bluuebunny/crossref_metadata_2025 huggingface dataset:
Classification of tropomyosin components into an α‐like or a β‐like family by partial peptide mapping
Authors: Kardami Elissavet , Fiszman Marc-Yves
Published: July 2002

Tropomyosins can be classified as belonging to an α‐like or a β‐like family depending on the absence or presence, respectively, in their protease‐V8 digestion pattern of two peptides with an apparent molecular mass of 21 kDa. Chicken cardiac tropomyosin and the 43 kDa component from gizzard tropomyosin are accordingly classified as α‐like tropomyosins, while the 33 kDa gizzard tropomyosin component is a β‐like tropomyosin. The 21 kDa peptides have an overall charge which is more positive than that of the intact tropomyosin or any other tropomyosin peptide and probably contain the ‐NH2 half of the molecule.
Connectivity of proton and carbon spectra of the blue copper protein, plastocyanin, established by two‐dimensional nuclear magnetic resonance
Authors: Kojiro Christopher L. , Markley John L.
Published: July 2002

NMR studies of plastocyanin have centered on the ligands to the copper atom at the active site, particularly histidines‐37 and ‐87. Heteronuclear (13C, 1H) J‐connectivity spectroscopy has enabled cross assignment of 1H and 13C NMR resonances from the two copper‐ligated histidines. In addition to providing assignments of the 13C resonances, the two‐dimensional Fourier transform NMR results require the reversal of the original 1H NMR assignments to the ring protons of histidine‐37. The line widths of the ring protons of histidine‐87 are field‐dependent leading to determination of the reduced lifetime of the proton on the Nδ atom (about 400 μs).
Modulation of β‐oxidation and proton conductance pathway of brown adipose tissue in hypo‐ and hyperinsulinemic states
Authors: Seydoux J. , Trimble E.R. , Bouillaud F. , Assimacopoulos-Jeannet F. , Bas S. , Ricquier D. , Giacobino J.P. , Girardier L.
Published: July 2002

The metabolic capacity of interscapular brown adipose tissue of hypoinsulinemic (diabetic) rats is decreased and a reduced β‐oxidative capacity contributes to this metabolic alteration. It was thus of interest to compare, in diabetic and in chronically (8 days) insulin‐infused rats, the β‐oxidative capacity and indices of the thermogenic state (GDP‐binding and 32 000 M
r protein) in this tissue. Mitochondrial GDP‐binding and 32 000 M
r protein were both decreased in diabetic rats compared to appropriate controls and markedly increased as was also the β‐oxidative capacity in hyperinsulinemic rats.
Modification of the chloride requirement for photosynthetic O2 evolution
Authors: Andersson Bertil , Critchley Christa , Ryrie Ivan J. , Jansson Christer , Larsson Christer , Anderson Jan M.
Published: July 2002

In thylakoid preparations from spinach and the halophyte Avicennia marina a correlation is observed between functional O2 evolution at low chloride concentrations and the presence of the 23 kDa protein. Addition of spinach 23 kDa protein to polypeptide‐depleted halophyte inside‐out thylakoid vesicles lowers their chloride requirement for optimal O2 evolution activity from 250 to 5 mM. It is suggested that the specific role for the 23 kDa protein is to increase the affinity of the water oxidation site for chloride.
Cell‐free synthesis of putative precursors for envelope membrane polypeptides of spinach chloroplasts
Authors: Flügge U.I. , Wessel D.
Published: July 2002

Protein synthesis in the presence of [35S]methionine was carried out in a wheat germ cell‐free system programmed with spinach leaf poly(A)+ RNA. After translation, identification of the putative precursors of the envelope membrane polypeptides was performed by immonoprecipitation using antibodies against 3 envelope membrane polypeptides; M
r 22 000, 29 000 (phosphate translocator) and 36 000. The identities of the immunoprecipitated precursors are established by performing competition studies. The results show that the precursor of the M
r 22 000 envelope membrane polypeptide is of M
r ∼ 10 000, that of the phosphate translocator (M
r 29 000) of M
r ∼ 11 000 and that of M
r 36 000 polypeptide of M
r ∼ 2000 larger than the authentic protein.
Inactivation of beef heart mitochondrial F1‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides
Authors: Fernandes de Melo Dirce , Satre Michel , Vignais Pierre V.
Published: July 2002

Beef heart mitochondrial F1‐ATPase was inactivated by the 2′,3′‐dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non‐specific labeling, the number of specifically bound [14C]oADP was 2–3 mol per mol F1. By SDS‐polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the α and β subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.
31P NMR as a tool for monitoring detergent solubilization of sarcoplasmic reticulum membranes
Authors: Roux Michel , Champeil Philippe
Published: July 2002

Sarcoplasmic reticulum vesicles were solubilized stepwise by the nonionic detergent octaethyleneglycol monododecyl ether; 31P NMR enabled the extent of phospholipid solubilization to be monitored by following the conversion of the broad resonance peak characterizing the phospholipids inserted in the bilayer to the narrow resonance signal characterizing phospolipids inserted into a mixed micelle. Up to 0.25 g detergent/g protein could be incorporated into the membrane without solubilization. Higher detergent concentrations of up to 1.5–2 g detergent/g protein led to gradual solubilization. Although the method allows us to monitor the extent of solubilization of individual phospholipid classes, there was no evidence of either preferential solubilization or retention of a specific class of phospholipids.
Two distinct quinone‐modulated modes of antimycin‐sensitive cytochrome b reduction in the cytochrome bc1 complex
Authors: Robertson Dan E. , Giangiacomo Kathleen M. , de Vries Simon , Moser Christopher C. , Dutton P.Leslie
Published: July 2002

Reduction of cytochrome b‐560 (analogous to cyt b‐562 of mitochondria) via an antimycin‐sensitive route has been revealed in chromatophores of the photosynthetic bacterium, Rhodopseudomonas sphaeroides Ga. Indeed, the results suggest that two reductive mechanisms can be operative. One is consistent with the idea that the quinol generated at the reaction center QB site enters the Q pool and, via the Qc site, equilibrates with cytochrome b‐560. The other reductive mode circumvents redox equilibrium with the pool; we consider that this could result from a direct encounter of the reaction center with the bc
1 complex perhaps involving a direct QB‐QC site interaction. This latter reaction is suppressed by occupancy of the QC site, not only by antimycin but by ubiquinol and ubiquinone.
Limulus ventral photoreceptors contain a homologue of the α‐subunit of mammalian Ns
Authors: Malbon Craig C. , Kaupp U.B. , Brown J.E.
Published: July 2002

Membranes from ventral photoreceptors of Limuluswere incubated with cholera toxin and [32P]NAD+. Cholera toxin catalyzes a specific ADP‐ribosylation of a 43‐kDa peptide from Limulus ventral photoreceptors. Possible homologies between the 43‐kDa peptide of Limulus and the α‐subunits of mammalian stimulatory, guanine nucleotide‐binding regulatory component of adenylate cyclase (Ns) were investigated by comparing the electrophoretic patterns of proteolytic fragments derived from each of these peptides that are radiolabeled by [32P]NAD+ and cholera toxin. Evidence is provided for structural homology between this invertebrate peptide and mammalian Ns.
The isolation and sequencing of human gastric inhibitory peptide (GIP)
Authors: Moody Alister J. , Thim Lars , Valverde Isabel
Published: July 2002

Human GIP 1–42 and fragments of human GIP corresponding to GIP 10–42, GIP 11–42, and GIP 17–42 were isolated from acid‐ethanol extracts of human small intestines with the aid of an anti‐GIP serum specific for the extreme C‐terminal portion of the GIP molecule. The full sequence of human GIP has been established by Edman degradation of these peptides and fragments thereof by automatic gas‐phase sequencing. Human GIP differs from porcine GIP at residues 18 and 34. The sequence of human GIP is thus: Tyr‐Ala‐Glu‐Gly‐Thr‐Phe‐Ile‐Ser‐Asp‐Tyr‐Ser‐Ile‐Ala‐Met‐Asp‐Lys‐Ile‐His‐Gln‐Gln‐Asp‐Phe‐5 10 15 20 Val‐Asn‐Trp‐Leu‐Leu‐Ala‐Glu‐Lys‐Gly‐Lys‐Lys‐Asn‐Asp‐Trp,Lys‐His‐Asn‐Ile‐Thr‐Gln. Amino acid 25 30 35 40 residues 18 and 34 are Arg and Ser, respectively, in porcine GIP.
Lateral diffusion of lipids and proteins in bilayer membranes
Authors: Vaz Winchil L.C. , Goodsaid-Zalduondo Federico , Jacobson Ken
Published: July 2002

Lipid bilayers
Lateral diffusion
Membrane proteins
Fluorescent probes
Photobleaching
A simple modification converts the spinning cup protein sequencer into a vapour‐phase sequencer
Authors: Brandt Wolf F. , Alk Herman , Chauhan Madhu , von Holt Claus
Published: July 2002

Spinning cup protein sequencer
Vapour‐phase sequencer
Protein structure
Diheme cytochrome c‐554 from Nitrosomonas
Authors: Andersson Kristoffer K. , Babcock Gerald T. , Hooper Alan B.
Published: July 2002

The diheme cytochrome c‐554 which participates in ammonia oxidation in the chemoautotroph, Nitrosomonas europaea has been studied by Soret excitation resonance Raman spectroscopy. The Raman spectrum of reduced cytochrome c‐554 at neutral pH is similar classical 6‐coordinate low‐spin ferrous mammalian cytochrome c. In contrast, the spectrum of ferric cytochrome c‐554 suggests a 5‐coordinate state which is unusual for c hemes. The oxidized spectrum closely resembles that of horseradish peroxidase (HRP) or cytochrome c peroxidase (CcP) at pH 6.4. The narrow linewidth of the heme core‐size vibrations indicates that both heme irons of c‐554 have similar geometries.
Urea‐gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products
Authors: Vilanova M. , Burgos F.J. , Cuchillo C.M. , Avilés F.X.
Published: July 2002

Monomeric procarboxypeptidase A (PCPA) and isolated proproteinase E (PPE), both from pig pancreas, were shown by means of electrophoresis on transverse urea gradients (0–9 M) to form a very stable complex, identical to their natural binary complex. Although the complex is maintained by the interaction of both active regions, the activation segment of PCPA participates directly in the binding. Procarboxypeptidase B (PCPB) also associates with PPE, but in this case the complex shows low stability. In contrast with carb‐oxypeptidases A that strongly bind to their corresponding severed activation segments, no interaction was observed between carboxypeptidase B and its severed activation segment. The above results give some insight into several characteristics of the structure and activation properties of pancreatic PCPA and PCPB.
Further comments on the logic of the application of uncoupler‐inhibitor titrations for the elucidation of the mechanisms of energy coupling
Authors: Pietrobon Daniela , Roy Caplan S.
Published: July 2002

Following the appearance of two papers in this journal commenting on the logic of the application of uncoupler‐inhibitor titrations as a means of discriminating between ‘delocalized’ and ‘localized’ chemiosmotic mechanisms [(1984) FEBS Lett. 176, 79‐82; (1985) FEBS Lett. 186, 8‐10], and in contrast with the arguments presented there and elsewhere, we show that in a linear model the increase in ΔgmH which accompanies partial inhibition of the ATPases always leads to a relatively higher decrease of the rate of ATP synthesis by a given concentration of uncoupler in the presence of an ATPase inhibitor than in its absence. This is due to the fact that the same titre of uncoupler leads to a higher dissipative H+ flow in the presence of inhibitor, since the driving force ΔgmH is higher.
Amino acid sequence of heat‐stable enterotoxin produced by Vibrio cholerae non‐01
Authors: Takao Toshifumi , Shimonishi Yasutsugu , Kobayashi Makoto , Nishimura Osamu , Arita Michiko , Takeda Tae , Honda Takeshi , Miwatani Toshio
Published: July 2002

The amino acid sequence of heat‐stable enterotoxin, produced by Vibrio cholerae non‐01 and isolated from its culture supernatant, was determined by both Edman degradation of native and reductively carboxy‐methylated enterotoxin and also a combination of fast atom bombardment mass spectrometry and carboxy‐peptidase Y digestion of native enterotoxin to be as follows: Ile‐Asp‐Cys‐Cys‐Glu‐Ile‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Phe‐Gly‐Cys‐Leu‐Asn. This sequence is very similar, but not identical, to those of heat‐stable enterotoxins produced by enterotoxigenic Escherichia coli and Yersinia enterocolitica.
Prostaglandins E1 and E2 enhance the stimulation of superoxide release by 1‐oleoyl‐2‐acetylglycerol from human neutrophils
Authors: Penfield Adrienne , Dale M.Maureen
Published: July 2002

Superoxide release from human neutrophils was stimulated either by receptor activation (using fMet‐Leu‐Phe) or by activating, independently, each of the two pathways considered to be involved in signal transduction‐calcium mobilization (using the ionophore, A23187) and protein kinase C activation (using phorbol myristate acetate or 1‐oleoyl‐2‐acetylglycerol). Prostaglandin E1 (3 × 10−5 M) decreased fMet‐Leu‐Phe‐stimulated superoxide release, had no effect on superoxide release stimulated by A23187, or by phorbol myristate acetate, and markedly enhanced the superoxide release stimulated by 1‐oleoyl‐2‐acetylglycerol. Similar enhancement was obtained with prostaglandin E2.
Effect of spectrin dimer on actin polymerization
Authors: Strömqvist Mats , Backman Lars , Shanbhag Vithaldas P.
Published: July 2002

Spectrin dimer is shown to influence the polymerization behaviour of actin. The polymerization of both Mg2+‐ and Ca2+‐actin is regulated by an enhancement in the rate of nucleation and a fragmentation of preformed actin filaments. In addition, spectrin decreases the critical concentration of Ca2+‐actin but not that of Mg2+‐actin. This suggests that the two types of actin may differ in their interaction with spectrin dimer probably due to the different conformations. Band 4.1 elevates the effects of spectrin under non‐equilibrium conditions but its contribution is less at steady state.
Three‐dimensional structure of the ribonuclease t1 · 3'‐guanylic acid complex at 2.6 Å resolution
Authors: Sugio Shigetoshi , Oka Ken-ichi , Ohishi Hirofumi , Tomita Ken-ichi , Saenger Wolfram
Published: July 2002

X‐ray crystallography
Ribonuclease T1

3'‐Guanylic acid
Ribonuclease T1 · 3'guanylic acid complex
Specific recogniton
Disordered ribose
Phorbol ester (TPA) potentiates noradrenaline and acetylcholine‐evoked amylase secretion in the rat pancreas
Authors: Singh Jaipaul
Published: July 2002

The effect of the phorbol ester 12‐O‐tetradeconyl‐phorbol‐13‐acetate (TPA) on noradrenaline (NA) and acetylcholine (ACh)‐evoked amylase secretion in isolated segments of rat pancreas was investigated. TPA alone evoked a relatively small increase in amylase output. However, when combined with either noradrenaline or ACh, the phorbol ester markedly enhanced the secretagogue‐induced amylase secretion. These effects were dose related. TPA also enhanced the amylase secretion evoked by either the Ca2+ ionophore A23187 or dibutyryl cyclic AMP. This potentiation by TPA of noradrenaline, ACh, Ca2+ ionophore A23187 and dibutyryl cyclic AMP‐evoked amylase output may suggest the existence of a third pathway controlling enzyme secretion in the pancreas.
pH‐induced change in nucleotide binding geometry in the ribonuclease T1‐2'‐guanylic acid complex
Authors: Sugio Shigetoshi , Amisaki Takashi , Ohishi Hirofumi , Tomita Ken-ichi , Heinemann Udo , Saenger Wolfram
Published: July 2002

At pH 4.0, the RNase T1‐2'GMP complex (1) crystallizes isomorphously with the isoenzyme complex (2) (Heinemann, U. and Saenger, W., 1982, Nature 299, 27‐31). The X‐ray structure of 1 was refined with 1.9 Å data to R = 0.195. Polypeptide folding is similar in 1 and 2. However, the sugar pucker of 2'‐GMP is 2'‐endo (3'endo in 2), and guanine binding involves four hydrogen bonds in 1, which all differ from the two bonds in 2. Phosphate contacts Glu58, Arg77, Tyr38 in 1, but His40 in 2. These changes are not due to differences in sequence between the mother‐ and isoenzyme (Gln25‐Lys) but are associated with pH changes leadingto an inactive enzyme structure.
The complete amino acid sequence of the bacteriochlorophyll c binding polypeptide from chlorosomes of the green photosynthetic bacterium Chloroflexus aurantiacus
Authors: Wechsler Thomas , Suter Franz , Fuller R.Clinton , Zuber Herbert
Published: July 2002

The BChlc polypeptide was isolated from chlorosomes of the green bacterium Chloroflexus aurantiacus on Sephadex LH‐60. The complete amino acid sequence of this 5.6 kDa polypeptide (51 amino acid residues) was determined. Most probably the 5.6 kDa polypeptide forms an α‐helix between Trp 5 and Ile 42 with an asymmetrical (bipolar) distribution of polar amino acid residues along the helix axis: (i) At one side of the α‐helix 5 Gln and 2 Asn residues are the possible binding sites for 7 BChlc molecules, (ii) On the other side Ser, Thr, His residues seem to be polypeptide‐polypeptide interaction sites within the BChlc‐protein complexes. It appears that the BChl‐protein complex (chlorosome subunit, 5.2 × 6 nm) composed of 12 5.6 kDa polypeptides corresponds to the 'globular units' found by electron microscopy within the chlorosomes.
Human tissue‐type plasminogen activator is related to albumin and alpha‐fetoprotein
Authors: Baker Michael E.
Published: July 2002

Tissue‐type plasminogen activator
Albumin
Alpha‐fetoprotein
Epidermal growth factor
Protein evolution
Isolation from bovine brain of tropomyosins that bind to actin filaments with different affinities
Authors: Keiser Thomas , Wegner Albrecht
Published: July 2002

Tropomyosin was isolated from bovine brain using mild conditions thereby avoiding heat precipitation. Separation by DEAE ion exchange chromatography yielded a 33 kDa tropomyosin and a mixture of 30 and 32 kDa tropomyosin. Binding of the tropomyosins to actin filaments was measured by a newly developed method. The binding was assayed by the retarding effect of tropomyosin on actin polymerization. The 33 kDa tropomyosin was found to bind to actin filaments with considerably higher affinity than the 30 and 32 kDa tropomyosin.
Expression of the human apolipoprotein AI gene fused to the E. coli gene for β‐galactosidase
Authors: Lorenzetti R. , Sidoli A. , Palomba R. , Monaco L. , Martineau D. , Lappi D.A. , Soria M.
Published: July 2002

The human apoAI gene was expressed in E. coli by in‐frame fusion to a modified β‐galactosidase gene present in plasmid pUR291. The fused β‐galactosidase‐apoAI gene product was expressed at a high level and was recognized by an anti‐human apoAI antiserum. Besides the fused protein, at least one degradation product having an M
r similar to that of β‐galactosidase was present in high amounts in bacterial extracts. These results and those of a pulse‐chase experiment indicate that degradation took place only in the apoAI moiety of the chimeric protein.
Inhibition of lipid transport in insects by a factor secreted by the parasite, Blepharipa sericariae
Authors: Hayakawa Yoichi
Published: July 2002

A parasitoid fly secretes a factor into hemolymph of the host silkworm, which inhibits lipid transport from the host's fat body by lipophorin. The available data indicate that the factor is a small peptide with a molecular mass of less than 3500 Da.
Inactivation of ribulose‐bisphosphate carboxylase by limited proteolysis
Authors: Gutteridge Steven , Millard Barbara N. , Parry Martin A.J.
Published: July 2002

Limited proteolysis of ribulose‐bisphosphate carboxylase with trypsin or endopeptidase Lys C causes the loss of the carboxylase and oxygenase activities, without disrupting the binding of bisphosphates or the reactions normally associated with activation of the enzyme. Gel electrophoresis of the non‐denatured enzyme indicates that the L8S9 quaternary structure of large and small subunits is unaffected by this treatment. However, electrophoresis in denaturing conditions shows that the L subunit is smaller as a result of the removal of two short polypeptides, each approx. 1000 Da. The loss of activity is associated with the removal of the second peptide. The amino acid composition of the isolated peptides indicates that both originate from the N‐terminus of the L subunit. Over protracted periods of exposure to the proteases a third smaller peptide is also released from the N‐terminus of the L subunit. The S subunit is not affected by these treatments. The carboxylase isolated from a number of photosynthetic species exhibits a differential response to this limited proteolysis.
Type IX collagen is a potent inducer of PGE2 and interleukin 1 production by human monocyte macrophages
Authors: Dayer Jean-Michel , Ricard-Blum Sylvie , Kaufmann Marie-Thérèse , Herbage Daniel
Published: July 2002

Type IX collagen and its collagenous fragments are potent stimulatory agents on human blood mononuclear cells for the production of prostaglandin E2 and interleukin 1/mononuclear cell factor. Type IX collagen is 2–4‐fold more potent that type I and II and 1α‐, 2α‐ and 3α‐collagens. This property may be important in the destructive process of cartilage in inflammatory diseases.
A tobacco chloroplast DNA sequence possibly coding for a polypeptide similar to E. coli RNA polymerase β‐subunit
Authors: Ohme M. , Tanaka M. , Chunwongse J. , Shinozaki K. , Sugiura M.
Published: July 2002

DNA sequencing has revealed a long open reading frame (ORF) in the large single‐copy region of tobacco chloroplast DNA. This ORF consists of 1070 codons and its deduced amino acid sequence shows about 39% homology to that of the β‐subunit of E. coli RNA polymerase. This finding raises a possibility that some of the chloroplast RNA polymerase subunits are coded for by the chloroplast genome.
Specificity studies on anti‐histone H1 antibodies obtained by different immunization methods
Authors: Srebreva Ljuba , Zlatanova Jordanka
Published: July 2002

Antibodies were elicited against chromatographically purified histone H l subfractions or against their complexes with RNA and their specificity studied by enzyme‐linked immunosorbent assay. The results show that complexing of the pure protein with RNA (i) does not lead to any significant increase in the antibody titer and (ii) results in obtaining antibodies predominantly against the common antigenic determinants present in the HI histone class. On the other hand, using pure histone fractions for immunization gives rise to antibody populations reacting mainly with the subfraction‐specific determinants on the histone molecule. In view of these results the literature data should be interpreted with caution.
Expression of amyloplast DNA in suspension‐cultured cells of sycamore (Acer pseudoplatanus L.)
Authors: Macherel David , Kobayashi Hirokazu , Valle Estela , Akazawa T.
Published: July 2002

(Sycamore cell)
amyloplast DNA
Gene expression
Transcription
Methylation
Direct immunological identification of full‐length cDNA clones for plant protein without gene fusion to E. coli protein
Authors: Nakamura Kenzo , Hattori Tsukaho , Asahi Tadashi
Published: July 2002

By immunological screening of a cDNA library constructed from potato tuber poly(A)+ RNA and Escherichia coli expression vector pUC8 by the vector‐primer and linker procedure of Okayama and Berg [(1982) Mol. Cell Biol. 2, 161‐170], nearly full‐length cDNA clones for patatin, a major protein of potato tuber, were identified. The cDNA carrying part of the 5'‐noncoding region of the patatin mRNA, in addition to entire coding and 3'‐noncoding regions, expressed prepatatin in E. coli cells by translational initiation inside cDNA. These results suggest that nearly full‐length cDNA clones with entire coding region can be identified directly by immunological screening without gene fusion to E. coli proteins at least for some plant mRNAs.
Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase
Authors: Champness J.N. , Stammers D.K. , Beddell C.R.
Published: July 2002

The structure of the complex between E. coli (RT500) form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X‐ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. We discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme.
Isolation and amino acid sequence of the ‘Rieske’ iron sulfur protein of beef heart ubiquinol:cytochrome c reductase
Authors: Schägger H. , Borchart U. , Machleidt W. , Link T.A. , Von Jagow G.
Published: July 2002

The sequence of the ‘Rieske’ iron sulfur protein from the bc
1 complex of beef heart mitochondria has been determined by solid phase Edman degradation of the whole protein and of various proteolytic fragments. The protein consists of 196 amino acid residues. The molecular mass of the apoprotein was calculated to be 21 536 Da, that of the holo‐protein including the Fe2S2 cluster as 21 708 Da. The protein is mainly hydrophilic with a polarity index of 42.9% and 25% of charged residues. It contains a hydrophobic membrane anchor which is predicted to form a ‘hairpin’ structure. The iron sulfur cluster is bound near the C‐terminus of the protein between a hydrophobic and a more amphipathic domain. This reflects the fact that the cluster is located near the outer surface of the inner mitochondrial membrane. A folding pattern describing all known features of the protein is proposed.
Investigation of the solution structures and mobility of oxidised and reduced cytochrome b5 by 2D NMR spectroscopy
Authors: Veitch Nigel C. , Concar David W. , Williams Robert J.P. , Whitford David
Published: July 2002

Two dimensional 1H NMR spectroscopy is used to examine the structure and mobility of cytochrome b
5 in solution. The assignment of many residues and the interpretation of nuclear Overhauser effects (NOEs) in both redox states allow definition of secondary structural elements. Comparison with X‐ray diffraction data shows that differences between crystal and solution structures are small. The dynamics of the protein are examined and the protein is shown to be more mobile than cytochrome c. The relationship of the structure and dynamics to the electron transfer function of cytochrome b
5 is discussed.
Analysis of guanine nucleotide bound to ras protein in PC12 cells
Authors: Satoh Takaya , Endo Masami , Nakamura Shun , Kaziro Yoshito
Published: July 2002

The ras gene product (p21) specifically binds GDP or GTP. In analogy with the reaction mechanism of other GTP‐binding proteins, only the GTP‐bound conformation is believed to be the biologically active one. Previously, we reported that not only oncogenic p21 (Val‐12) but also proto‐oncogenic p21(Gly‐12) could induce morphological differentiation in rat pheochromocytoma PC12 cells when microinjected in the complexed form with GTPγS [(1987) Mol. Cell. Biol. 7, 4553–4556]. In the present report we transformed PC12 cells with the oncogenic ras gene placed under the metallothionein I promoter. It was found that the transformed cells, when induced with Cd2+, differentiated in the absence of NGF. Then we analyzed the guanine nucleotide bound to p21 in the intact PC12 cells. It was found that conditionally induced p21(Val‐12) was mostly present in the GTP‐bound form, whereas the endogenous p21(Gly‐12) was in the GDP‐bound form. These results indicate again that p21·GTP induces the morphological differentiation of PC12 cells.
Isolation and partial characterization of prothymosin α from porcine tissues
Authors: Economou M. , Seferiadis K. , Frangou-Lazaridis M. , Horecker B.L. , Tsolas O.
Published: July 2002

Prothymosin α, an immunoactive polypeptide of 12 kDa, has been isolated from porcine thymus, spleen, lung and kidney. It lacks aromatic and sulfur‐containing amino acids and has a high content of glutamic and aspartic acids. Tryptic digestion of porcine thymus prothymosin α yielded peptides which on separation, amino acid analysis and alignment with the known sequence of prothymosin α from rat and man showed that the amino terminal portion of the molecule is conserved and the few differences present are confined to the car☐y terminal.
N6‐Methyladenine in mitochondrial DNA of higher plants
Authors: Vanyushin B.F. , Alexandrushkina N.I. , Kirnos M.D.
Published: July 2002

After incubation of etiolated wheat seedlings in the presence of [8‐14C]adenine the radioactive N
6‐methyladenine (m6A) has been detected in the newly synthesized mitochondrial DNA (mtDNA) (ML = 100·m6A/(m6A+A)=0.4–0.6). This DNA is a low molecular mass (7.7 S) fraction of the mtDNA population. The detection of N
6‐methyladenine in the mtDNA of wheat seedlings indicates the presence of adenine DNA methylase in mitochondria of higher plants. The presence of m6A in plant mtDNAs makes them distinct from animal mtDNAs in which, as we know, this additional base has not been found.
Time‐resolved fluorescence studies on the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: Hanemaaijer Roeland , Masurel Remco , Visser Antonie J.W.G. , de Kok Arie , Veeger Cees
Published: July 2002

The dihydrolipoyl transacetylase (E2) component of A. vinelandii PDC and its lipoyl domain shows similar dynamic properties as revealed with fluorescence anisotropy decay of lipoyl‐bound IAANS. The lipoyl domain (32.6 kDa), containing three almost identical subdomains shows a mode of rotation characteristic for a protein of about 30 kDa. A similar rotation is found in E2, indicating an independent rotational mobility of the whole domain in the multimeric E2 core (1.6MDa). No independent rotation of a single lipoyl subdomain (10 kDa) is observed. The E1 component, in contrast to the E3 component, shows interaction with the lipoyl domain.
Exploration of the catalytic site of endopeptidase 24.11 by site‐directed mutagenesis Histidine residues 583 and 587 are essential for catalysis
Authors: Devault Alain , Sales Valérie , Nault Christiane , Beaumont Ann , Roques Bernard , Crine Philippe , Boileau Guy
Published: July 2002

Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His‐142 and His‐146) and a third histidine (His‐231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His‐583, His‐587 and His‐637) was explored by site‐directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS‐1 cells. Substitution of either His‐583 or His‐587 of NEP for Phe completely abolished the activity and Zn‐directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His‐ 142 and His‐146 of thermolysin as zinc ligands. In contrast, substitution of His‐637 for a phenylalanine residue was without effect on enzyme activity.
Single crystal absorption spectra of ascorbate oxidase from green zucchini squash
Authors: Merli Angelo , Rossi Gian Luigi , Bolognesi Martino , Gatti Giuseppina , Morpurgo Laura , Finazzi-Agro Alessandro
Published: July 2002

Single crystal polarized absorption spectra of the dimeric multicopper enzyme ascorbate oxidase from green zucchini squash indicate that its most relevant functional and structural properties are maintained in the crystalline state. Since the polarized absorption spectra of crystalline ascorbate oxidase are very similar, in the visible region, to those of crystalline plastocyanin, we expect that structural data will show similar orientation of the type 1 Cu2+ center with respect to the crystal axes. The selective removal of type 2 Cu2+ from the crystal has been realized and has a potential value for the identification of the copper centers in the crystallographic analysis of the enzyme. Evidence is presented for an azide binding site formed by type 2 and type 3 Cu3+, similar to the trinuclear copper center suggested to be present in laccase.
N‐terminal amino acid sequence of a major allergen of Japanese cedar pollen (Cry j I)
Authors: Taniai Madoka , Ando Shunsaku , Usui Mitsuko , Kurimoto Masashi , Sakaguchi Masahiro , Inouye Sakae , Matuhasi Tyoku
Published: July 2002

A purified preparation of a major allergen of Japanese cedar pollen, sugi basic protein (SBP, Cry jI), was separated into 5 subtractions of 50‐45 kDa. All of the SBP subtractions were confirmed to be reactive to IgE antibodies from patients with Japanese cedar pollinosis, and also to mouse anti‐SBP monoclonal antibodies. The sequences of 20 N‐terminal amino acids of these 5 subtractions were found to be identical. Peptide mapping analyses of the SBP subtractions showed similar patterns, with some differences which might in part be due to the existence of an N‐linked carbohydrate chain. The N‐terminal amino acid sequence of SBP was identical to the reported sequence of an allergen of mountain cedar which vegetated in North America.
Iron‐ and manganese‐containing superoxide dismutases can be distinguished by analysis of their primary structures
Authors: Parker Michael W. , Blake Colin C.F.
Published: July 2002

The iron‐ and manganese‐containing superoxide dismutases have very similar three‐dimensional structures but can be distinguished by various biochemical means. The primary structures of six manganese‐containing and three iron‐containing superoxide dismutases are known. Analysis of the aligned amino acid sequences of these enzymes taken together with structural data from X‐ray diffraction studies demonstrates that the two classes of enzyme can be distinguished on the basis of a small number of single‐site substitutions that are positioned in and close to the active site of the enzyme.
Isolation of the calmodulin‐dependent protein kinase system from rabbit skeletal muscle sarcoplasmic reticulum
Authors: Tuana Balwant S. , MacLennan David H.
Published: July 2002

A calmodulin‐dependent protein kinase system from the sarcoplasmic reticulum was dissolved in Nonidet P40, adsorbed to a CaM affinity column in the presence of Ca2+ and eluted in the presence of EGTA. The purified fraction contained major proteins of 60 and 20 kDa and minor components of 89 and 34 kDa, all of which were phosphorylated with dependencies on Ca2+, CaM, ATP and pH similar to those observed in the sarcoplasmic reticulum. Differences in the phosphopeptides produced by partial proteolysis of the individual phosphoproteins indicated that they are distinct entities. 125I‐CaM labeled only the 60 kDa protein, suggesting that it is a kinase.
Phenylalanyl‐tRNA synthetase from Thermus thermophilus HB8 Purification and properties of the crystallizing enzyme
Authors: Ankilova V.N. , Reshetnikova L.S. , Chernaya M.M. , Lavrik O.I.
Published: July 2002

Phenylalanyl‐tRNA synthetase from Thermus thermophilus HB8 was isolated, characterized and crystallized. The enzyme is a tetramer of α2β2‐type structure, its molecular mass being 264 kDa. Molecular masses of the enzyme subunits are 40 (α) and 92 (β) kDa. The optimal temperature conditions of the tRNAPhe aminoacylation, catalyzed by this enzyme, are close to 80°C. K
M values for tRNAPhe from E.coli, for tRNAPhe from T. thermophilus HB8, for phenylalanine and ATP, as well as their temperature dependencies were determined. The enzyme crystals were grown by the hanging drop technique at 4°C in the presence of ammonium sulfate.
Analysis of murine interferon‐γ binding to its receptor on intact cells and solubilized membranes Identification of an 80 kDa receptor
Authors: Cofano F. , Landolfo S. , Appella ]E. , Ullrich S.J.
Published: July 2002

The receptor for murine‐interferon‐γ (Mu‐IFN‐γ) has been characterized for its molecular size and equilibrium binding constant on a thymoma cell line, EL‐4. Binding of 125I‐IFN‐γ to intact cells and their solubilized membranes has shown a single class of receptor with K
d values of 1.9 × 10−9 M and 1.3 × 10−8 M, respectively. It was shown that solubilization of the Mu‐IFN‐γ receptor with a Zwitterionic detergent (Chaps) preserves its binding activity. A direct comparison of the molecular mass of the Mu‐IFN‐γ receptor on intact cells versus detergent‐solubilized membranes was performed using a radiolabeled photoactivated crosslinking reagent and direct hybridization with 125I‐labeled IFN‐γ on Western blots of solubilized receptor. The results indicate that both types of receptors have an identical molecular mass of ∼80 kDa.
Independence of triacylglycerol‐containing compartments in cultured fibroblasts from Wolman disease and multisystemic lipid storage myopathy
Authors: Salvayre Robert , Nègre Anne , Radom Jean , Douste-Blazy Louis
Published: July 2002

The functional relationship between the two subcellular compartments involved in catabolism of triglycerides, i.e. lysosomes and lipid‐containing cytoplasmic vacuoles, has been investigated using cultured fibroblasts from patients affected with two different genetic lipid (triacylglycerol) storage disorders: Wolman disease and multisystemic lipid storage myopathy. As shown by metabolic studies in intact cultured cells, lysosomal degradation of exogenous labelled triacylglycerols (incorporated into lipoproteins and internalized via the apo B/E receptor pathway) was blocked in Wolman cells, whereas catabolism of endogenously biosynthesized triacylglycerols was in the normal range. In contrast, in fibroblasts from multisystemic lipid storage myopathy, the degradation of endogenous triacylglycerols was blocked, whereas that of exogenous triacylglycerols (i.e. from lipoproteins) was normal. This comparative study demonstrates that the lysosomal and cytoplasmic compartments are functionally independent. Enzymatic studies allow one to discriminate clearly between 3 lipases and 2 carboxylesterases the role of which is discussed.
Lipid dependence of peptide‐membrane interactions
Authors: Stankowski Stefan , Schwarz Gerhard
Published: July 2002

Membrane incorporation and aggregation of the peptide alamethicin have been investigated as a function of lipid type. Head group and acyl chain regions both contribute to modulate alamethicin incorporation. Specifically, the peptide prefers thin membranes and saturated chains; incorporation is reduced by the presence of cholesterol. Aggregation of the peptide in the bilayer is virtually insensitive to changes in lipid composition. These findings show some analogies to results obtained with intrinsic membrane proteins and cast doubt on the use of global membrane parameters for interpreting lipid‐peptide interactions.
Improved resolution in 1H‐detected 1H‐15N correlation experiments
Authors: Norwood T.J. , Boyd J. , Campbell I.D.
Published: July 2002

The determination of protein structure by NMR is restricted at molecular masses above 10 kDa by overlapping resonances. One way of overcoming this problem is to label the protein with 15N. The conventional way to record 15N spectra is to use heteronuclear multiple‐quantum coherence. We present here an alternative approach based on 15N single‐quantum coherence. This is shown to have substantial advantages over the multiple‐quantum method, including better F1 resolution.
Monitoring the transport and phosphorylation of 2‐deoxy‐D‐glucose in tumor cells in vivo and in vitro by 13C nuclear magnetic resonance spectroscopy
Authors: Navon Gil , Lyon Robbe C. , Kaplan Ofer , Cohen Jack S.
Published: July 2002

We describe the use of 2‐deoxy‐D‐[6‐13C]glucose to follow simultaneously, by 13C NMR, both transport and phosporylation to its 6‐phosphate form, in MCF‐7 breast cancer cells in vitro and in vivo in subcutaneous tumors in nude mice.
Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface
Authors: Borisova G.P. , Berzins I. , Pushko P.M. , Pumpen P. , Gren E.J. , Tsibinogin V.V. , Loseva V. , Ose V. , Ulrich R. , Siakkou H. , Rosenthal H.A.
Published: July 2002

Insertion of foreign oligopeptide sequences (40–50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunogically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre‐S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine‐rich, 39 amino acid‐long C terminus of HBcAg.
Alkaloid homoharringtonine inhibits polypeptide chain elongation on human ribosomes on the step of peptide bond formation
Authors: Tujebajeva R.M. , Graifer D.M. , Karpova G.G. , Ajtkhozhina N.A.
Published: July 2002

The aim of the present study was to investigate homoharringtonine alkaloid effect on: (i) the nonenzymatic and eEF‐1‐dependent Phe‐tRNAphe binding to poly(U)‐programmed human placenta 80 S ribosomes; (ii) diphenylalanine synthesis accompanying nonenzymatic Phe‐tRNAphe binding; and (iii) acetylphenylalanyl‐puromycin formation. Neither nonenzymatic nor eEF‐1‐dependent Phe‐tRNAphe binding were noticeably affected by the alkaloid, whereas diphenylalanine synthesis and puromycin reaction were strongly inhibited by homoharringtonine. It has been proposed that the site of homoharringtonine binding on 80 S ribosomes shouldoverlap or coincide with the acceptor site of the ribosome.
Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+‐sensitive fluorescence dye indo‐1
Authors: Halachmi D. , Eilam Y.
Published: July 2002

Ca2+, cytosolic; Yeast; Saccharomyces cerevisiae; Indo‐1
1H‐NMR study of endothelin, sequence‐specific assignment of the spectrum and a solution structure
Authors: Saudek Vladimir , Hoflack Jan , Pelton John T.
Published: July 2002

Endothelin; NMR; Conformation; Molecular modeling
Deuterons cannot replace protons in active transport processes in yeast
Authors: Kotyk Amošt , Dvořáková Milada , Koryta Jiří
Published: July 2002

Replacement of ordinary water with heavy water causes a sharp reduction of the rates of both primary hydrogen ion transport (at the plasma membrane ATPase) and secondary symports (H+‐associated transports of sugars and amino acids) in several species of yeast. At the same time, the hydrolytic activity of the ATPase is affected only very little. Likewise, the membrane potential, the ΔpH and, correspondingly, the accumulation ratios of the various symported solutes are altered much less. This serves as evidence that H+ or 113,0 + ions are direct participants in the various active transports of nutrients in yeast.
A putative AP‐2 binding site in the 5' flanking region of the mouse POMC gene
Authors: Bishop John F. , Rinaudo Mario S. , Ritter Joseph K. , Chang Annie C.-Y. , Conant Katherine , Gehlert Donald R.
Published: July 2002

Using extracts of AtT‐20 cell nuclei, protein binding sites on the POMC gene 5'‐flanking region were examined with an exonuclease protection approach. One such binding site, located from −119 to −106 bp upstream from the mouse POMC gene transcription initiation site, which exhibited a close homology to the aetivator protein‐2 (AP‐2) site [1]. A double‐stranded oligonucleotide containing this site was subsequently used in gel shift assays to demonstrate AP‐2 consensus sequence binding activity in extracts of AtT‐20 cell nuclei. Gel shift competition experiments using both homologous and heterologous competitor DNA sequences revealed that the AP‐2 like factor(s) exhibited specific binding to the mouse AP‐2 consensus sequence. Furthermore, AP‐2 factor binding was also modulated by a CTF/NFl‐like factor. Pretreatment of AtT‐20 cell nuclear extracts with alkaline phosphatase prior to inclusion in gel shift assays led to a reduction in the intensities of AP‐2 factor‐specific bands, indicating a potential involvement of protein phosphorylation in AP‐2 factor binding in AtT‐20 cells.
The purification and amino acid sequence of the lethal neurotoxin Tx1 from the venom of the Brazilian ‘armed’ spider Phoneutria nigriventer
Authors: Diniz Carlos Ribeiro , do Nascimento Cordeiro Marta , Junor Leonides Rezende , Kelly Patrick , Fischer Stefan , Reimann Frank , Oliveira Eduardo B. , Richardson Michael
Published: July 2002

A lethal neurotoxic polypeptide of Mr 8kDa was purified from the venom of the South American ‘armed’ or wandering spider Phoneutria nigriventer by centrifugation, gel filtration on Superose 12, and reverse phase FPLC on columns of Pharmacia PepRPC and ProRPC. The purified neurotoxin Tx1 had an LD50 of 0.05 mg/kg in mice following intracerebroventricular injection. The complete amino acid sequence of the neurotoxin was determined by automated Edman degradation of the native and S‐carboxymethylated protein in pulsed liquid and dual phase sequencers, and by the manual DABITC/PITC double coupling method applied to fragments obtained after digestions with the S. aureus V8 protease and trypsin. The neurotoxin Tx1 consists of a single chain of 77 amino acid residues, which contains a high proportion of cysteine. The primary structure showed no homology to other identified spider toxins.
Formation of a cytosolic ganglioside‐protein complex following administration of photoreactive ganglioside GM1 to human fibroblasts in culture
Authors: Chigorno Vanna , Valsecchi Manuela , Acquotti Domenico , Sonnino Sandro , Tettamanti Guido
Published: July 2002

A GM1 ganglioside derivative bearing a photoreactive nitrophenyl azide group and tritium labeled at the acetyl group of N‐acetylneuraminic acid, has been administered to cultured human fibroblasts. With photolabeling experiments we found that a portion of the ganglioside in the cell cytosol was associated with a soluble protein of about 30 kDa.
Hemorphins derived from hemoglobin have an inhibitory action on angiotensin converting enzyme activity
Authors: Lantz Ingrid , Glämsta Eva-Lena , Talbäck Lena , Nyberg Fred
Published: July 2002

The hemorphins are opioid active peptides, which are enzymatically released from the beta‐chain of hemoglobin. In this paper we report an inhibitory effect of these peptides on angiotensin converting enzyme (ACE) activity, known to be involved in blood pressure regulation. The hemorphins were found to be quite stable in tissue extracts containing ACE, and their importance as naturally occurring ACE inhibitors is discussed.
Rabbit skeletal muscle myosin unfolded carboxyl‐terminus and its role in molecular assembly
Authors: Maeda Kayo , Rösch Andrea , Maéda Yuichiro , Kalbitzer Hans Robert , Wittinghofer Alfred
Published: July 2002

We have expressed in E. coli segments of the rod portion of rabbit skeletal fast muscle myosin and compared physical properties of two different species, LMM‐30 and LMM‐30C′. LMM‐30 consists of 263 amino acids including the original C‐terminus of myosin heavy chain. LMM‐30C′ is colinear with LMM‐30, but is devoid of 17 residues at the C‐terminus. 1H NMR spectroscopy indicates that the C‐terminus of LMM‐30, but not of LMM30C′ is unfolded and freely mobile. Furthermore, the present results show that the unfolded C‐terminus is essential for molecular assembly of LMM‐30; at pH 8.0 LMM‐30, but not LMM‐30C′, formed aggregates upon decreasing the ionic strength.