors: JAKOBS Karl H. , BAUER Silvia , WATANABE Yasuhiro
Published: March 2005

The influence of the phorbol ester, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), a direct avtivator of the Ca2+‐activated, phospholipid‐dependent protein kinase (protein kinase C), was studied on regulation of human platelet adenylate cyclase. Intact platelets were pretreated with the phorbol ester and, thereafter, membranes were prepared and the regulation of the hormone‐sensitive adenylate cyclase in these membranes was studied. The following data were obtained: The TPA treatment applied had apparently no effect on the activity of the catalytic moiety of the platelet adenylate cyclase nor on the stimulatory Ns protein nor on stimulatory hormone receptors (prostaglandin E1) and the mutual interactions of these components of the stimulatory hormone‐sensitive pathway. However, the TPA treatment of intact platelets largely impaired the GTP‐dependent, hormone‐sensitive inhibitory pathway to the adenylate cyclase, involving inhibitiory Ni protein. The pretreatment led to a large reduction or loss of adenylate cyclase inhibition by GTP itself and by the inhibitory agonists, epinephrine and thrombin, inhibiting the untreated enzyme via separate receptors by an Ni‐mediated process. In contrast, platelet adenylate cyclase inhibition not involving the Ni protein was not affected by the TPA treatment. The observed effects of TPA were very rapid in onset and were not shared by a derivative of TPA which did not activate protein kinase C. The data obtained suggest than protein kinase C activated by the phorbol ester interferes with the platelet adenylate cyclase system, leading to a specific alteration of the Ni‐protein‐mediated signal transduction to the adenylate cyclase.
Protein kinase C phosphorylates the inhibitory guanine‐nucleotide‐binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase
Authors: KATADA Toshiaki , GILMAN Alfred G. , WATANABE Yasuhiro , BAUER Silvia , JAKOBS Karl H.
Published: March 2005

Human platlet membrane proteins were phosphorylated by exogenous, partially purified Ca2+‐activated phospholipid‐dependent protein kinase (protein kinase C. the phosphorylation of one of the major substrates fro protein kinase C (Mr= 41000) wa specifically suppressed by the β subunit of the inhibitory guaninenucleotide‐binding regulatory component (G1, Ni) of adenylate cyclase. The free α subunit of Gi (Mr= 41000) also served as an excellent substrate for the kinase (<0.5 mol phosphate incorporated per mol of subunit), but the Gi oligomer (α·β·γ) did not. Treatment of cyc‐ S49 lymphoma cells, which are dificient in Gs/Ns(the stimulatory component) but contain functional Gi/Ns, with the phorbol ester, 12‐O‐tetradecanoylophorbol 13‐acetate, a potent activator of protein kinase C, did not alter stimulation of adenylate cyclase catalystic activity by forskolin, whereas the Gi/Ni‐mediated inhibition of the cyclase by the hormone, somatostatin, was impaired in these membranes. The results suggest that the α subunit of the inhibitory guanine‐nucleotide‐binding regulatory component of adenylate cyclase may be a physiological substrate for protein kinase C and that the function of the component in transducing inhibitory hormonal signals to adenylate cyclase is altered by its phosphorylation.
Synthesis and degradation of unassembled polypeptides of the coupling factor of photophosphorylation CF1 in 70S ribosome‐deficient rye leaves
Authors: BIEKMANN Siegbert , FEIERABEND Jürgen
Published: March 2005

The formation of polypeptides of the coupling factor CF1 was investigated in 70S ribosome‐deficient rye leaves generated by growing the plants at a non‐permissive elevated temperature of 32°C, in order to analyse mechanisms coordinating subunit accumulation. Antibodies were raised in rabbits against total CF1 as well as against its five individual subunits purified from chloroplast thylakoids from rye leaves. Several immunological techniques applying these antibodies (immunoprecipitation, immunoblotting, antibody affinity chromatography) were unable to detect the presence of any of the CF1 subunits in heat‐treated 70S ribosome‐deficient leaves. After in vivo labeling with l‐[35S]methionine and subsequent immunoprecipitation, however, radioactivity was found to be incorporated into the subunits γ and δ, but not into α, β and ɛ, in 70S ribosome‐deficient leaves, demonstrating the cytoplasmic synthesis of CF1‐γ and CF1‐δ. Chase experiments after in vivo labeling with L‐[35S]methionine indicated that the unassembled subunits γ and δ were rapidly and preferentially degraded, while they were stabilized when integrated into the complete CF1 complex in normal green leaves from permissive growth conditions. The apparent half‐times of the unassembled subunits were 2 h for CF1‐γ and 4 h for CF1‐δ in 32°C‐grown leaves. Several other, stromal, plastid proteins of cytoplasmic origin were stable in 32°C‐grown leaves during the period of chase. In etiolated leaves total CF1, including all subunits, appeared to be less stable than in green leaves grown under permissive temperature conditions in light. Rapid degradation of the excess of unassembled subunits is regarded as an important mechanism ensuring a constant stoichiometry and apparently synchronous development of CF1 subunits.
Equilibrium binding of thioredoxin FB to chloroplastic fructose bisphosphatase
Authors: SOULIÉ Jean‐Michel , BUC Jean , RIVIÉRE Mireille , RICARD Jacques
Published: March 2005

Thioredoxin fB, the protein activator of chloroplastic fructose 1,6‐bisphosphatase, strongly binds its target enzyme with a stoichiometry of one protein dimer per enzyme tetramer. The thioredoxin binding site is distinct from the active site and the dissociation constant of the protein‐enzyme complex has the extremely small value of 769 nM at pH 7.5. This interaction involves both ionic and hydrophobic contributions and is enhanced by a pH increase from 7 to 8. These results suggest that the above molecular properties may be involved in the light activation of chloroplastic fructose bisphosphatase.
Weakly basic amines inhibit the proteolytic conversion of proalbumin to serum albumin in cultured rat hepatocytes
Authors: ODA Kimimitsu , IKEHARA Yukio
Published: March 2005

Effects of weak amines on the proteolytic conversion of proalbumin to serum albumin were studied in primary culture of rat hepatocytes. In control culture proalbumin was converted to serum albumin before discharge into the medium. However, in the presence of chloroquine the conversion to serum albumin was inhibited and proalbumin per se was released into medium. A similar inhibition of the processing was also observed in the presence of other amines such as methylamine and NH4Cl. Thus weak amines mimic the carboxylic ionophore monensin with regard to the effect on proalbumin conversion [Oda & Ikehara (1982) Biochem. Biophys. Res. Commun. 105, 766–772]. Since proteolytic conversion of proalbumin is believed to occur at the Golgi complex, these results suggest that weakly basic amines perturb the Golgi complex in addition to lysosomes and endosomes.
Translation kinetics in cultured mouse hepatoma cells
Authors: LEDFORD Barry E. , JACOBS Donna F.
Published: March 2005

The synthesis of albumin in the liver has been shown to correlate with the availability of essential amino acids in the diet. We have investigated this phenomenon in the highly differentiated mouse hepatoma cell line, Hepa. Cells were grown for three days in complete medium with daily changes. The cells were then incubated for 22 h in media containing varying concentrations of individual essential amino acids. The deficient media were then changed; 1.5 h later the cells were labeled for 0.5 h with [3H]leucine. Albumin was immunoprecipitated and total protein was acid‐precipitated from postribosomal supernatants of detergent‐solubilized cells. With the exception of isoleucine, the relative rates of albumin synthesis decreased as a function of amino acid concentration from 4.3% in complete medium to 2.5% in totally deficient media. This specific reduction in albumin synthesis was confirmed by analysis of labeled Hepa proteins displayed on sodium dodecyl sulfate/polyacrylamide gels. Essential amino acid limitation reduced total protein synthesis by 50%. This is the result of a decrease in the translation efficiency of total mRNA from 5 to 3 polypeptides/message min−1 and is consistent with a reduction in the initiation rate. In contrast, the 70% decrease in albumin synthesis was a result of a reduced number of functional albumin messages/cell. The translation efficiency of these albumin messages remained unchanged at 1.
DNA sequence analysis of the oli1 gene reveals amino acid changes in mitochondrial ATPase subunit 9 from oligomycin‐resistant mutants of Saccharomyces cerevisiae
Authors: OOI Beng Guat , NOVITSKI Charles E. , NAGLEY Phillip
Published: March 2005

The nucleotide sequence of the oli1 gene encoding mitochondrial ATPase subunit 9 (76 amino acids) has been determined for five oligomycin‐resistant mutants of Saccharomyces cerevisiae. Three of the mutations affect amino acids in the vicinity of the glutamic acid residue 59 at which dicylohexyl carbodiimide binds. Two other mutations lead to substitution of amino acid 23, which would lie very close to residue 59 in the folded hairpin conformation that this protein is thought to adopt in the inner mitochondrial membrane. The apposition of residues 23 and those adjacent to residue 59, lying respectively in the two hydrophobic membrane‐spanning arms of subunit 9, is considered to constitute an oligomycin‐binding domain. By consideration of the amino acid substitutions in those mutants cross‐resistant to venturicidin, a domain of resistance for venturicidin is defined to lie within the oligomycin‐binding domain, also centred on residues 23 and 59. These data also clarify the genetic recombination behaviour of alleles previously defined to form part of the oli3 locus (mutants characterized by resistance to both oligomycin and venturicidin) together with alleles defined to form part of the oli1 locus (mutants not cross‐resistant to venturicidin). The oli1 and oli3 loci can now be seen to form two overlapping extended groups within the oli1 gene, with sequenced oli3 mutations being as far apart as 125 nucleotides within the subunit 9 coding region of 231 nucleotides.
Demonstration of an Mg2+‐induced conformational change by photoaffinity labelling of the high‐affinity ATP‐binding site of (Na++ K+)‐ATPase with 8‐azido‐ATP
Authors: SCHEINER‐BOBIS Georgios , SCHONER Wilhelm
Published: March 2005

8‐Azido‐ATP (8‐N3ATP) is a substrate of (Na++ K+)‐ATPase from pork kidney and photoinactivates it by binding to the Mr= 100 000 α‐subunit. The photoinactivation requires the presence of Mg2+ even though 8‐azido‐ATP is recognized by the high‐affinity ATP binding site (Kd= 3.1 μM). K+ ions protect the enzyme against photoinactivation as does excess ATP.

To see whether the Mg2+‐requirement of the photoinactivation is due to the action of free Mg2+ or to the existence of an Mg · 8‐azido‐ATP complex, the action of the stable Mg · ATP complex analogue, chromium · 8‐N3ATP (Cr · 8‐N3ATP), was studied. Cr · 8‐N3ATP photoinactivates (Na++ K+)‐ATPase in the absence of Mg2+, but the photoinactivation is enhanced by Mg2+, indicating that the formation of a Mg · ATP complex is an absolute requirement for photoinactivation. However, the interaction of Mg2+ with a low‐affinity site also enhances the photoinactivation. It is therefore concluded that interactions with MgATP and free Mg induce conformational changes in the purine subsite of the high‐affinity ATP binding site.

Controlled trypsinolysis of the [α‐32P]8‐N3ATP‐phostolabelled enzyme in the presence of K− results in the formation of an Mr= 56000 radioactive peptide, whereas trypsinolysis of a [γ‐32P]Cr · ATP‐labelled enzyme under identical conditions forms an Mr= 41000 radioactive peptide. Extensive trypsinolysis of the [α‐32P] 8‐N3ATP‐photolabelled α‐subunit leads to the formation of a radioactive peptide of Mr= 1800.
Structure and function of platelet membrane glycoproteins Ib and V
Authors: WICKI Andreas N. , CLEMETSON Kenneth J.
Published: March 2005

Treatment of platelets with human leukocyte elastase causes a rapid loss in response to von Willebrand factor and a biphasic loss in response to thrombin, first rapid then more slowly. The rapid phase corresponds to cleavage of a 45‐kDa glycopeptide from the extracellular end of membrane glycoprotein GPIb. Longer treatment removes 80‐kDa and 90‐kDa glycopeptides and a glycopeptide corresponding to the major part of GPV. The 45‐kDa and 90‐kDa species could be obtained by elastase treatment of glycocalicin, the major proteolytic cleavage product of GPIb. Polyclonal rabit antibodies against the purified 45‐kDa glycopeptide had the same effect on the action of von Willebrand factor and thrombin on platelets as cleavage of GPIb by elastase. These results indicate that both the von Willebrand factor and thrombin binding sites on GPIb are located in the same region on the outside of the molecule. Thrombin activation of platelets involves at least two receptors, one on the 45‐kDa glycopeptide, which when occupied causes an increase in the speed of response of the platelets to the cleavage of the second. GPV, a candidate for the second receptor, is a hydrophobic glycoprotein that is cleaved from the platelet membrane by several proteases. Proteases that do not activate platelets but degrade the second receptor remove larger fragments from GPV than do proteases such as thrombin or trypsin which activate platelets.
The influence of ganglioside insertion into brain membranes on the rate of ganglioside degradation by membrane‐bound sialidase
Authors: SCHEEL Günther , SCHWARZMANN Günter , HOFFMANN‐BLEIHAUER Peter , SANDHOFF Konrad
Published: March 2005

Microsomal membranes isolated from calf brain contain a sialidase which cleaves ganglioside substrates naturally occurring within these membranes as well as exogenously added [3H]ganglioside GD1a. Micelles of [3H]ganglioside GD1a bind to the microsomal membranes in two steps. The first step, called adsorption, is fast and reversible by treatment with trypsin; the second step, called uptake, is slower and not reversible. The product of the enzymic degradation, [3H]ganglioside GM1, is exclusively located in the ganglioside pool taken up by the sialidase‐bearing membranes, and not in the trypsin‐releasable pool. Electron spin resonance (ESR) studies using a spin‐labelled analogue of ganglioside GD1a indicate that the ganglioside uptake by microsomal membranes is accompanied by the disappearance of the micellar structure and by the ‘dilution’ of the probe molecules with membrane lipids. These findings suggest that exogenously added ganglioside substrate inserts into the microsomal membrane before it is recognized as substrate by the membrane‐bound sialidase. Therefore, the influence of pH, ionic strength and membrane‐fluidizing agents on the degradation rate measured with exogenous ganglioside GD1a does not only reflect kinetic parameters of the enzymic reaction itself but also the velocity of ganglioside insertion. Increasing ionic strength reduces the degradation rate. The acceleration of insertion with falling pH values shifts the measured pH optimum of the ganglioside degradation to lower values (pH 3.6) and masks the substantial residual sialidase activity at pH 5 – 7. The membrane‐fluidizing alcohol n‐hexanol greatly accelerates ganglioside insertion as well as ganglioside degradation. The latter was clearly demonstrated by studying the hydrolysis of endogenous ganglioside substrates, and is due to a decrease of the apparent Km value and an increase in the Vmax value. The Vmax value was also enhanced by freezing and thawing of the microsomal membranes.
Characterization of the purified molybdate‐stabilized glucocorticoid receptor from rat liver
Authors: IDZIOREK Thierry , FORMSTECHER Pierre , DANZE Pierre‐Marie , SABLONNIERE Bernard , LUSTENBERGER Patrick , RICHARD Claude , DUMUR Viviane , DAUTREVAUX Michel
Published: March 2005

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three‐step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high‐performance size‐exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone‐acetonide–receptor complex was obtained in 20% yield with an overall 11800‐fold purification. The dissociation rate constant of this complex was 1.6 × 10−4 min−1. The purified receptor sedimented at 8.3 S in high‐salt and 9.4 S in low‐salt sucrose gradients containing molybdate. A 7.0‐nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high‐salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90000‐Mr band. Three minor bands with Mr of 78000, 72000 and 48000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high‐performance ion‐exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90000‐Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA‐binding state after molybdate removal followed by warming 30 min at 25°C in presence of 0.2% bovine serum albumin: 50–78% transformation yield could be demonstrated by DNA‐cellulose chromatography. Partial transformation could also be obtained at 0°C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.
Isolation and characterization of a blue copper protein from Thiobacillus versutus
Authors: van HOUWELINGEN Tiny , CANTERS Gerard W. , STOBBELAAR George , DUINE Johannis A. , FRANK Jzn Johannes , TSUGITA Akira
Published: March 2005

The blue copper protein induced during growth of Thiobacillus versutus on methylamine was purified and characterized. It is an acidic protein (isoelectric point 4.7), contains one Cu2+ ion/enzyme molecule, is a monomeric protein (molecular mass about 14 kDa), has a maximum in its absorption spectrum at 596 nm (molar absorption coefficient 3.9 × 103 M−1 cm−1), shows an axial type‐I electron paramagnetic resonance spectrum (g‖= 2.239. g‖= 2.046 and A‖= 5.6 mT) and has a redox potential (E0) of + 260 mV. In view of these properties and in view of the fact that the protein is active as an electron carrier between methylamine dehydrogenase and cytochrome c, it is concluded that it is similar to the amicyanins isolated from Methylomonas sp. strain J and Pseudomonas sp. strain AM1.