bluuebunny/crossref_metadata_2025 huggingface dataset:
Hormone‐sensitive adenylate cyclase of prolactin‐producing rat pituitary adenoma (GH4C1) cells: molecular organization
Authors: GORDELADZE Jan O. , SLETHOLT Kjersti , THORN Niels A. , GAUTVIK Kaare M.
Published: March 2005

Hormonal activation and inhibition of the GH4Cl1 cell adenylate cyclase complex is delineated. In the presence of the guanyl nucleotide GTP, enzyme activity was enhanced twofold by thyroliberin, sixfold by vasoactive intestinal peptide (VIP), twofold by prostaglandin E2 and twofold by isoproterenol. The diterpene, forskolin, increased, the activity 14‐fold. In the presence of high GTP (400 μM) and NaCl (150 mM) concentrations, somatostatin inhibited (ED50= 0.5 μM) the cyclase activity by 40%.

In the presence of 10 μM somatostatin, the ED50 values (5 nM) for thyroliberin‐ and VIP‐stimulated adenylate cyclase activities were shifted to 20 nM. Forskolin‐elicited activation was, however, not affected by somatostatin. Cholera‐toxin and pertussis‐toxin pretreatment of the enzyme brought about some 20‐fold and twofold activation, respectively. Inhibition by somatostatin was abolished upon pre‐exposure to pertussis toxin.

Mild alkylation by N‐ethylmaleimide increased basal and hormone‐activated adenylate cyclase while somatostatin again failed to express its inhibitory potential. Further alkylation caused a gradual decline and convergence of hormone‐modulated cyclase activities towards zero. The N‐ethylmaleimide‐induced attenuation of thyroliberin‐elicited activity was paralleled by a decrease in [3H]thyroliberin binding.

Trifluoperazine and an anti‐calmodulin serum reduced basal and net thyroliberin‐, VIP‐ and forskolin‐enhanced cyclase activities by some 30%, 100%, 70% and 80%, respectively. The Vmax of basal and thyroliberin‐stimulated adenylate cyclase was diminished by 65%, leaving the apparent Km values (7.2 mM and 2.6 mM, respectively) for Mg2+ unaltered. Finally, the phorbol ester 12‐O‐tetra‐decanoyl‐phorbol 13‐acetate (TPA) doubled the activity. This effect was counteracted by the protein kinase C inhibitor, polymyxin B, while thyroliberin‐enhanced adenylate cyclase remained unaffected.

In summary, we have described an adenylate cyclase with stimulatory (Rs) and inhibitory (Ri) receptors coupled to a calmodulin‐sensitive holoenzyme through the Gs and Gi type of GTP‐binding proteins. The ratio of the Gs to Gi is high. It appears that the GH4C1 cell adenylate cyclase is also activated by protein kinase C by interference with Gi. Apparently, thyroliberin activates the cyclase both directly through Gs and indirectly via protein kinase C stimulation.
Spectroscopic characterization of a high‐potential monohaem cytochrome from Wolinella succinogenes, a nitrate‐respiring organism
Authors: MOURA Isabel , LIU Ming Y. , COSTA Cristina , LIU Ming C. , PAI Govind , XAVIER António V. , LeGALL Jean , PAYNE William J. , MOURA José J. G.
Published: March 2005

When purified, a high‐potential c‐type monohaem cytochrome from the nitrate‐respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between‐two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem‐methionine coordination (low‐spin form) coexisting with a high‐spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid‐point redox potential measured by visible redox titration of the low‐spin form was approximately +100 mV. Binding cyanide (Ka= 5 × 105 M−1) resulted in the displacement of the methionyl axial residue, and full conversion to a low‐spin, cyanide‐bound form.

Structural features were studied by 300‐MHz 1H‐NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5–10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high‐spin/low‐spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25°C) was extremely temperature‐dependent [δ(323K) –δ(273K) = 7.2 ppm] and was assigned to the S‐CH3 group of the axial methionine. In the ferrous state only a low‐spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned.

Detailed monitoring of the NMR‐redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 × 106 M−1 s−1 at 303 K and pH = 6.31.

A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.
An IncY plasmid‐encoded single‐stranded DNA‐binding protein from Escherichia coli shows the identical pattern of stacked tryptophan residues as the chromosomal ssb gene product
Authors: CASAS‐FINET Jose R. , JHON Nam‐In , KHAMIS Mustafa I. , MAKI August H. , RUVOLO Peter P. , CHASE John W.
Published: March 2005

In an extension of earlier studies on the Escherichia coli plasmid‐encoded single‐stranded DNA‐binding proteins pIP71a SSB, F SSB and R64 SSB [Khamis, M. I., Casas‐Finet, J. R., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) Biochemistry 26, 3347–3354; Casas‐Finet, J. R., Khamis, M. I., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) J. Biol. Chem. 262, 8574–8593], we have investigated the binding of pIP231a SSB to natural and heavy‐atom‐derivatized single‐stranded homopolynucleotides. Fluorimetric equilibrium binding isotherms indicate that pIP231a SSB has a greater solubility at low ionic strength than any other plasmid SSB protein investigated. Furthermore, its complex with mercurated poly(uridylic acid) [poly(Hg5U)] shows a greater resistance to disruption by salt than the other plasmid SSB complexes. Essentially complete binding of pIP231a SSB to poly(Hg5U) could be achieved, and time‐resolved optically detected triplet‐state magnetic resonance (ODMR) techniques could be applied to the complex. These methods allowed complete resolution of the three Trp chromophores of pIP231a SSB. Comparison of wavelength‐selected ODMR results with those obtained for the poly(Hg5U) complex of a point‐mutated chromosomal ssb gene product (Eco SSB) carrying substitutions of Phe for Trp [Khamis, M. I., Casas‐Finet, J. R., Maki, A. H., Murphy, J. B. & Chase, J. W. (1987) J. Biol. Chem. 262, 10938–10945] confirm that Trp40 and Trp54 of pIP231a SSB are stacked in the complex, while Trp88 is not. This is the same distribution of stacked Trp residues found in Eco SSB. These results are confirmed further by specific effects observed on the ODMR signals of pIP231a SSB upon binding to poly(Br5U) and poly(dT), which are known to be caused by the stacking of Trp54 with nucleic acid bases.
Tritium thermal activation study of bacteriorhodopsin topography
Authors: TSETLIN Victor I. , ALYONYCHEVA Tatyana N. , SHEMYAKIN Vladimir V. , NEIMAN Lev A. , IVANOV Vadim T.
Published: March 2005

The action of thermally activated tritium on the purple membrane and delipidated bacteriorhodopsin fragments has been studied, tritium incorporation into specified amino acid residues being quantified by Edman degradation. The membrane environment was found to affect the accessibility of amino acid residues for tritium. Bacteriorhodopsin fragments 14–31, 45–63, 81–89, 171–179, and 210–225 were localized to the membrane interior while fragments 4–12, 32–44, 64–65, 73–80, and 156–170 should lie outside or close to membrane surface. It was demonstrated that the peptide fragments joining transmembrane rods are not fully exposed to the solution.
Secondary structure of the Arg‐Gly‐Asp recognition site in proteins involved in cell‐surface adhesion
Authors: REED Jennifer , HULL William E. , von der LIETH Claus‐Wilhelm , KÜBLER Dieter , SUHAI Sandor , KINZEL Volker
Published: March 2005

The primary sequence Arg‐Gly‐Asp has been found in a number of proteins which bind to cell surface receptors. Studies with synthetic peptides have shown that the presence of charged side chains alone is not sufficient to confer binding activity. Application of folding algorithms to proteins and peptides having similar sequences indicates that binding activity is strongly correlated with the presence of two or more closely spaced residues that each have a high probability of initiating a β‐bend. Circular dichroic studies on the hexapeptide GRGDSP, whose sequence is contained in fibronectin and which also shows binding activity, demonstrate that it adopts an unusual conformation in aqueous solution. 1H‐NMR spectra of the peptide in aqueous solution show that the two amide hydrogens of Asp4 and Ser5 exchange very slowly. Computer‐assisted modeling using restrained molecular dynamics and energy minimization results in conformations that include two β‐bends of type III‐III or III‐I (hydrogen bonds 4 → 1 and 5 → 2), fully consistent with constraints imposed by 1H‐ and 13C‐NMR data. It is suggested that this unusual secondary structure provides an additional specificity determinant.
Three‐dimensional structure of proteinase K at 0.15‐nm resolution
Authors: BETZEL Christian , PAL Gour P. , SAENGER Wolfram
Published: March 2005

The crystal and molecular structure of proteinase K was determined by X‐ray diffraction data to 0.15‐nm resolution. The enzyme belongs to the subtilisin family with an active‐site catalytic triad Asp39–His69–Ser224 but is a representative of a subgroup with a free Cys73 close to and ‘below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34–Cys123 and Cys178–Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel β‐sheet decorated by six α‐helices, three short antiparallel β‐sheets, 18 β‐turns and involving several internal, structurally important water molecules.

Proteinase K exhibits two Ca2+‐binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate‐recognition site (Ser132–Gly136), probably associated with a cis → trans isomerization of cis Pro171.

Several water molecules are located in the active site. One, W335, is positioned in the ‘oxyanion hole' and is displaced by the C=O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three‐stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132–Leu133–Gly134 on one side and Gly100–Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39–His69–Serr224 triad. These latter residues display low thermal motion corresponding to well‐defined geometry and are hardly accessible to solvent molecules. whereas the recognition‐site amino acids are more flexible and partially exposed to solvent.
Differences in the binding of H1 variants to DNA
Authors: CLARK David J. , THOMAS Jean O.
Published: March 2005

A study of the complexes formed between short linear DNA and three H1 variants, a typical somatic H1, and the extreme variants H5, from chicken erythrocytes, and spH1 from sea urchin sperm, has revealed differences between H1, H5 and spH1 that have implications for chromatin structure and folding.

All three histones bind cooperatively to DNA in 35 mM NaCl forming similar, but not identical, rod‐like complexes. With sufficiently long DNA the complexes may be circular, circles forming more easily with H5 and spH1 than with H1.

The binding of H5 and spH1 to DNA is cooperative even in 5 mM NaCl, resulting in well‐defined thin filaments that appear to contain two DNA molecules bridged by histone molecules. In contrast, H1 binds distributively over all the DNA molecules in 5 mM NaCl, but forms short stretches similar in appearance to the thin filaments formed with H5 and spH1. Rods appear to arise from the intertwining of regular thin filaments containing cooperatively bound histone molecules on raising the NaCl concentration to 35 mM.

The compositions of the rods correspond to one histone molecule for about every 47 bp (H1), 81 bp (H5) and 112 bp (spH1), suggesting average spacings of 24 bp (H1), 41 bp (H5) and 56 bp (spH1) in the component thin (double) filaments. Strikingly, these values are proportional to the linker lengths of the chromatins in which the particular H1 variant is the main or sole H1.
Involvement of a serine protease in tumour‐necrosis‐factor‐mediated cytotoxicity
Authors: SUFFYS Philip , BEYAERT Rudi , VAN ROY Frans , FIERS Walter
Published: March 2005

We investigated the effect of various protease inhibitors on the anti‐proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells.

The following serine‐type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, Nα‐p‐tosyl‐L‐lysine chloromethane, Nα‐p‐tosyl‐L‐phenylalanylchloromethane, Nα‐p‐tosyl‐L‐arginine methyl ester, L‐leucine methyl ester, DL‐phenylalanine methyl ester, N‐acetyl‐DL‐phenylalanine‐β‐naphthyl ester, p‐nitrophenyl p′‐guanidino‐benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF.

Inhibition of TNF‐mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide.

TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1, 3‐3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with Nα‐p‐tosyl‐L‐lysine chloromethane did not influence its biological activity.

The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage.

Protease inhibitors did not influence TNF binding, internalization or metabolization.

No increase in supernatant protease activity or in cell‐associated protease activity could be detected after treatment of L929 cells with TNF.

Our results document the involvement of protease activity, acting quite late during the cytolytic and growth‐inhibiting processes induced by TNF.
Nomenclature of tetrapyrroles
Authors: MOSS Gerard P.
Published: March 2005

In the revised recommendations two new trivial names (isobacteriochlorin and sirohydrochlorin) are defined. Isobacteriochlorin is 2,3,7,8‐tetrahydroporphyrin and is isomeric with bacteriochlorin. An example of an isobacteriochlorin is sirohydrochlorin, an intermediate in the biosynthesis of corrinoids. The names of linear tetrapyrroles are amended. The name bilin is now restricted to the fully oxidized parent, and less oxidized forms are named bilane, bilene, and biladiene. Names are now provided for the analogous dipyrrole system based on the parent dipyrrin. Tables are provided to show the structures of the more commonly encountered compounds using the Fischer system for denoting isomers (the fifteen isomers of mesoporphyrin defined by Fischer, and some isomers of biliverdin). Experience has shown that these names continue to be useful because of their brevity.
Infrared spectroscopic studies of Acholeplasma laidlawii B membranes
Authors: MANTSCH Henry H. , YANG Paul W. , MARTIN Adèle , CAMERON David G.
Published: March 2005

The physical state of the membrane lipids in the plasma membranes of intact, live Acholeplasma laidlawii B cells was probed by Fourier‐transform infrared spectroscopy and compared with that in isolated membranes. Infrared spectra of live A. laidlawii B cells, enriched biosynthetically in the presence of avidin, with saturated deuterated and unsaturated non‐deuterated fatty acids have been recorded at a variety of temperatures. The results indicate that within the temperature range of the gel to liquid‐crystal phase transition, the live cells are able to keep the ‘fluidity’ of their plasma membranes at a considerably higher value compared to that in the isolated membranes at the same temperature. While this is a generally valid observation, the degree by which live and isolated membranes differ in their liquid‐crystal‐phase content at a given temperature depends on the nature of the exogenous fatty acid and the temperature of growth.
Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide
Authors: SIMONCSITS Andras , TJÖRNHAMMAR Marie‐Louise , KALMAN Miklos , CSERPAN Imre , GAFVELIN Guro , BARTFAI Tamas
Published: March 2005

Synthetic genes coding for elongated precursors of the vasoactive intestinal polypeptide (VIP) were synthesized and cloned in a highly efficient Escherichia coli expression vector. The synthetic genes code for VIP with its methionine (at position 17) replaced by leucine and elongated at the C‐terminus by Gly (vasoactive intestinal polypeptide‐Gly, i.e. VIPa) or by Gly‐Lys‐Arg (vasoactive intestinal polypeptide‐Gly‐Lys‐Arg, i.e. VIPb). The synthetic genes fused to the N‐terminal part of the E. coliβ‐galactosidase gene were expressed to yield high amounts of fusion proteins reaching upon induction at least 60% of the total cellular protein. The fusion proteins of 314 and 316 amino acids carrying in their C‐terminal portion either the 29 or 31 amino acids long VIP precursor polypeptide were shown to be immunoreactive with VIP antisera and were further purified and cleaved by CNBr. The resulting purified peptide precursors (VIPa and VIPb) were recognized by VIP receptors in rat liver plasma membranes and by antibodies to porcine VIP in a radioimmunoassay. Both precursors activated adenylate cyclase in rat liver membranes and stimulated pancreatic secretion in the cat. The affinity and potency of the cloned precursors is close to that of VIP purified from porcine intestine, suggesting that the elongated VIP precursors may act even without being converted into the C‐terminal amide form of the peptide. The elongated VIP precursors expressed in E. coli may provide a cheap, large‐scale source of experimental material for studies on VIP actions.
Fluorescence‐quenching‐resolved spectra of fluorophores in mixtures and micellar solutions
Authors: WASYLEWSKI Zygmunt , KASZYCKI Pawel , GUZ Andrzej , STRYJEWSKI Wieslaw
Published: March 2005

The application of a new fluorescence‐quenching‐resolved spectroscopic method [Wasylewski, Z., Koloczek, H. and Waśniowska, A. (1988) Eur. J. Biochem. 172, 719–724] for resolving fluorescence emission spectra of a mixture of fluorophores into components is described. Contrary to fluorescence lifetime measurements, in this method the overlapping spectra can be decomposed even if the components have similar or the same fluorescence lifetimes, but differ in bimolecular‐rate‐quenching constants. Using this technique, we have resolved the emission spectra of a two‐component mixture of fluorescein and riboflavin, which have very similar fluorescence lifetimes. To illustrate the utility of this approach in the study of fluorophores in compartmentalized biological systems such as lipid bilayers, we have also used the method to resolve the emission spectra of a two‐component mixture of fluorophores commonly used in biological studies which undergo partition between water and a micellar phase.
Distances between functional sites in cardiac sarcoplasmic reticulum (Ca2++ Mg2+)‐ATPase
Authors: JOSHI Nanda B. , SHAMOO Adil E.
Published: March 2005

The high‐affinity Ca2+‐binding sites of cardiac sarcoplasmic reticulum (Ca2++ Mg2+)‐ATPase have been probed using trivalent lanthanide ions. Non‐radiative energy‐transfer studies, using luminescent probe Eu3+ as a donor and Nd3+ or Pr3+ as acceptor, were carried out to estimate the distance between two high‐affinity Ca2+‐binding/transport sites. Eu3+ was excited directly with pulsed laser light and the energy‐transfer efficiency to Nd3+ or Pr3+ was measured, under the conditions in which most donor‐acceptor pairs occupied the high‐affinity Ca2+ sites. The distance between two high‐affinity Ca2+ sites is about 0.89 nm. In the presence of ATP the distance between the high‐affinity sites is about 0.855 nm, whereas in the presence of adenosine 5′‐[β,γ‐methylene]triphosphate or adenosine 5′‐[β,γ‐imino]triphosphate the distance is about 0.895 nm. To estimate the distance between the high‐affinity Ca2+ sites and ATP‐binding/hydrolytic site, we have measured the energy‐transfer efficiency between Eu3‐ and Cr3+‐ATP with Eu3+ at the high‐affinity Ca2+ sites and Cr3+‐ATP at the ATP‐binding/hydrolytic site. Our results show that ATP‐binding/hydrolytic site is separated by about 2.2 nm from each high‐affinity Ca2+ site.
Kinetic evidence for a heart mitochondrial pore activated by Ca2+, inorganic phosphate and oxidative stress
Authors: CROMPTON Martin , COSTI Andreas
Published: March 2005

Evidence that the Ca2+‐induced permeabilization of mitochondria is attributable to a reversible Ca2+‐activated pore [Al Nasser & Crompton (1986) Biochem. J. 239, 19–29] has been further investigated. Permeabilization is induced in a wholly synergistic manner by either Ca2+ plus phosphate or Ca2+ plus tert‐butyl hydroperoxide. When permeabilization is complete, extramitochondrial [14C]sucrose equilibrates with the matrix space with a half‐time of about 800 ms; [14C]mannitol equilibrates at least threefold faster. Permeabilization is essentially fully reversed on Ca2+ chelation with EGTA, when the half time for [14C]sucrose equilibration is increased 600–1400‐fold (to 550–1150 s). A pulsed‐flow [14C]solute‐entrapment technique has been developed to measure the kinetics of EGTA‐induced resealing. The technique incorporates a suitable choice of [14C]solute and an appropriate model for data analysis, and is competent to measure permeation state changes occurring in 100 ms. The data obtained are consistent with exponential resealing of mitochondria in which pores of any single mitochondria close with a high degree of synchrony. The rate of resealing is increased about eight‐fold by ADP (half‐time ∼ 1 s; Km∼ 30 μM). CoA, Mg2+, AMP and also ATP, when account is taken of ADP arising by hydrolysis, are essentially ineffective. It is concluded that heart mitochondria do contain a pore whose permeation state is controlled over an approximate 1000‐fold range by Ca2+ and other factors including phosphate, oxidative stress and ADP. The possible involvement of the pore in reoxygenation‐induced injury in heart is discussed.
Oxidation‐reduction thermodynamics of the acceptor quinone complex in whole‐membrane fragments from Chloroflexus aurantiacus
Authors: VENTUROLI Giovanni , ZANNONI Davide
Published: March 2005

Oxidation‐reduction thermodynamic equilibria involving the quinone‐acceptor complex have been examined in whole‐membrane fragments from Chloroflexus aurantiacus. The primary quinone acceptor was titrated by monitoring the amount of cytochrome c554 photooxidized by a flash of light as a function of the redox potential. In contrast to previous data obtained in purified plasma membranes, in which the primary quinone acceptor exhibited a midpoint potential equal to ‐50 mV at pH 8.2, in whole‐membrane fragments it titrated at −210 mV (pH 8.0), with a pH dependence of −60 mV/pH up to a pK value of 9.3. o‐Phenanthroline, an inhibitor of electron transfer from the primary to the secondary quinone acceptor, shifted the Em/pH curve of the primary acceptor to higher redox potentials. The midpoint potential of the secondary quinone acceptor and its dependence on pH has been determined by comparing the kinetics of the charge recombination processes within the reaction center complex in the presence and in the absence of o‐phenanthroline. It is concluded that both the primary and the secondary quinone acceptors interact with a proton, with pK values of 9.3 and of approximately 10.2 respectively. At physiological pH the electron appears to be stabilized on the secondary with respect to the primary quinone acceptor by approximately 60 meV.
The large glycoprotein subunit of the skeletal muscle voltage‐sensitive calcium channel
Authors: BURGESS Alison J. , NORMAN Robert I.
Published: March 2005

Deglycosylation was used to assess the size of the core polypeptide of the large α2‐glycoprotein subunit of the 1,4‐dihydropyridine‐sensitive calcium channel from rabbit skeletal muscle. The extent of glycosylation was assessed by measuring the shift in apparent molecular mass of the α2 component following electrophoresis in sodium dodecyl sulphate/polyacrylamide gels, using anti‐(α‐subunit) monoclonal antibody staining of immunoblots. Chemical deglycosylation with trifluoromethanesulphonic acid produced a shift in apparent molecular mass of the α2 component from Mr 140 000 to Mr 105 000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments were insufficient to deglycosylate the α2 subunit fully, possibly due to the inaccessibility of glycosidic bonds to enzyme attack. Enzymatic deglycosylation procedures did, however, reduce the 1,4‐dihydropyridine‐binding activity of transverse‐tubule membranes. Neuraminidase alone or together with endo‐β‐N‐acetylglucosaminidase (endoglycosidase F) reduced the number of sites for (–)[3H]PN 200–110 by 73 ± 2% and 77 ± 5% respectively, with no change in apparent dissociation constant, implying a possible role for the glycosylated subunits in the binding of 1,4‐dihydropyridines to the calcium‐channel complex. The development of the α2 component in rat skeletal muscle was shown to be indistinguishable from the appearance of 1,4‐dihydropyridine binding activity consistent with the involvement of the α2 subunit in the calcium‐channel complex at all stages of development.
Cell‐surface heparan sulfate and heparan‐sulfate/chondroitin‐sulfate hybrid proteoglycans of mouse mammary epithelial cells
Authors: DAVID Guido , VAN DEN BERGHE Herman
Published: March 2005

The hydrophobic cell‐surface proteoglycans of mouse mammary epithelial cells were purified by gel filtration, ion‐exchange chromatography, and liposome incorporation. The size of the proteoglycans appeared to be directly proportional to the size of their heparan‐sulfate chains, larger proteoglycans vielding larger chains. The chondroitin sulfate chains, in contrast, showed no size heterogeneity. Digestion of 125I‐labeled proteoglycans with heparitin‐sulfate lyase and chondroitin ABC lyase yielded core proteins of ∼ 93 kDA, ∼ 85 kDa, and ∼ 38 kDa. Comparison with single enzyme digestions identified the 93‐kDa and 85‐kDa cores as components of hybrid proteoglycans that carried both heparan‐sulfate and chondroitin‐sulfate chains. Immunoblotting indicated that the 93‐kDa and 85‐kDa cores shared the epitope defined by monoclonal antibody 281‐2. The 38‐kDa core, in contrast, carried only heparan‐sulfate chains and lacked the 281‐2 epitope. Preparations enriched in heparan sulfate or in heparan‐sulfate/chondroitin‐sulfate hybrid proteoglycans were obtained by N‐desulfation and ionexchange chromatography. Hybrid proteoglycans accounting for the bulk of the chondroitin‐sulfate and nearly half of the heparan‐sulfate residues of the proteoglycans showed a similar polydispersity of heparan‐sulfate chain sizes as found in proteoglycans that carried only, or predominantly, heparan‐sulfate chains. These hybrids contained heparan‐sulfate and chondroitin‐sulfate chains in similar molar amounts. Analysis of 125I‐labeled proteoglycans suggested that typical hybrid proteoglycans were composed of a 85‐kDa core protein that carries a single chondroitin‐sulfate chain and a single heparan‐sulfate chain of variable length. A minority of hybrids seemed characterized by the variant, but possibly structurally related 93‐kDa core protein. The other half of the hydrophobic proteoglycans were composed of the 38‐kDa core and carried only heparan‐sulfate chains. The significance of the co‐existence of hybrid and heparan‐sulfate proteoglycans at the cell surface and possible relationships between the proteoglycans need to be further clarified.
Structural studies on the amino‐acid‐linked teichuronic acid of Bacillus subtilis AHU 1219 cell walls
Authors: IWASAKI Hiroyoshi , ARAKI Yoshio , KAYA Shunji , ITO Eiji
Published: March 2005

Structural studies were carried out on a teichuronic acid isolated from a mild acid extract of Bacillus subtilis AHU 1219 cell walls. The teichuronic acid contained D‐glucuronic acid, D‐glucose, D‐galactose, l‐serine and L‐threonine in a molar ratio of 1:1:1:0.5:0.5. Results of analyses of the polysaccharide by Smith degradation, methylation and 1H‐NMR and 13C‐NMR spectroscopy, in combination with data on analyses of oligosaccharides obtained by partial acid hydrolysis and alkaline hydrolysis of the polymer, led to the most likely structure for the repeating unit, →4)(l‐Ser/l‐Thr)‐d‐GlcA(β1 → 3)‐D‐Glc(β1 → 4)‐d‐Gal(α1 →. In each unit, either amino acid is linked to the glucuronic acid residue through an amide bond.
Heme may not be a positive regulator of cytochrome‐P450 gene expression
Authors: SRIVASTAVA Gopesh , BAWDEN Michael J. , HANSEN Antony J. , MAY Brian K.
Published: March 2005

It has been proposed that transcription of cytochrome‐P450 genes is positively regulated by heme, the prosthetic group of cytochrome‐P450 proteins. We have investigated this proposal in rats treated with succinylacetone, a known specific inhibitor of the heme biosynthetic pathway. While 2‐allyl‐2‐isopropylacetamide, phenobarbitone, dexamethasone, β‐naphthoflavone and clofibrate induced specific cytochrome‐P450‐mRNA species in rat liver, the levels of these induced mRNAs were not affected by succinylacetone administration. Synthesis of the first enzyme of the heme biosynthetic pathway, 5‐aminolevulinate synthase, is known to be regulated by the end‐product heme, with heme inhibiting 5‐aminolevulinate‐synthase‐gene transcription. Hepatic 5‐aminolevulinate‐synthase mRNA was induced by drugs and the level increased further by succinylacetone. Furthermore, treatment of rats with succinylacetone alone resulted in elevated levels of 5‐aminolevulinate‐synthase mRNA but did not affect cytochrome‐P450‐mRNA levels.

The results show that while lowered heme levels lead to an increase in 5‐aminolevulinate‐synthase‐mRNA synthesis there is no effect on cytochrome‐P450‐mRNA levels, implying that heme is not required for the cytochrome‐P450‐gene transcription.
Mechanism and binding specificity of β‐glucosidase‐catalyzed hydrolysis of cellobiose analogues studied by competition enzyme kinetics monitored by 1H‐NMR spectroscopy
Authors: BOCK Klaus , SIGURSKJOLD Bent W.
Published: March 2005

The application of high‐resolution 1H‐NMR spectroscopy to monitor substrate and product time dependencies in progress curve enzyme kinetics is described with β‐glucosidase‐catalyzed hydrolyses of cellobiose analogues as examples. It is demonstrated that inhibition patterns, relative binding specificities and catalytic rates can be inferred from competition experiments with two or more substrates. It could be concluded from competition experiments that substrates which form less stable enzyme‐substrate complexes than methyl β‐cellobioside are hydrolyzed faster than this reference substrate when they are the sole substrate, due to a lower activation energy in the catalytic step, but that they are hydrolyzed slower than the reference compound in direct competition, due to the formation of the less stable enzyme‐substrate complex in the binding step.
Characterization and application of a thermostable primary transport system: Cytochrome‐C oxidase from Bacillus stearothermophilus
Authors: DE VRIJ Wim , HEYNE Rene I. R. , KONINGS Wil N.
Published: March 2005

Cytochrome‐c oxidase from Bacillus stearothermophilus has been purified to homogeneity by detergent extraction followed by DEAE‐cellulose, hydroxyapatite‐ and gel‐filtration chromatography. The enzyme is a typical cytochrome‐aa3‐type oxidase which binds carbon monoxide and is sensitive to classical oxidase inhibitors like cyanide and azide. The purified enzyme is composed of three different subunits (57, 37 and 22 kDa). The subunit with intermediate molecular mass contains a covalently attached heme‐c moiety. The enzyme appeared to be extremely thermostable (inactivation temperature = 81°C). Highest turnover rates of the reconstituted enzyme were obtained with Saccharomyces cerevisiae cytochrome c or reduced forms of non‐physiological electron donors like N,N,N′,N′‐tetramethyl‐p‐phenylenediamine and phenazine methosulphate. The reconstituted enzyme can generate a proton‐motive force consisting of a high membrane potential and trans‐membrane pH gradient. The high electro‐motive force of the enzyme (Δp=–180 to –200 mV) indicates that this enzyme functions as a high‐capacity electrogenic proton pump.

Liposomes containing the purified thermostable and thermoactive cytochrome‐c oxidase were fused with membranes from the fermentative bacterium Clostridium acetobutylicum. In the hybrid system a high protonmotive force can be generated upon oxidation of reduced N,N,N′,N′‐tetramethyl‐p‐phenylenediamine by the incorporated oxidase which subsequently can be used to drive secondary transport of amino acids. This demonstrates the applicability of the cytochrome‐c oxidase to study solute transport in membranes of fermentative bacteria.
Conserved epitopes on plant H1 histones recognized by monoclonal antibodies
Authors: MAZZOLINI Laurent , VAECK Mark , Van MONTAGU Marc
Published: March 2005

A series of monoclonal antibodies specific for distinct regions of H1 histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme‐linked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.
The Aspergillus toxin restrictocin is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells
Authors: CONDE Francisco P. , ORLANDI Rosaria , CANEVARI Silvana , MEZZANZANICA Delia , RIPAMONTI Marina , MUÑOZ Saturnino M. , JORGE Pablo , COLNAGHI Maria I.
Published: March 2005

The protein toxin restrictocin, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28‐S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non‐glycosylated polypeptide of Mr 16836, exhibiting in cell‐free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restrictocin toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later.

After derivatization with N‐succinimidyl 3‐(2‐pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell‐free system. This derivatized toxin was then coupled to monoclonal antibodies (MBr1, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites internalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.
Factors involved in preferential transcription of the fibroin gene
Authors: TAKIYA Shigeharu , SUZUKI Yoshiaki
Published: March 2005

Transcription factors for class II genes from a tissue source, the posterior silk glands of Bombyx mori, were fractionated by stepwise elution on a phosphocellulose column into five fractions: A (0.04 M KCI flow through), A′ (0.1 M KCI eluate), B (0.3 M KCI eluate), C (0.5 M KCI eluate) and D (1 M KCI eluate). The minimal requirement for reconstruction of accurate initiation of transcription of the fibroin gene, as well as of the adenovirus 2 major late promoter, was the combination of fractions A, B and D, suggesting that transcription factors from B. mori can recognize general signals of the promoters for class II genes and that basic transcription factors are conserved even in distantly diverged species of eukaryotes. To detect activities stimulating the transcription governed by the promoter of fibroin gene, each fraction was tested for its function by supplementing a basal amount of HeLa cell extract. When circular templates were used, stimulatory activities specific for the fibroin gene were detected in fraction D. This preferential transcription is composed of at least three activities; the first and the second dependent on the upstream elements and the third dependent on the sequence downstream from the TATA box. However, when linear templates were used the preference for the fibroin gene was apparently lost and transcription activation by fraction D became general.
Chronic stimulation‐induced changes of myosin light chains at the mRNA and protein levels in rat fast‐twitch muscle
Authors: KIRSCHBAUM Bernhard J. , SIMONEAU Jean‐Aimé , BÄR Angelika , BARTON Paul J. R. , BUCKINGHAM Margaret E. , PETTE Dirk
Published: March 2005

Transitions in the expression of the myosin light chains (LC) were investigated in fast‐twitch muscles of the rat during chronic (10 h/day), low‐frequency (10 Hz) stimulation. Changes were followed at the mRNA level by Northern blot analysis and in vitro translation, as well as at the protein level by electrophoresis under denaturing and nondenaturing conditions. In vivo synthesis of the light chains was assessed by measuring the incorporation of intramuscularly injected [35S]methionine. Chronic stimulation induced a transition in the isomyosin pattern with an increase of FM3, a concomitant decrease in FM1 and, after longer stimulation periods, the appearance of low concentrations of the slow isomyosin. These changes were accompanied by an elevated LC1f/LC3f ratio and increases in the amounts of both the LC1sb and, to a lesser degree, LC2s proteins. Alterations in the amounts of specific mRNAs were the same whether determined by Northern blot analysis or by in vitro translation of total RNA preparations from the same muscles. Generally, the changes in the relative concentrations of fast and slow light‐chain proteins agreed with the changes detected at the mRNA level and the alterations in protein synthesis detected with the use of an in vivo labeling assay. An exception was the elevated tissue content of LC2s where no changes were detectable in the concentration of its mRNA as determined by in vitro translation or in vivo synthesis. The increase in LC2s protein may, therefore, have been due to reduced degradation. In addition, the decrease in LC3f was more pronounced at the protein level than at the mRNA level. This might indicate an increased turnover of LC3f or the existence of additional post‐transcriptional regulations of LC3f expression.
The membrane‐bound hydrogenase from Paracoccus denitrificans
Authors: KNÜTTEL Karlheinz , SCHNEIDER Klaus , SCHLEGEL Hans Günter , MÜLLER Achim
Published: March 2005

The membrane‐bound hydrogenase from Paracoccus dentitrificans was purified 68‐fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 μmol H2 min−1 (mg protein)−1 (methylene blue reduction). Purification involved solubilzation by Triton X‐114, phase separation, chromatography on DEAE‐Sephacel, ammonium‐sulfate precipitation and chromatography on Procion‐red HE‐3B‐Sepharose. Gel electrophoresis under denatruing conditions revealed two non‐identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane‐bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half‐life for inactivation under air at 200C was 8 h.

The Praracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6‐dichloriondophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V= 161.9 U/mg; Km= 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively).

The native hydrogenase from P. denitrificans cross‐reacted with purified antibodies raised aginst the membrane‐bound hydrogense from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.
Human plasma inter‐α‐trypsin inhibitor is encoded by four genes on three chromosomes
Authors: DIARRA‐MEHRPOUR Maryam , BOURGUIGNON Jeannette , SESBOÜE Richard , MATTEÏ Marie‐Geneviève , PASSAGE Edith , SALIER Jean‐Philippe , MARTIN Jean‐Pierre
Published: March 2005

Human inter‐α‐trypsin inhibitor is a plasma protein of Mr 180000 which has long been described as a single polypeptide chain. However, we have previously demonstrated that it is synthesized in liver by two different mRNA populations coding for heavy or light polypeptide chains [Bourguignon, J. et al. (1983) FEBS Lett. 162, 379–383] and cDNA clones for the heavy or light chains have recently been isolated and characterized [Bourguignon, J. et al. (1985) Biochem. Biophys. Res. Commun. 131, 1146–1153; Salier, J. P. et al. (1987) Proc. Natl Acad. Sci. USA 84, 8272–8276].

In the present study, we show that human poly(A)‐rich RNAs hybrid‐selected with various heavy‐chain‐encoding cDNA clones translate three different heavy chains, designated H1 (Mr 98000) and H3 (Mr 107000). We previously characterized two heavy‐chain cDNA clones. We now report that they correspond to H1 and H2 chains. We have also determined the sequence of an additional cDNA clone which codes for H3 chain. Its insert size is 1.79 kb with a single open reading frame and a poly(A) tail. The deduced amino acid sequence of the H3 chain is highly similar to those of the H1 (54%) and H2 (44%) chains. Northern analysis of human liver poly(A)‐rich RNAs with the three heavy‐chain cDNAs as probes clearly identified a single major mRNA population of 3.3 ± 0.1 kb.

Chromosomal localization by in situ hybridization shows that inter‐α‐trypsin inhibitor genes are located on three different human chromosomes. The H1 and H3 genes are located in the p211–p212 region of chromosome 3, whereas the H2 gene resides in the p15 band of chromosome 10. The light‐chain gene is located in the q32–q33 region of chromosome 9.

These results indicate that heavy and light chains of inter‐α‐trypsin inhibitor are encoded by at least four functional genes.
Metabolites controlling the rate of starch synthesis in the chloroplast of C3 plants
Authors: PETTERSSON Gösta , RYDE‐PETTERSSON Ulf
Published: March 2005

The extent to which different stromal metabolites affecting ADPglucose pyrophosphorylase control the rate of photosynthetic starch production in the chloroplast of C3 plants has been examined by kinetic model studies. The results indicate that ATP, glucose 1‐phosphate, 3‐phosphoglycerate, fructose 6‐phosphate, and orthophosphate may provide significant contributions to the starch synthesis rate changes induced by variation of the external concentration of orthophosphate, the detailed control situation being dependent on the actual concentration of the external metabolite.
ADP‐ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis
Authors: GEIPEL Udo , JUST Ingo , SCHERING Beate , HAAS Dagmar , AKTORIES Klaus
Published: March 2005

ADP‐ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin increased the rate of exchange of actin‐bound [γ‐32P]ATP by unlabelled ATP about twofold. Increased exchange rates were observed with ATP and ATP[γS], much less with ADP but not with AMP or NAD. ADP‐ribosylation of skeletal muscle actin reduced “basal” and Mg2+ (1 mM)‐induced ATP hydrolysis by about 80%. Similar inhibition of ATP hydrolysis was observed with liver actin ADP‐ribosylated by Clostridium botulinum C2 toxin. The data indicate that ADP‐ribosylation of actin at Arg‐177 largely affects the ATP‐binding and ATPase activity.
Phosphorylation of bovine hormone‐sensitive lipase by the AMP‐activated protein kinase
Authors: GARTON Andrew J. , CAMPBELL David G. , CARLING David , HARDIE D. Grahame , COLBRAN Roger J. , YEAMAN Stephen J.
Published: March 2005

Hormone‐sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP‐activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca2+/calmodulin‐dependent protein kinase II also phosphorylates hormone‐sensitive lipase predominantly at this site, whilst cyclic‐GMP‐dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic‐AMP‐dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone‐sensitive lipase by the cyclic‐AMP‐dependent and cyclic‐GMP‐dependent protein kinases, indicating that site‐2 phosphorylation may have an antilipolytic role in vivo.