bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Homology of Lens Crystallins
Authors: ARGOS Patrick , SIEZEN Roland J.
Published: March 2005

The X‐ray crystallographic structure of bovine γ‐crystallin shows four similar folding motifs each composed of about 42 residues arranged as four topologically sequential, anti‐parallel β‐strands. Since the β and γ‐crystallin sequences show good homology, proposals for a four‐motif β‐crystallin model have been made. The other bovine eyelens protein species, α‐crystallins, are not homologous to β or γ‐crystallin in primary structure. In the present work, smoothed plots of amino acid sequence number versus a residue characteristic (e.g. hydrophobicity) were calculated for the various crystallins. Cross‐correlation coefficients were then determined between pairs of crystallin plots for various registers of the curves. The correlation plots were then combined for several characteristics and for pairwise comparisons between β or γ‐crystallin and the α‐crystallins. The resulting plots showed four peaks separated by about 42 residues for the α‐crystallins, suggesting that they also possess a four‐motif β‐barrel structure. The physical parameter comparison technique appears generally applicable in suggesting a structural and functional relationship amongst proteins that show no primary sequence homology.
Effects of Substrates on the Heat Stability and on the Reactivities of Thiol Groups of 3‐Phosphoglycerate Kinase
Authors: CSERPÁN Imre , VAS Mária
Published: March 2005

Pig muscle 3‐phosphoglycerate kinase contains seven cysteine residues/molecule enzyme. Two of them react with Ellman's reagent (Nbs2) in a second‐order reaction [k= (1.1 ± 0.1) × 103 M−1 s−1]; the reaction of the other five thiols are limited by a first‐order protein structural change [k= (2.0 ± 0.4) × 10−4 s−1] in 0.1 M Tris/HCl buffer, pH 7.5 at 20°C.

Blocking the rapidly reacting thiols with Nbs2 inactivated the enzyme (these two –SH groups are not equivalent in this respect), but it does not abolish substrate‐binding ability.

The rapidly reacting thiol groups readily participate in intermolecular disulfide formation following their partial blocking with Nbs2. This type of aggregation of 3‐phosphoglycerate kinase molecules also leads to inactivation.

The order of effectivity of substrates in inhibiting the reaction of the slowly reacting thiols is very similar to the order of their protective effect against heat inactivation. Both phenomena presumably reflect the structure‐stabilizing effect of substrates.
Protein Synthesis Directed by Polyadenylated and Non‐polyadenylated RNA
Authors: BARAHONA Isabel , GALEGO Lisete , RODRIGUES‐POUSADA Claudina
Published: March 2005

Free and membrane‐bound polysomes were prepared from the protozoa Tetrahymena pyriformis using a procedure which gives good recovery and practically no cross‐contamination. Polysomes are intact as analysed by sedimentation analysis. Poly(A)‐rich RNA and poly(A)‐free RNA, isolated from both populations of polysomes, show similar electrophoretic patterns. These RNAs were translated in the rabbit reticulocyte lysate cell‐free system and the translation products were analysed by one‐dimensional and two‐dimensional gel electrophoresis. The most striking differences were found in the two‐dimensional electrophoretic analysis namely: (a) a group of polypeptides (10) is synthesized mainly on membrane‐bound polysomes, (b) a second abundant group is synthesized mainly in free polysomes (c) and a third class of polypeptides is synthesized on both kinds of polysomes.

Poly(A)‐free RNAs, isolated from free polysomes, are also able to promote synthesis of some polypeptides.

The results are discussed taking into account the fact that T. pyriformis is a non‐secretory cell.
Polymorphism in High‐Affinity Calcium‐Binding Proteins from Crustacean Sarcoplasm
Authors: WNUK Wlodzimierz , JAUREGUI‐ADELL Juan
Published: March 2005

The sarcoplasmic calcium‐binding proteins (SCP) from crayfish, lobster and shrimp myogen have been purified to homogeneity. These proteins exist as dimers and dissociate in the presence of sodium dodecyl sulfate or urea in subunits of 22000 molecular weight. During the last step of purification (DEAE‐cellulose chromatography), SCP emerges in three peaks in the ratio of 14:1.5:1 for crayfish, of 7:2:1 for lobster and of 3:2:1 for shrimp. Gel electrophoresis and isoelectrofocusing experiments, under native and denaturing conditions, indicate that among the three SCP isotypes there are only two different polypeptide chains, α and β, which appear in the form of three dimers: α2, αβ and β2. The α and β subunits differ slightly in polypeptide chain composition as found by amino acid analyses of the crayfish and lobster SCPs, and also by comparison of tryptic peptides for crayfish SCPs. The polymorphism observed in crustacean SCPs, which is increased by their ability to form dimers, contrasts with the situation prevailing among other invertebrate SCPs and vertebrate parvalbumins where only monomeric isotypes are found. Equilibrium binding studies show that all three SCP isotypes from both crayfish and lobster display the same metal‐binding properties. They have in their dimeric form six Ca2+‐binding sites: two calcium‐specific sites, two Ca/Mg sites that interact with positive cooperativity and two Ca/Mg sites that interact with negative cooperativity. Interactions between the two subunits of SCP seem to result in cooperative binding of Ca2+, which in turn may control more efficiently Ca2+ fluxes in crustacean muscle.
Differential Determination of the 3‐Deoxy‐d‐mannooctulosonic Acid Residues in Lipopolysaccharides of Salmonella minnesota Rough Mutants
Authors: BRADE Helmut , GALANOS Chris , LÜDERITZ Otto
Published: March 2005

Different applications of the thiobarbituric acid assay were used to determine C4/5‐unsubstituted, heptosyl‐substituted and total 3‐deoxy‐d‐mannooctulosonic acid (dOclA) in lipopolysaccharides of Salmonella minnesota rough mutants. C4/5‐unsubstituted dOclA is determined without prior hydrolysis of the lipopolysaccharides by periodate oxidation near neutral pH at 4°C using the methyl ketoside of dOclA as a standard. C4/5‐unsubstituted dOclA, and dOclA which is substituted by other dOclA residues, is quantified after hydrolysis in 0.1 M acetate buffer pH 4.4 at 100°C/1 h. Total dOclA, including heptosyl‐substituted dOclA, is measured after hydrolysis in 1 M HCl at 100°C for 1–4 h. For S. minnesota R595, 760–790 nmol/mg lipopolysaccharide are determined independently of the hydrolysis conditions employed. In R7, one half, and in R345, two thirds of the total dOclA content are detected after mild acid hydrolysis. Hydrolysis in 1 M HCl at 100°C for 1 h (R7) and 4 h (R345) are required to liberate the heptosyl‐substituted dOclA residues. For the three lipopolysaccharides tested, determination of C4/5‐unsubstituted dOclA revealed that about one third of the total dOclA is reactive in this application of the thiobarbituric acid assay. By strong acid hydrolysis, a 3‐deoxy‐2‐ketoaldonic acid is identified in the lipopolysaccharide of Vibrio cholerae. Upon mild and strong acid hydrolysis, a 3‐deoxy‐2‐ketoaldonic acid is identified in the lipopolysaccharide of Vibrio cholerae. Upon mild and strong acid hydrolysis, dOclA undergoes degradation which is recognized by (a) decreased reactivity in the thiobarbituric acid assay (b) changed ultraviolet spectra and (c) development of compounds which are reactive in the thiobarbituric acid assay after reduction with sodium borohydride. The rate of degradation depends on the strength of the acid but is the same for reference and lipopolysaccharide‐liberated dOclA under a given hydrolysis condition.
Two α‐3‐d‐Galactosyltransferases in Rabbit Stomach Mucosa with Different Acceptor Substrate Specificities
Authors: BETTERIDGE Alan , WATKINS Winifred M.
Published: March 2005

Homogenates of rabbit stomach mucosa were examined for enzymes catalysing the transfer of d‐galactose from UDP‐d‐galactose to various low‐molecular‐weight acceptors of known structure. Treatment of the products with α and β‐d‐galactosidases revealed that d‐galactose was transferred in both α and β‐anomeric linkages. The β‐d‐galactosyltransferase used N‐acetylglucosamine and compounds containing terminal nonreducing β‐anomeric linkages. The β‐d‐galactosyltransferase used N‐acetylglucosamine and compounds containing terminal nonreducing β‐N‐acetylglucosaminyl residues as acceptor substrates. The compounds accepting d‐galactose in α‐anomeric linkage had unsubstituted terminal non‐reducing β‐d‐galactosyl units or a fucose substituent on the carbon‐2 position of a subterminal β‐d‐galactosyl unit. Methylation analysis of the products formed with N‐acetyllactosamine [β‐d‐Galp(1 → 4)d‐GlcNAcp] and 2'fucosyllactose [α‐l‐Fucp(1 → 4)d‐Glcp] revealed that d‐galactose was transferred to the carbon‐3 position of the β‐d‐galactosyl residue in both of these acceptor substrates. Competition experiments with the two substrates indicated that the transfer of d‐galactose was catalysed in each case by a different α‐3‐d‐galactosyltransferase. Differences were also observed in the solubility properties of the enzymes: the α‐3‐d‐galactosyltransferase using acceptor substrates with unsubstituted β‐d‐galactosyl residues was more realdily soluble both in the presence and absence of detergents than the transferase using β‐d‐galactosyl residues substituted at carbon‐2 with l‐fucose. These findings demonstrate that rabbit stomach mucosa has two disfinct α‐3‐d‐galactosyltransferases: one, which is more tightly membrane‐bound, resembles the human B‐gene‐specified transferase in its acceptor specificity, and the second, which is a more soluble enzyme, transfers d‐galactose to the same positional linkage in unsubstituted β‐d‐galactosyl residues.
An Electron Microscopic Approach to the Quaternary Structure of Mitochondrial F1‐ATPase
Authors: TIEDGE Henri , SCHÄFER Günter , MAYER Frank
Published: March 2005

The three‐dimensional structure of F1‐ATPase from beef heart mitochondria was investigated by electron microscopic techniques. The presence of high concentrations of nucleotides is essential for preservation of the quaternary structure. When in vistigated under such coinditions, monodisperse F1‐ATPase could not be distinguished from the membrane‐bound enzyme. At low resolution, the particle shape resembles an oblate ellipsoid of revolution with an axial ratio of about 2:1.

From several lines of evidence (including field micrographs at higher magnifications, Markham rotational analysis, and tilting experiments), two conclusions may be drawn concerning the three‐dimenwsional fine structure of F1‐ATPase.

1. At the periphery of the molecule, six globular protein masses are orientated in a way similar to the chair conformation of cyclohexane. This array is interpretated to be made up of an alternating sequence of α and β subunits.

2. Part of the central space is occupied by a seventh protein mass, protrusions of which are likely to be in contact with some of the outer subunits. A γ subunit is supposed to be constituent part of this central protein mass.

As a consequence, this model favours a stoichiometry oif
Binding of MgATP to the Nitrogenase Proteins from Azotobactor vinelandii
Authors: CORDEWENER Jan , HAAKER Huub , VEEGER Cees
Published: March 2005

Binding of MgATP to the MoFe and Fe proteins from Azotobacter vinelandii has been studies, By means of the flow dialysis technique it was demonstrated that one molecule of reduced Fe peotein binds one molecule of MgATP, with a dissociation ocnstant of 0.56 ± 0.11 mM. The oxidized binds two molecules of MfATP, with identical intrinsic dissociation constants of 0.29 ± 0.05 mM. The binding of MgATO to the Fe protein was also studied by equilibrium dialysis. It was found that during dialysis of reduced Fe protein in the presence of MgATP, dishionite was oxidized. Moreover, in the presence of MgATP both reduced and oxidized Fe protein were inactivated during the dialysis. These observations demonstrate that binding of MgATP to the Fe protein can only be measured by a relatively fast method.

With the same methods as used for the Fe protein, no binding of MgATP to the MoFe protein of A. Vinelandii could be demonstrated.

The redox properties of the Fe protein in the presence and absence of MgATP are sicussed with respect to the observed binding properties of MgATP for the Fe protein. The implications of these results are discussed with respect tothe present models for the inhteractins between the Fe and MoFe proteins of nitrogenase.
B‐Z Transition in Methylated DNA
Authors: VAN LEIR John J. C. , SMITS Marieke T. , BUCK Henk M.
Published: March 2005

On the basis of modified neflect of differntial overlap (MNDO) quantum‐chemiocal calculations on nucleoside systems, we descrive the effect of methylation on the energies calculated for the rotation around the glycosidic C(1′)‐N bond. We found a high anti‐syn activation energy in the case of the pyrimidine nucleosides C and m5C, Whereas for the purine nucleosides G, m6G, m7 and m8 only moderate anti‐syn energetic barriers were claculated. This result is conssitent with the experimentally observed preference for d(G‐C)3 and d(G‐m5C)3 duplexes to adopt Z‐DNA stuctures, in which the syn conformation of guanine is favoured. Enhanced anti‐syn activation energy with respect to the unmethylated derivative was calculated in the cases of m5G. This result is rationalized on the basis of steric and electronic factors. In addition, an increased stabilization of the syn conformer due to selective methylation of guanine was calculated. The data obtained are in good corrspondence with the experimentally observed B‐Z transition iun synthetic methylated DNA duplexes with alternating dC‐dG sequence.

Our work concerning the initiating step in the B‐Z transistion which involves rotation around the C(4′)‐C(5′) bond induced by P(V) trigonal bipyramidal intermediates is discussed. In combination with the rotation around the glycosidic C(1′)‐N bond, it can be shown that the phosphate withinh thge dpC structure is selectively sctivated.
Stability of the Ca2+‐Specific and Ca2+‐Mg2+ Domains of Troponin C
Authors: INGRAHAM Richard H. , SWENSON Charles A.
Published: March 2005

The unfolding of rabbit fast skeleal muscle troponin C has been examined as a function of pH using differential scanning calorimetry as a probe. Studies were conducted between pH 7.2 and 5025 in the absence of both Ca2+ and Mg2+. At pH 7.2, a single unfolding transition with a tm of 58°C, is observed. Decreasing the pH to 5.5 increases the tm for this transition to 67°C. At values ≦5.5, a second unfolding transition is observed, which has a tm of 51°C. These studies suggest that troponin C consists of two types of structural domains: one has a compac structure throughout the pH range examined; the other has a poorly ordered structure at pH 7.2 which becomes more compact as the pH is decreased. We conclude that stabilization of both types of domains occurs through protonation of carboxylate groups clustered in each of the four Ca2+‐binding loops. Our data are consistent with assignment of the higher‐temperature transition to unfolding of N‐terminal protion of the molecule, which contains the Ca2+‐specific domains and of the lower‐temperature transition to unfolding of the C‐terminal portion, which contains the Ca2+‐Mg2+ domains.
Low‐Molecular‐Weight Polypeptides of Vicilin from Vicia faba L. are Product of Proteolytic Breakdown
Authors: SCHOLZ Günter , MANTEUFEEL Renate , MÜNTZ Klaus , RUDOLPH Armin
Published: March 2005

Vicilin, the main 7‐S globulin of Vicia faba L., undergoes cleavage during prolonged treatment at room temperature, which can be inhibited by protease inhibitors such as 1 μM leupptin. The cleavage products show identical electrophoretic mobilities with the polypeptides normally visible after sodium dodecylsulphate gel electrophoresis of vicilin prepared from mature seeds. N‐terminal amino acid analysis of electrophoretically prepared polypeptides reveals serine as common N terminus of the two largest polypeptides of Mr∼ 50000 and 35000. According to serological experiments and peptide mapping the low‐molecular‐weight polypetides (Mr∼ 35000; 31000; 19000 and below) have antigenic determinants and amino acid sequences, respectively, that are similar to each other and are all contained within the structure of the large polypetide of Mr∼ 50000. This leads of the conclusion that polypetides of Mr∼ 35000 and below are derived by proteolysis from one (or a few closely related) polypeptide(s) of Mr∼ 50000 and that proteolysis starts soon after biosynthesis as a post‐translational process within the developing seed. Some experiments indicate the existence of ‘nicking’ points within the vicilin polypetides, which are the major cleavage sitesssss during preparation. These observations strongly support the view that ‘native’ vicilin is a trimeric or tetrameric globulin with polypetides of Mr∼ 50000.
Baker's Yeast Adenlate Kinase
Authors: TOMASSELLI Alfredo G. , NODA Lafayette H.
Published: March 2005

The diastereomers of adenosine 5′‐O‐(1‐thiotriphosphate) ATPαS) and adenosine 5′‐O‐(2‐thiotriphosphate) (ATPβS) were used to determine the stereochemical mode of coordination of Mg2+ to ATP when bound in the yeast adenylate kinase complex. ATPαS isomer A is the preferred substrate either in the presence of Mg2+ or Cd2+; ratios of the rate of reaction at infinite concentration of substrate, V, for A to B isomers of ATPαS with Mg2+ were 457/51.4 = 8.9 and with Cd2+ 124/12.8 = 9.7. Thus there is no reversal of stereospecificity on substituting Cd2+ for Mg2+ and this supports the view that metal does not chelate with the α‐P. In contrast the V ratios of ATPβS(A) to ATPβS(B) with Mg2+ were 1.33/0.15 = 8.9 and with Cd2+ 0.18/5.1 = 0.035. Thus this reversal of stereospecificity when Cd2+ is substituted for Mg2+ (in view of the fact that Mg2+ binds to O and Cd2+ to S) leads to the conclusion that a metal chelation must occur via the β‐phosphate. Since the enzyme prefers MgATPβS(A) and CdATPβS(B) (both isomers have Δ‐screw sense) to MgATPβS(B) and CdATPβS(A) (both isomers have Δ‐screw sense) and Mg2+ also coordinates with the γ‐phosphate (by analogy with other adenylate kinase isozymes), the interpretation is that baker' yeast adenylate kinase binds MgATP as Δ, β, γ. The fluorescence of the two residues of yeast adenylate kinase has been used to investigate conformational changes of the enzyme on binding its substrates. Fluorescene is maximal at 334 nm (λexx 295 nm, pH 7.5) and is reduced 10% with no shift in λmax in the presence of ATP and reduced a further 15% on the addition of Mg2+ or other metal acitvators to E‐ATP. In contrast non‐activating metals such as Ba2+ and Sr2+ increase quenching only slightly. With ITP, UTP, CTP, GTP the quenching is 4.1%, 5.2%, 8.7% and 6.6% with or without Mg2+. Differential conformational changes of yeast adenylate kinase on binding MgATP, ATP, and other nucleotides are shown by (a) fluorescence changes, (b) previously reported kinetic and binding studied and (c) by the fact that the Km for AMP in the presence of MgATP is 17 times less than in the presence of MgGTP. These differential conformational changes result in differing tendencies of AMP to bind to the enzyme. Ultraviolet difference spectra obtained on binding of ATP to the enzyme (shoulder at 271 nm and peak at 276 nm) have been compared with the difference spectra of the interaction of ATP, adenosine and 1‐methyl adensoine respectiively with methyl mercuric hydroxide and formaldehyde. On the basis of these comparisons, the shoulder at 271 nm reflects the interaction of N(1) and the peak at 276 nm the interaction of tthe 6‐amino group of ATP with the enyzme.

The following mechanism of reaction is proposed: upon binding MgATP the enzyme undergoes a conformational change which closes the active‐site crevice by bringing the two lobes closer, expelling water from the crevice. AMP is bound with higher affinity and the α‐oxygen and the α‐oxygen attacks the γ‐phosphate of ATP in a nucleophilic reaction.
Determination of the Turn‐Off Reaction for the Epinephrine‐Inhibited Human Platelet Adenylate Cyclase
Authors: JAKOBS Karl H.
Published: March 2005

Epinephrine inhibits human platelet adenylate cyclase by an α2‐adrenoceptor‐mediated and GTP‐dependent process. The turn‐off reaction for this epinephrine‐inhibited enzyme was studied by measuring the rate of cyclic AMP formation upon addition of the α2‐adrenoceptor antagonist, yohimbine, or upon addition of an excess of the stable GDP analog, guanosine 5′‐O‐(2‐thiodiphosphate) (GDPβS), which competitively inhibited the action of GTP in the epinephrine‐induced inhibition. The decay of the inhibited state of the adenylate cyclase was used to calculate the rate constant of the turn‐off reaction. With both methos, almost identical koff values of 0.6–0.7 min−1 at 25°C were obtained for the epinephrine‐inhibited platelet enzyme. Cholera toxin, which does not inhibit the epinephrine‐induced GTPase stimulation in platelet membranes, did not affect this turn‐off reaction. In contrast, the turn‐off rate of the prostaglandin‐E1‐stimulated human platelet adenylate cyclase, measured with GDPβS, was reduced from about 9 min−1 to 2 min−1 at 25°C by pretreatment of the membranes with cholera toxin, which inhibits the prostaglandin‐E1‐stimulated GTPase activity. The data strongly that the guanine nucleotide regulatory site, mediating epinephrine‐induced adenylate cyclase inhibition is activated and inactivated by similar mechanisms as is the site mediating adenylate cyclase stimulation, and that cholera toxin affects only the stimulatory site. The findings furthermore suggest that the activity states of these two regulatory sites control the activity of the adenylate cyclase.
Post‐Translational Fate of Variant MOPC 315 λ Chains in Xenopus Oocytes and Mouse Myeloma Cells
Authors: VALLE Giorgio , BESLEY Jayne , WILLIAMSON Alan R. , MOSMANN Timothy R. , COLMAN Alan
Published: March 2005

The post‐translational fates of three immunoglubulin λ chain variants of MOPC 315 were investigated in mouse plasmactyoma cell lines and in mRNA‐microinjected Xenopus oocytes.

Quite unexpectedly we found that one non‐secretory variant chain (λ‐43) underwent extensive post‐translational N‐glycosylation; however the presence of the oligosaccharide moiety did not account for the non‐secretory phenotype nor did it affect the rate of degradation of this λ chain. Another variant chain (λ‐47) at first believed to be non‐secretory, was found to be secreted from oocytes at a very low level, but mostly as a λ‐λ dimer. In myeloma cells a low level of λ‐47 chain was secreted and again λ‐λ dimers were the favoured secretory form. The secretory λ‐48 chain also formed λ‐λ dimers, whereas λ‐43, which was never secreted, was only found as a monomeric λ chain in both oocytes and myeloma cells.

A similar relationship between assembly and secretion was found when oocytes were coinjected with MOPC 21 heavy (γ1) chain mRNA and MOPC 315 λ chain mRNAs. The wild type λ chain (λ‐48) was able to assemble with the γ chain in a covalently bound tetramer (γγλλ). The variant λ‐47 chain was also able to form γγλλ tetramers, whereas the λ‐43 was nott, even when glycosylation was prevented by tunicamycin. Both types of tetramer were secreted.

These data reinforce the idea that conformational changes play a major role in the routing of secretory proteins and that the cellular mechanisms by which these changes are recognized are not cell‐type specific.
Partial Purification and Characterisation of an Acetylcholine Receptor with Nicotinic Properties from the Supraoesophageal Ganglion of the Locust (Schistocerca gregaria)
Authors: FILBIN Marie T. , LUNT George G. , DONNELLAN John. F.
Published: March 2005

An α‐bungarotosin‐binding component has been partially purified from the supraoesophageal ganglion of the locust, (Schistocerca gregaria). The component binds α‐bungarotosin with a Kd of about 1.7 nM and this value changes little throughout the purification procedure. The specific binding activity ranges from 1.18 pmol α‐bungarotoxin bound/mg protein for the membrane‐bound site up to a maximum of 230 pmol bound/mg protein for the partially purified component. The pharmacological properties of the membrane‐bound site are predominantly nicotinic. Affnity labelling of the binding species with 4‐(N‐maleimido)‐[3H]benzyltrimethylammonium suggests that the binding is associated with a peptide of Mr 58000. Polyacrylamide gel electrophoresis of the partially purified of binding component shows three major bands corresponding to Mr of 60000, 41000 and 25000. We suggest that the binding component can be tentatively identified as a nicotinic acetylcholine receptor.
Binding of Photobilkirubin to Human Serum Albumin
Authors: LAMOLA Angelo A. , FLORES Jorge , BLUMBERG William E.
Published: March 2005

Results of experiments bases upon circular dichroic spectra suggest that configuratinally (Z → E) isomerized bilirubin (photobilirubin) binds to human serum albumin at the primary bilirubin biding site withan affinity only 2–3‐times lower than that of biliruin. The high affinity of photobiliruin fopr albumin, comparable to that of bilirubin, supports the roles of albumin in the stablization and transport of the isomerized pigment in vivo and stronlgy suggests that albumin also functions to sequester photobilirubin effectively, reducing its toxic potential. The high affinity of photobilirubin for albumin predicts that the isomerized pigment, formed in large amounts during phototherapy ofr neonatal hyperbilirubinemia, should not appear in the fast‐diazo‐reacting (‘dirct’) bilirubin pool nor should it interfere wth nonspectroscopic biliruin binding tests. These predicions were confirmed for the Evelyn and Malloy diazo assay for ‘direct’ bilirubin and a Sephadex chromatography method for assessing ‘loosely bound’ plasma bilirubin.
Spin‐Label Study of Histone H1‐DNA Interaction
Authors: GURARDET Jean‐Luc , LAWRENCE Jean‐Jacques
Published: March 2005

As shown in a previous paper [J. J. Lawrence et al. (1980) Eur. J. Biochem. 107, 263–269], convalent spin labeling of basis residues in histone H1 allows the study of the interaction of this protein with DNA. Using a step gradient dialysis procedure to reconstitute chromatin from labeled H1 and stripped chromatin, it is shown that the process of interaction of the lysine residures and DNA is the same whether histone H1 is bound to linear purified DNA or to H1‐depleted chromatin.

In contrast, spin labeling of the unique tyrosine of histone H1 located in the globular part of the molecule shows that this part is more involved in the interaction with chromatin than it is with linear DNA (as judged from the lengthening of the rotational correlation time).

These data are interpreted as reflecting defferent roles for th N and C termini of the molecule of H1 and the central globular part. A model, based on these observations together with examination of the primary structures of histones H1, is proposed which accounts for the H1 involvement in the chromatosome structure.
Structure of Teichoic‐ Acid—Glycopeptide Complexes from Cell Walls of Bacillus cereus AHU 1030
Authors: SASAKI Yasuo , ARAKI Yoshio , ITO Eiji
Published: March 2005

From lysozyme digests of N‐acetylated cell walls of Bacillus cereus AHU 1030, two acidic polymer fractions with molecular weights of about 24000 and 45000 were isolated by ion‐exchange chromatography and gel chromatography. These polymer fractions, containing glycerol, phosphorus and glucose in a molar ration of 1.00: 1.00: 0.85 together with small amounts of glycopeptide components and mannosamine, were characterized as teichoic‐acid—glycopeptide complexes with one and two teichoic acid chains made of 60–65 repeating glycerol phosphate units that were mostly glucosylated. Mild alkali treatment of the complexes yielded a disaccharide‐linked glycopeptide. The disaccharide was liberated from the glycopeptide by mild acid treatment and identified as N‐acetylmannosaminyl(β1 → 4) N‐acetylglucosamine. On the other hand, the same disaccharide linked to the teichoic acid chain was obtained by direct heating of the cell walls at pH 2.5. These results lead to a conclusion that in the cell walls of this strain the glycerol teichoic acid chain is attached to the glycan chain of peptidoglycan through this disaccharide unit. The disaccharide is linked at its reducing and nonreducing ends to the glycan chain and the teichoic acid chain, respectively, through phosphodiester bridges.
Ribosomal Proteins cross‐Linked to 28‐S and 18‐S rRNA Separated by Sedimentation After Ultraviolet Irradiation of Rat‐Liver Ribosomes
Authors: UCHIUMI Toshio , TERAO Kazuo , OGATA Kikuo
Published: March 2005

Rat liver 80‐S ribosomes, irradiated with 254nm ultraviolet light for 15–60 min (0.63–2.5*105 quanta/ribosome), were treated with sodium dodecyl sulfate; 18‐S and 28‐S RNA fractions were isolated by sucrose density‐gradient centrifugation. The protein moieties of the 18‐S and 28‐S rRna fraction were labeled with 125I. After digestion of the rRNA fractions with RNase A and T1, the cross‐linked proteins were analyzed by two‐dimensional acrylamide gel electrophoresis and then autoradiography. Cross‐linkid proteins were examined further by didecyl sulfate/adcurylamide gel electrohphoresis of individual radioactive spots of proteins and their proteolytic peptides on two‐dimensional gels. The results showed that S2 and S15 were cross‐linked to 18‐SrRNA, and L5, L6 and L8 to 28‐S rRNA. Similar resulys were obtained when isolated 40‐S or 60‐S subunits were irradiated.
Calcium Modulates Fatty Acid Dynamics in Rat Liver Plasma Membranes
Authors: SCHROEDER Friedhelm , SOLER‐ARGILAGA Carlos
Published: March 2005

Modulation of free fatty acid binding in isolated rat liver plasma membrances was evaluated using the fluorecent fatty acids trans‐parinaric and cis‐parinaric acid as analogues for saturated and unsaturated fatty acids, respectigvely. Binding of trans‐parinarate but not cis‐parinarate was inhibited by physiological levels of Ca2+. The effect was revedrsed by addition of excess EGTA. Calcium decreased the aqueous to lipid partition coefficient, Kp, of trans‐parinaric acid for liver plasma membrances while increasing the Kp for trans‐parinaric acid. In addition, Ca2+ also altered the fluorescence lifetime, the quantum yield, and the relative partitioning of trans‐parinaric and cis‐parinaric acid into fluid and solid phases. Calcium and EGTA did not affect the binding of 1, 6‐diphenyl‐1,3,5‐hexatriene. The effect of Ca2+ on the liver plasma membrane structure was to increase the rigidity of the membrane, primarily the solid domain. The fluorescence polarization of trans‐parinarate, cis‐parinarate, and 1,6‐diphenyl‐1,3,5‐hexatriene at 24°C in liver plasma membranes in the absence of Ca2+ was 0.295 ± 0.008, 0.253 ± 0.007, and 0.284 ± 0.005, respectively. Calcium (2.4 mM) increased the polarization of these probe molecules in liver plasma membrances by 8–10%. EGTA (3.4 mM) reversed or abolished the increase in polarization. Thus, the fluorescent fatty acids trans‐parinarate and cis‐parinarate may be used to monitor fatty acid binding by isolated membranes, to evaluate factors such as Ca2+ which modulate fatty acid binding, and to investigate the microenvironemtn in which the fatty acids reside. The data suggest that Ca2+ may be an important regulator of fatty acid uptake by the liver plasma membrane, and therby interact with intermediary metabolism of lipids at a step not involging lipolytic or synthetic enzymes.
Phosphorylated Intermediate of a Transport ATPase and Activity of Protein Kinase in Membranes from Corn Roots
Authors: SCALLA René , AMORY Antoine , RIGAUD Jacqueline , GOFFEAU André
Published: March 2005

A maize‐root microsomal fraction was enriched in ATPase by treatment with Triton X‐100. This activity, which reached 1.2–2.0/μmol Pi× min−1× gm protein−1, was specific for ATP, very slightly stimulated by K+, inhibited by orthovanadate and diethylstibestrol, resistant to oligomycin and azide, and had a Km of 1.2 mM MgATP.

Incubation of th emicrosomal fraction with [y32‐P]ATP followed by electrophoresis in acid conditions revealed the presence of several phosphoproteins. The phosphorylation of a 110000‐M4 polypeptide reached the steady‐state level in less than 5 s and rapidly turned over the phosphate group. The phosphorylation level was an hyperbolic function of the [ATP] with a Km of 0.6 mM, suggesting that the rat e of Pi production was proportional to the phosphoprotein concentration. The extent of phosphoprotein was decreased by vanadate and diethylstilbestrol. The phosphorylation level was 30% decreased by 50 mM K+ or Na+ while the ATPase activity was slightly stimulated (12% and 5%. respectively). The polypeptide could not bephosphorylated in reverse by Pi. This phosphorylated intermediate from maize‐root microsomes exhibits molecular properties characteristic of transport ATPases such as the yeast plasma membrance H+ ‐translocating ATPase. This similarity indicates existence of a transport ATPase in plant plasma membranes.

Three other plant microsomal polypeptides (Mr= 52000, 17000 and 16000) and a low molecular weight component (Mr < 1000) were phosphorylated much more slowly, were not undergoing a rapid turnover and were not hydrolysed by hydroxylamine. These phosphoproteins and the Mr < 1000 phosphorylated component were inhibited by vanadate and diethylstilbestrol. These properties are similar to those of the protein kinase activity recently described in yeast plasma membranes.
Interaction of tRNAPhe and tRNAVal with Aminoacyl‐tRNA Synthetases
Authors: VLASSOV Valentin V. , KERN Deniel , ROMBY Pascale , GIEBÉ Richrd , EBEL Jean‐Pierre
Published: March 2005

The alkylation by ethylnitrosourea of phyosphodiester bonds in tRNAPhe from veast and in tRNAVal from yeast and from rabbit liver and that by 4‐N‐2‐chloroethyl‐n‐methylamino)‐benzylamine of N‐7 atoms of guanosine residues in yeast tRNAVal have been used to study the interaction of these tRAAs with anminoacyl‐tRNA synthetases. The modifications occurring at low yield were carried out on 3′ and'or 5′ end‐labelled tRNAs either free or in the presence of cognate or non‐cognate synthetases. After splitting of the tRNAs at the alkylated positions, the position of the modification sites in the tRNA sequences were detected by acrylamide gel electrophoresis. It was found that the synthetases protect against alkylation certain phosphate or guanosine residues in their cognate tRNAs. Non‐cognate synthetases failed to protedct efficiently specific positions in tRNA against modification.

In yeast tRNAPhe the cognate phenylalanyl‐tRNA synthetase protects certain phosphates located in all four stems and in the anticodon and extra‐loop of the tRMA. Particularly strong protections occur on phosphate 34 in the anticodon loop and on phosphates 23, 27, 28, 41 in the D and anticodon stems.

In yeast tRNAVal complexed with yeast valyl‐tRNA synthetase the protected phosphates are essentialy located in the tRNA. Three guanosine residues, located in the D stem, and another one in the 3′ part of the anticodon stem were also found protected by the synthetase. In mammalian tRNAVal, complexed with the congnate but heterologous yeasst valyl‐tRNA synthetase, the protected phosphates lie in the anticodon stem, in the extra‐loop andd in the T ψ arm.

The location of the protected residues in the structure of three tRNAs suggests some common features in the binding of tRNAs to aminoacyl‐tRNA synthetases. These results will be discussed in the light of infsormations of interaction sites obtained by nucleases digestion and ultravilet cross‐linking methods.
An Aspartic Proteinase from Fusarium moniliforme
Authors: KOLACZKOWSKA Maria K. , WIEVCZOREK maciej , POLANOWSKI Antoni
Published: March 2005

An acd proteinase from the culture filtrate of Fusarium moniliforme was purified by acetone precipitation, gel filtration and ion‐exchange chromatography. The final enzyme preparation wa homogeneous on polyacrylamide gel electrophoresis. The apparent molecular weight was estimated to be 38000, based on sodium dodecylk sulfate gel electrophoresis and an isoelectric point of pH 4.90. The optimum pH was found to be 3.2 when measured with denatured hemoglobin as substrate. The enzyme showed maximum stability in the pH range 3.0–5.5. Pepstatin, 1,20epoxy‐3‐(p‐nitrophenoxy)propane and diazoacetyl‐dl‐norlecine methyl exster strongly inhibited the proteinase activity, whereas thiol reagents, serine‐type proteinase inhibitors and metal‐chelating reagent had no signigficant effect. The data indicate that the proteinase from F. moniliforme is an aspartic proteinase which possesses milkclotting activity.
Succinate Dhydrogenase Mutants of Bacillus Suybtilis Lacking Covalently Bound Flavin in the Flavoprotien Subunit
Authors: HEDERSTEDT Lars
Published: March 2005

Succinate dehydrogenase consist of two unequal subunits; Fp and Ip. An FAD group is covalently linked to a histidyl residue in the Fp subunit. The mechanisms by which flavin is attached to protein is not know. Covalently bound flavin was studied in wild‐type and succinate‐dehydrogenase‐negatige Bacillus subtilis. The Fp subunit of succinate dehydrogenase was fount to be the only (major) flainlated protein in the cell. Mutanst lacking convaletnly bound flavin and still containing the Fp polypeptide are descrbed. It is shown that flavin is not essential for assembly and membrane binding of succinate dehydrogenase in B. subtilis.
Chemical Modification of the Mitochodrial bc1 by N,N′ ‐Dicyclohexylcarbodiimide Inhibits Proton Translocation
Authors: PRICE Brendan D. , BRAND Martin D.
Published: March 2005

We report here that N,N/i′‐dicyclohexylcarbodiimide (DCCD) decreases the H2e stoichiometry of the mytochrome bc1 complex from 308 ± 0.2 (10) to 201 ± 0.1 (8) but has only a minimal effect on the H2e ratio of cytochrome oxidase under the relatively mild condition used. The veffect on the bc1 complex connot be explained by uncoupling, by inhibition of electron transport or by selective mitochondrial damage.

We conclude that DCCD is an inhibitor of proton translocation within ther bc1 complex. There are three possible explanation of this effect: (a) DCCD could alter the pathway of electron flow, (b) DCCD could prevent one of the proton translocation reations but not electrton transport, (c) DCCD could prevent the couduction of the translocated proton to the external phase.
Hydrolysis of Mixed Monomolecular Filme of phosphatidulchoiline/Triacylglycerol by Pancereatic Phospholipase A2
Authors: PIERONI Gérard , VERGER Robret
Published: March 2005

We studied the effect of glycerides on pancreatic phospholopase A2 hydrolysis of mixed monomoecular films of trioctanoylglyderol/1, 2‐didodecanoyl‐sn‐ glycero‐3‐phosphocholine with the technique of Piéroni and Verger [(1979) J. Biol. Chem. 254, 10090–10094]. The quantity of enzyme adsorbed to the interface was concomitantly determined with [3H] amidinated phospholipase. At phospholipid packing above the critical penetration pressure, triacylglycerol stimulates phosphatidylcholone hydrolysis to a great extent. On th eother hans, the activity of pancreatic phospholipase A2 on a mixed film is inhibited by the action o fpandreatic lipase. Interface binding of phospholipase A2 to the lipid sustrate does not imply activity.
A New Myofibrillar Protein Characteristic of Type I Human Skeletal Muscle Fibres
Authors: HEIZMANN Claus W. , CELIO Marco R. , BILLETER Rudolf , REIST Ursula , MORET Maria‐Luisa , ARNOLD Eva M.
Published: March 2005

Analysis of a single human type I (slow‐twitch) skeletal muscle fibres by two‐dimensional gel electrophoresis shows that they contain a specific protein of subunit M, 34 × 103 and PI 6.0,6.1 and 6.2 (three spots) which is absent from type IIA and IIB muscle fibres. This type I protein is located in the I‐band of isolated myofibrils as demonstrated by the indirect immunofluorescence using specific antisera against the denatured form of type I protein. Type I protein could only be extracted from myofibrils with high salt solutions (0.6 ‐ 1 M KCl), conditions which also coextract actin and myosin, demonstrating its firm attachment.

Rat and cat type I muscle fibres as well as the slow‐tonic fibres from chicken anterior latissimus dorsi also contained a protein homologous to the human type I protein.

The human type I protein was found to be different from the known regulatory and contractile muscle proteins by electrophoretic and immunological criteria, and may therefore be a new myofibrillar protein in type I muscle fibres.
Activity of Human Blood Platelets in Prothrombin and in Factor X Activation Induced by Ionophore A23187
Authors: VAN RIJN Jan , ROSING Jan , VAN DIEIJEN Gerbrand
Published: March 2005

Two reactions of the blood coagulation cascade are highly stimulated by membranes containing negatively charged phospholipids. These are the activation of prothrombin to thrombin by factor Xa with factor Va as cofactor, and the conversion of factor X into Xa by factor IXa with factor VIIIa as cofactor. It is thought that under physiological conditions these reactions take place with the clotting factors adsorbed at the surface of blood platelets.

The role of blood platelets in the two reactions was studied using two assay systems consisting of highly purified clotting factors. The rates of formation of thrombin or factor Xa, measured with specific chromogenic substrates, were dependent on the quantity of negatively charged phospholipid present in the reaction mixture. Phospholipid vesicles with platelet phospholipid composition were highly active in the two reaction systems. When the purified lipids were substituted by disrupted platelets, containing the same quantity of lipids, the same high activities were found. With intact human blood platelets a low but significant activity was observed. Incubation of the platelets with the calcium ionophore A23187, resulted in a 50–60‐fold activity increase in formation of thrombin and factor Xa. Platelet disruption increases procoagulant activity, since the negatively charged phospholipids, originally situated in the inner leaflet of the lipid bilayer of the platelet membrane become exposed upon platelet disruption. Ionophore activation of platelets was however not due to lysis since discharge of platelet lactate dehydrogenase was less then 3%. The activity of A23187‐activated platelets in formation of thrombin and factor Xa was completely abolished after incubation with phospholipase A2, which is to be expected if negatively charged lipids are present at the platelet outer surface. In contrast to platelets, erythrocytes could not be activated by the ionophore.

As found by other workers, A23187 induces release of ATP and factor V(a) and results in platelet aggregation. The appearance of procoagulant activity, ATP and factor V(a) release were all dependent on the A23187 concentration and were too rapid to study the time course. All platelet activities were induced between 0.2 μM and 0.7 μM A23187, suggesting a relationship between the release processes and the appearance of procoagulant activity.
Calcium‐Independent Activation of Adenylate Cyclase by Calmodulin
Authors: KILHOFFER Marie‐Claude , COOK G. HOPE , WOLFF J.
Published: March 2005

Adenylate cyclase of Bordetella pertussis is stimulated by calmodulin by two distinct interactions. At low activator concentrations (∼ 1 nM) the process is Ca2+‐dependent (i.e. inhibited by EGTA added before calmodulin). High activator concentrations (∼ 0.1–10 μM) stimulate adenylate cyclase also in the presence of EGTA, an effect not accounted for by residual Ca2+ or low concentrations of Ca · calmodulin, which thus appears to be due to calcium‐free calmodulin. Some calmodulin dose‐response curves show both phases of stimulation, separated by a plateau of activity, and half‐maximal activating concentrations differ by 100–300‐fold. Both effects are on the V and not the Km for ATP and are not mimicked by 105‐fold greater concentrations of parvalbumin or by various polyanions.

In addition, adenylate cyclase stimulation at high calmodulin concentrations is greater in the presence of EGTA than in its absence. This enhancement is also produced by 1,10‐phenanthroline and 8‐hydroxyquinoline but not by non‐chelating isomers. These compounds are poor Ca2+ chelators, stimulate at any calmodulin concentration (unlike EGTA), and suggest regulation of this adenylate cyclase by a second metal ion.
Nuclear Matrix: A Cell‐Cycle‐Dependent Site of Increased Intranuclear Protein Phosphorylation
Authors: HENRY Sally M. , HODGE Lon D.
Published: March 2005

Evidence is presented that the nuclear matrix is a cell‐cycle‐dependent site of increased intranuclear protein phosphorylation. The incorporation of radioactive phosphate (32P) is highest during the premitotic (G2) phase and 40–50% less in the postmitotic phase (G1). This is observed for both total matrix protein and for several individual polypeptides ranging in molecular mass from > 200 kDa to 19 kDa. The phenomenon can be demonstrated when the matrix is isolated from orthophosphate‐labeled intact cells, as well as when the matrix is isolated and then incubated in vitro in a protein kinase reaction mixture. The ability of the isolated matrix to mimic the events in vivo indicates the presence of endogenous protein phosphokinase activity and physiological substrates in this isolated nuclear fraction. Further evidence for such mimicry was obtained when amino acid phosphorylation sites were determined. Phosphoserine is the most abundant phosphoamino acid in the matrix labelled both in vitro and in vivo, although phosphothreonine and phosphotyrosine are also present. On the basis of several pieces of data, the endogenous matrix activity appears to be due to multiple protein phosphokinases. Since the maximum phosphorylation coincides with premitosis, the phosphoproteins may play a role in mitotic events. These observations extend and expand the application of this fraction to the study of nuclear structure/function relationships, particularly at the time of mitosis.
Further Characterization of the Three Polypeptide Chains of Bovine and Human Short‐Chain Collagen (Intima Collagen)
Authors: JANDER Renate , RAUTERBERG Jürgen , GLANVILLE Robert W.
Published: March 2005

Short‐chain collagen was isolated from bovine and from human placenta after limited pepsin digestion. By reductive cleavage of disulfide bonds under non‐denaturing conditions, mainly non‐collagenous domains were cleaved off yielding a uniform component composed of three polypeptide chains with molecular masses between 37 kDa and 48 kDa in a nearly equimolar ratio. The chains (SC1*, SC2* and SC3*) were isolated and characterized. By comparison of peptide patterns obtained after various cleavage procedures, they could be identified as more or less shortened forms of SC1, SC2 and SC3, the isolation of which from bovine short‐chain collagen has been described [Jander et al. (1981) Eur. J. Biochem. 114, 17–25].

The peptide patterns; as well as N‐terminal sequence determination, give evidence for the genetic individuality of the three chains; the data so far available suggest that together they form one triple‐helical structure which represents the collagenous domain of the basic molecular unit of short‐chain collagen.