bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential phosphorylation of the light‐harvesting chlorophyll—protein complex in appressed and non‐appressed regions of the thylakoid membrane
Authors: Andersson Bertil , Åkerlund Hans-Erik , Jergil Bengt , Larsson Christer
Published: July 2002

It is now established that the light‐harvesting chlorophyll—protein complex (LHCP) of chloroplasts becomes phosphorylated in the light. In this study subfractionation of phosphorylated intact chloroplasts has been carried out to compare the phosphorylation of LHCP in non‐appressed and appressed thylakoid regions. The results show around 10‐times higher relative phosphorylation in the non‐appressed regions than in the appressed ones. Since the non‐appressed thylakoids also contain almost all photosystem 1, this region is likely to be the site for energy transfer from LHCP to photosystem 1 under phosphorylated conditions.
The interaction between pancreatic lipase and colipase: a protein‐protein interaction regulated by a lipid
Authors: Erlanson-Albertsson Charlotte
Published: July 2002

Pancreatic lipase readily adsorbs to a triglyceride droplet. In the intestine the triglyceride droplets are covered with bile salt and phospholipids which will prevent the adsorption of lipase. In this situation the activity of lipase is restored by colipase, another pancreatic protein. Lipase and colipase in solution form a 1:1 molar complex. I emphasize the fact that the binding and conformation of the two proteins in the complex is dependent on the type of lipids present and suggest that this lipid‐determined structure of the complex is responsible for the actual function of lipase/colipase. It determines whether colipase assists lipase in binding to the bile salt‐covered triglyceride droplet as is the case with tributyrin as substrate, and whether colipase in addition activates lipase as is the case with a mixed trioctanoin/lecithin monolayer substrate. In other words, lipase activity is regulated by the combined action of colipase and the lipid substrate.
NMR solution conformation of gramicidin A double helix
Authors: Arseniev Alexander S. , Bystrov Vladimir F. , Ivanov Vadim T. , Ovchinnikov Yuri A.
Published: July 2002

The conformation of species 3 of Val‐gramicidin A in dioxane has been determined by two‐dimensional NMR spectroscopy. It is presented by the left handed ⇅ππ5.6
LD double helix, a suitable model of an ion permeable pore across the membrane matrix.
Role of intestinal brush border membrane aminopeptidase N in dipeptide transport
Authors: Antonov V.K. , Vorotyntseva T.I. , Bessmertnaya L.Ya. , Mikhailova A.G. , Zilberman M.I.
Published: July 2002

The kinetics of uptake of radioactive label from [U‐14C]Gly, L‐[4,5‐3H]Leu and the dipeptide [14C]Gly—L‐[4,5‐3H]Leu by the brush border membrane vesicles of porcine small intestine have been studied. The effect of aminopeptidase N inhibitors and leucine‐binding protein on accumulation rates has also been tested. Comparison of the kinetic parameters for uptake and hydrolysis of Gly—L‐Leu makes it possible to conclude that the dipeptide transfer includes two conjugated steps, viz., hydrolysis catalysed by aminopeptidase N and transport of the resultant free amino acids by a specific carrier.
Interproton distance measurements in solution for a double‐stranded DNA undecamer comprising a portion of the specific target site for the cyclic AMP receptor protein in the gal operon
Authors: Clore G.Marius , Gronenborn Angela M.
Published: July 2002

Using the proton‐proton nuclear Overhauser effect, a large number (150) of interproton distances, both intra‐ and internucleotide, are determined in solution for a non‐self‐complementary DNA undecamer comprising a portion of the specific target site for the cAMP receptor protein in the gal operon. It is shown that these distances are very similar to those expected for classical B DNA (RMS difference of 0.5 Å) but are significantly different from those expected for classical A DNA (RMS difference 1.1Å). Glycosidic (χ) and C4'‐C3' (δ) bond torsion angles are obtained by model building on the basis of the intranucleotide interproton distances. Whereas the sugar pucker exhibits little base‐to‐base variation with δ lying in the range 120 ± 10°, the glycosidic bond torsion angles of the pyrimidine and purine residues are significantly different, with χpyr = −120 ± 10° and χpur = − 90 ± 10°.
Histamine release by pharmacological agents in the absence of external free Ca2+
Authors: Pintado E. , Carrasco M. , Goberna R.
Published: July 2002

The involvement of extracellular free Ca2+ in histamine release was investigated in rat peritoneal mast cells. Incubation of non‐antigenized cells in a media with high extracellular potassium did not increase histamine release. Secretion induced by A23187 and compound in the presence of Ca2+ requires metabolic energy. In the absence of external free Ca2+ (2.5 μM) histamine release induced by A23187 is reduced but not abolished. Secretion induced by compound is independent of extracellular Ca2+. These results lead us to suggest that mast cell plasma membranes probably lack voltage‐gated Ca2+ channels and that external Ca2+ may not be an absolute requisite for histamine secretion.
Effect of hydrocortisone on the desialylation of intestinal brush‐border enzymes of the rat during postnatal development
Authors: Kraml Jiří , Kolínská Jiřina , Kadlecová Libuše , Zákostelecká Marie , Lojda Zdeněk
Published: July 2002

Hydrocortisone acetate or hemisuccinate (75 body mass) applied to rats i.m. and/or s.c. on the 9th and 10th postnatal days causes a precocious decrease of sialic acid content of the small intestinal brush‐border membrane. On the 15th postnatal day the bound sialic acid of the whole membrane fraction drops to almost half of the values of control animals and to one third of the control values for the papainsolubilized membrane proteins. The hydrocortisone effect is manifested on isoelectric focusing zymograms by a faster increase of pI of the solubilized brush‐border enzymes on the 12th and 15th postnatal days.
Meproadifen enhances activation and desensitization of the acetylcholine receptor‐ionic channel complex (AChR): single channel studies
Authors: Aracava Y. , Albuquerque E.X.
Published: July 2002

Meproadifen
Nicotinic acetylcholine receptor
Single channel current
Endplate region
Activation‐desensitization
Heme a induces assembly of rat liver cytochrome c oxidase subunits I‐III in isolated mitochondria
Authors: Wielburski Antek , Nelson B.Dean
Published: July 2002

Cytochrome oxidase assembly
Heme a requirement
(Rat liver mitochondria)
A calorimetric study of Ca2+ binding to two major isotypes of bullfrog parvalbumin
Authors: Tanokura Masaru , Yamada Kazuhiro
Published: July 2002

Microcalorimetric titrations of the two major isotypes of parvalbumin (PA1 and PA2) from bullfrog skeletal muscle with Ca2+ in the presence and absence of Mg2+ have been carried out at 25°C and pH 7.0. The observed enthalpy titration curves were analyzed by the least‐squares method. The measured enthalpy changes (ΔH) of Ca2+ binding are −33.2 (PA1) and −16.3 site (PA2), and the entropy changes (ΔS) are 28 (PA1) and 76 per K (PA2) in the absence of Mg2+. When 5 mM Mg2+ is present, the enthalpy change of PA2 (−26.7 ) is about twice as large as that in the absence of Mg2+ whereas that of PA1 (−34.6 ) is about the same. The entropy changes are 8 (PA1) and 29 per K (PA2). Both enthalpy and entropy changes are favorable for the Ca2+‐binding reactions of PA1 and PA2 irrespective of the presence of Mg2+.
Serum and thyroid hormones T3 and T4 regulate nerve growth factor mRNA levels in mouse L cells
Authors: Wion Didier , Barrand Plana , Dicou Eleni , Scott James , Brachet Philippe
Published: July 2002

Mouse L cells synthesize and secrete a neurotrophic factor related to the β subunit of the submaxillary gland nerve growth factor (NGF) of male mice. Use of a cDNA probe which encodes the β‐NGF mRNA demonstrated that L cells produce a transcript identical in size to that of the submaxillary gland. Moreover, target sites of restriction enzymes EcoRI, PstI and BamHI were not significantly rearranged in the β‐NGF gene locus of these cells. The abundance of the β‐NGF transcript was found to depend on culture conditions. Removal of serum depressed the cellular content of polyadenylated RNA by a factor of 1.7, and decreased specifically the pool of β‐NGF transcript by an additional factor of 4. The presence of 10−7 M testosterone in the serum‐free medium did not modify the level of β‐NGF mRNA, while addition of 10−7 M T3 (or T4) increased this level by a factor of 1.5. These data provide the first evidence that the β‐NGF mRNA of L cells is subjected to regulation, but in a way apparently different from that described for the submaxillary gland.
Sequence specificity of curved DNA
Authors: Diekmann Stephan
Published: July 2002

Anomalously slow migration of DNA fragments on polyacrylamide gels is interpreted as resulting from curvature of the DNA fragment. Different models have been suggested to explain DNA curvature. In this work a number of DNA fragments were synthesized, cloned, and electrophoretically characterized to distinguish between these models. Strong anomaly of migration is found for sequence stretches (dA)
n
repeated in phase with the helix turn with n at least 4. For n smaller than 4 only negligible anomaly is observed. The results contradict the purine‐clash hypothesis. The data can be explained by assuming longer stretches of As to be in a B'‐form, and that tilt of this structure might be the reason for its curvature.
Effect of 2,4‐dinitrofluorobenzene on the enzymatic properties of the b‐c1 complex isolated from beef heart mitochondria
Authors: Lorusso M. , Marzo M. , Gatti D. , Papa S.
Published: July 2002

A study is presented on the effect of 2,4‐dinitrofluorobenzene (DFNB) on the enzymatic properties of mitochondrial b‐c
1 complex. The chemical modification by DNFB strongly inhibits the reductase activity of the complex, this being accompanied by labelling by [3H]DNFB of core protein I, the apoprotein of b cytochromes and the 12 kDa subunit. Chemical modification by DNFB appears to alter, in particular, the domain of heme b‐562.
Inactivation of potassium‐evoked adrenomedullary catecholamine release in the presence of calcium, strontium or BAY‐K‐8644
Authors: Sala F. , Fonteriz R.I. , Borges R. , García A.G.
Published: July 2002

The rate of catecholamine release from cat adrenal glands perfused with Krebs solution containing 59 mM K declined exponentially during the first few minutes of depolarization. The rate of decline was considerably slower when Ca was substituted by Sr. The late addition of Ca, Sr or the Ca‐channel activator BAY‐K‐8644 evoked a revival of secretion when catecholamine release was inactivated by prior K depolarization; the revival of secretion was independent of the depolarization time. These data demonstrate that inactivation of catecholamine release is specifically dependent on Ca; the modulatory role of Ca on secretion seems to be exerted at a step distal to the transmembraneous Ca channel.
Autophosphorylation of calmodulin kinase II: functional aspects
Authors: Bronstein J.M. , Farber D.B. , Wasterlain C.G.
Published: July 2002

Autophosphorylation of purified calmodulin kinase II dramatically inhibited protein kinase activity and enhanced substrate selectivity. Inhibition was observed over a wide range of calmodulin concentrations but calmodulin binding was unaffected. Autophosphorylation of calmodulin kinase II may be a mechanism for limiting phosphorylation to physiological substrates and terminating some of calcium's actions in synaptic events.
Primary structure of a major sialyl‐saccharide alditol from human amniotic mucins expressing the tumor‐associated sialyl‐X antigenic determinant
Authors: Hanisch Franz-Georg , Egge Heinz , Peter-Katalinic Jasna , Uhlenbruck Gerhard
Published: July 2002

A major sialyl‐saccharide alditol isolated from mucins of human amniotic fluid was purified by high‐performance liquid chromatography followed by high‐performance thin‐layer chromatography of the methylated derivative and subjected to structural analyses which comprised fast atom bombardment and electron impact‐mass spectrometry, methylation analysis, exoglycosidase digestion and CrO3 oxidation. The structure of a disialylated pentasaccharide characterized by a sialyl‐X antigenic determinant was established:
Insulin stimulation of glucose uptake and the transmembrane potential of muscle cells in culture
Authors: Klip Amira , Ramlal Toolsie , Walker Denise
Published: July 2002

The membrane potential of L6 muscle cells was measured with the fluorescent dye bis‐oxonol. Hyperpolarizations of up to 15 mV were caused by gramicidin (in N‐methyl‐D‐glucamine+ medium), or by monensin or ionomycin. Depolarization was achieved with gramicidin (in Na+ medium), or with K+. Insulin did not change the resting membrane potential of − 70 mV, yet it effectively stimulated 2‐deoxy‐D‐glucose uptake. Conditions that hyperpolarize the cells did not alter the basal rate of hexose uptake. Moreover, insulin was still capable of stimulating hexose uptake in depolarized cells. It is concluded that modulation of the membrane potential is probably not a signalling event in insulin stimulaton of hexose uptake.
A high resolution 1H NMR study of the solution structure of human epidermal growth factor
Authors: Carver J.A. , Cooke R.M. , Esposito G. , Campbell I.D. , Gregory H. , Sheard B.
Published: July 2002

500 MHz 1H NMR studies of human epidermal growth factor are described. The backbone resonances of the 1–48 derivative of hEGF have been assigned using two‐dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects and the NH‐αCH spin‐spin coupling constants allowed prediction of the secondary structure. Aspects of the tertiary structure are also identified. A pair of antiparallel β‐sheets involving residues 18–23 and 28–34 is a dominant feature of the solution structure.
Folding kinetics of mammalian ribonucleases
Authors: Lang Kurt , Wrba Alex , Krebs Herbert , Schmid Franz X. , Beintema Jaap J.
Published: July 2002

The folding kinetics of seven different pancreatic ribonucleases are compared both under native conditions and within the unfolding transition. In general, the folding kinetics of these proteins are similar despite numerous amino acid substitutions. Ribonucleases with 4–6 proline residues show 80% slow‐folding species. For three ribonucleases with 7 prolines this number increases to 90%. Porcine ribonuclease with a unique Pro 114‐Pro 115 sequence folds significantly slower than other ribonucleases which do not show this sequence.
Purification and characterization of a β‐toxin from the venom of the African scorpion Leiurus quinquestriatus
Authors: Rack Michael , Richter Dieter , Rubly Norbert
Published: July 2002

The venom of the African scorpion Leiurus quinquestriatus was subjected to high‐performance ion‐exchange chromatography. Among a large number ( > 25) of Small proteins and other substances, a protein component of approx. 6500 Da was purified. The effect of this toxin was tested on single myelinated nerve fibres of the frog Rana esculenta. Toxin concentrations less than 10 nM produced clear effects. Activation rather than inactivation of the voltage‐dependent sodium channel was strongly affected. Thus, this toxin from an African scorpion acts like the β‐toxins present in the venom of North American scorpions.
Modification of the GABA/benzodiazepine receptor with the arginine reagent, 2,3‐butanedione
Authors: Widdows Kate B. , Kirkness Ewen F. , Turner Anthony J.
Published: July 2002

Treatment of either crude or purified preparations of the γ‐aminobutyrate (GABA)/benzodiazepine receptor complex with arginine‐specific reagents resulted in a time‐ and concentration‐dependent loss of [3H]muscimol binding activity. Following exposure to either 2,3‐butanedione or phenylglyoxal (⩽ 20 mM), [3H]muscimol binding was inhibited by up to 80%. [3H]Flunitrazepam binding was much less sensitive to the effects of the reagents. Scatchard analysis of the binding data indicated that treatment with butanedione resulted in a loss of [3H]muscimol binding sites with little effect on binding affinity. Considerable protection against inactivation was provided by arginine and by the endogenous receptor ligand, GABA. These results indicate that arginine residues play a critical role in maintaining the GABA receptor in a conformation capable of ligand binding, possibly by participating in the binding site through interaction with the carboxylate moiety of GABA.
Fractionation, biochemical characterization and lysosomal phospholipases of human liver
Authors: Löffler Bernd-Michael , Kunze Hans
Published: July 2002

Human liver was homogenised and fractionated by differential centrifugation, and the subcellular fractions were characterised biochemically. Absolute values and distribution patterns of protein and marker enzyme activities obtained from human liver have also been compared with those from rat liver. In addition, acid phospholipase activities have been studied in human liver. On the basis of product formation from stereo‐specifically radiolabeled phosphatidylethanolamine substrates, lysosomal phospholipases A1 and A2 with optimal activities at pH 4.7 have been identified in human liver. Acid phospholipase C and lysophospholipase activities, however, were not found in human liver. Cationic amphiphilic drugs inhibited the activities of the acid phospholipases A in human and rat liver lysosomes to about the same extent.
Raman spectroscopic study on the conformation of 11 S form acetylcholinesterase from Torpedo californica
Authors: Aslanian Dimitrina , Gróf Pál , Négrerie Michel , Balkanski Minko , Taylor Palmer
Published: July 2002

Vibrational Raman spectroscopy has been used to study the conformation of the 11 S form of acetylcholine‐sterase from Torpedo californica. Secondary structure analysis by the method of Williams [(1983) J. Mol. Biol. 166, 581–603] shows 49% α‐helical structure, 23% β‐sheets, 11% turns and 15% undefined structure. Secondary structure estimates obtained for this enzyme by Raman spectroscopy and circular dichroism have been analyzed.
19F NMR study of 5‐fluorotryptophan‐labeled bacteriorhodopsin
Authors: Arseniev A.S. , Kuryatov A.B. , Tsetlin V.I. , Bystrov V.F. , Ivanov V.T. , Ovchinnikov Yu.A.
Published: July 2002

19F NMR and CD spectra reveal that bacteriorhodopsin as well as its 5‐fluorotryptophan‐labeled analog solubilized in a CH3OH‐CHCl3 mixture (i) retains a secondary structure of the fully active chromoprotein in the purple membrane and (ii) possesses a folded structure in which modifications at the Lys‐216 bound retinal are sensed by sequentially remote tryptophan residues. Individual fragments isolated after limited proteolysis and NaBH4‐cleavage of bacteriorhodopsin keep the spatial structure of the intact polypeptide chain in the organic solvent.
Phosphorylation of smooth muscle caldesmon by three protein kinases: Implication for domain mapping
Authors: Vorotnikov Aleksander V. , Shirinsky Vladimir P. , Gusev Nikolai B.
Published: July 2002

Phosphorylation of duck gizzard caldesmon by Ca2+/phospholipid‐dependent protein kinase, Ca2+/calmodulin‐dependent protein kinase and casein kinase II has been investigated. The Ca2+/phospholipid‐dependent protein kinase incorporates more than 3 mol phosphate per mol (140 kDa) caldesmon. All phosphorylation sites are localized in the actin‐ and calmodulin‐binding peptide (40–45 kDa) supposed to be a part of the C‐terminal domain of caldesmon. Casein kinase II phosphorylates only one site located in a short (25–27 kDa) peptide, presumably in the caldesmon N‐terminal domain. The Ca2+/calmodulin‐dependent protein kinase phosphorylates two sites located in the N‐ and C‐terminal domains of caldesmon.
Unusual biochemistry of changes in neuron membrane permeability evoked by cAMP
Authors: Liberman Efim A. , Minina Svetlana V. , Myakotina Olga L. , Mamikonova Tatyana A. , Tsofina Lilia M. , Shklovskl-Kordi Nikita E.
Published: July 2002

Influence of different metabolic poisons on cAMP‐evoked neuron membrane permeability is investigated. Drugs preventing cAMP binding with R subunits of protein kinase decrease the cAMP‐evoked current, but the inhibitor of the C subunit, H8, has no effect. The cAMP‐dependent current is increased by uncouplers and decreased by inhibitors of glycolysis and oxidative phosphorylation. The mechanism of cAMP action on neuron permeability is discussed.
Purification and characterization of two GTP‐binding proteins of 22 kDa from human platelet membranes
Authors: Nagata Koh-ichi , Nozawa Yoshinori
Published: July 2002

Two GTP‐binding proteins (G‐proteins) of 22 kDa were purified to near homogeneity from a sodium cholate extract of human platelet membranes by successive chromatographies on DEAE‐Sephacel, Ultrogel AcA‐44, phenyl‐Sepharose CL‐4B, Mono Q HR5/5 and hydroxyapatite columns. They bound maximally 0.89 mol of [35S]guanosine 5′‐(3‐O‐thio)triphosphate per mol of both purified proteins, and this binding was inhibited by GTP and GDP but not by ATP and AppNHp. Their molecular masses were somewhat lower than that of ras p21 and they were not recognized by an anti‐v‐Ki‐ras p21 antibody. These results indicate that human platelet membranes contain at least two low‐molecular‐mass G‐proteins distinct from ras p21, in addition to the heterotrimeric G‐proteins, the α‐subunits of which possess molecular mass values of about 40 kDa.
Amino‐terminal sequence of a Saccharomyces cerevisiae nuclear protein, NHP6, shows significant identity to bovine HMG1
Authors: Kolodrubetz David , Haggren Wendy , Burgum Alex
Published: July 2002

Several nonhistone chromatin proteins (NHPs) have been isolated from Saccharomyces cerevisiae nuclei. They have molecular masses and amino acid compositions typical of the high mobility group (HMG) proteins from higher eukaryotic cells. Polyclonal antisera raised against two of the NHPs have been used in immunoblots of proteins from subcellular fractions of yeast to show that the NHPs are indeed nuclear. In addition, the amino‐terminal amino acid sequences of several of the NHPs were determined. Importantly, the amino‐terminal sequence of one of the proteins, NHP6, has significant (60%) identity with a stretch of amino acids in calf thymus HMG1.
Palmitate‐binding, serum albumin‐like proteins in salmonids
Authors: Davidson William S. , Birt Victoria L. , Birt Tim P. , Green John M.
Published: July 2002

There has been considerable controversy over the existence of serum albumin in fish. One of the physiological functions of albumin is to bind free fatty acids. This characteristic was used to screen the plasma of seven species of salmonids. Each species contains a protein fraction that (i) binds palmitate, (ii) has a molecular mass similar to that of human serum albumin, and (iii) is one of the most rapidly migrating proteins when salmonid plasma is subjected to anodal polyacrylamide gel electrophoresis. We conclude therefore, that salmonids have serum albumins that are homologous to the serum albumin of higher vertebrates.
Acute exercise increases the number of plasma membrane glucose transporters in rat skeletal muscle
Authors: Hirshman Michael F. , Wallberg-Henriksson Harriet , Wardzala Lawrence J. , Horton Elizabeth D. , Horton Edward S.
Published: July 2002

To determine whether increased glucose transport following exercise is associated with an increased number of glucose transporters in muscle plasma membranes, the D‐glucose inhabitable cytochalasin B binding technique was used to measure glucose transporters in red gastrocnemius muscle from exercised (1 h treadmill) or sedentary rats. Immediately following exercise there was a 2‐fold increase in cytochalasin B binding sites, measured in purified plasma membranes enriched 30‐fold in 5′‐nucleotidase activity. This increase in glucose transporters in the plasma membrane may explain in part, the increase in glucose transport rate which persists in skeletal muscle following exercise. Where these transporters originate, remains to be elucidated.
Thermodynamic parameters of the binding of the tight‐binding I12X86 lac repressor to operator and non‐operator DNA
Authors: Maurizot Jean Claude , Grebert Philippe
Published: July 2002

The thermodynamic parameters ΔH and ΔS corresponding to the binding of the tight‐binding double mutant lac repressor I12X86 with operator and non‐operator DNA fragments were determined using the nitrocellulose filter binding assay. In both cases the binding processes are entropically driven and accompanied by an unfavorable enthalpy variation. The differences between these parameters and those previously reported for the wild type lac repressor show that the strategy adopted by the mutant to interact with DNA is highly different from that of the wild type repressor and suggest more hydrophobic contacts between the mutant and DNA.
Microinjection of catalytic subunit of cyclic AMP‐dependent protein kinases triggers acute morphological changes in thyroid epithelial cells
Authors: Roger Pierre P. , Rickaert Fabienne , Huez Georges , Authelet Michèle , Hofmann Franz , Dumont Jacques E.
Published: July 2002

In dog thyroid epithelial cells in primary culture, thyrotropin acting through cyclic AMP induced rapid morphological changes associated with complete disruption of actin containing stress fibers. This modification preceded cell retraction and rounding up. These morphological effects were also induced by glass capillary microinjection of purified catalytic subunit of cAMP‐dependent protein kinase. This provides the first direct evidence in intact cells that catalytic subunit, which is released upon activation of cAMP‐dependent protein kinases, is responsible for cAMP‐dependent morphological transformation.
Protein secondary structure and homology by neural networks The α‐helices in rhodopsin
Authors: Bohr Henrik , Bohr Jakob , Brunak Søren , Cotterill Rodney M.J. , Lautrup Benny , Nørskov Leif , Olsen Ole H. , Petersen Steffen B.
Published: July 2002

Neural networks provide a basis for semiempirical studies of pattern matching between the primary and secondary structures of proteins. Networks of the perceptron class have been trained to classify the amino‐acid residues into two categories for each of three types of secondary feature: α‐helix or not, β‐sheet or not, and random coil or not. The explicit prediction for the helices in rhodopsin is compared with both electron microscopy results and those of the Chou‐Fasman method. A new measure of homology between proteins is provided by the network approach, which thereby leads to quantification of the differences between the primary structures of proteins.
Excitation‐contraction coupling in skeletal muscle fibers injected with the InsP3blocker, heparin
Authors: Pape P.C. , Konishi M. , Baylor S.M. , Somlyo A.P.
Published: July 2002

Heparin, an inhibitor of inositol trisphosphate (InsP3)‐induced Ca2+ release in smooth muscle and non‐muscle cells, was injected into intact frog skeletal muscle fibres. Ca2+ release from the sarcoplasmic reticulum was elicited by the normal action potential mechanism and monitored by both fura‐2 fluorescence and an intrinsic birefringence signal. Both optical signals, and hence Ca2+ release, were unaffected by high concentrations of heparin. This result argues against a major physiological role of InsP3 as a chemical messenger of excitation‐contraction coupling in skeletal muscle.
αB‐Crystallin is expressed in kidney epithelial cell lines and not in fibroblasts
Authors: Nagineni Chandrasekharam N. , Bhat Suraj P.
Published: July 2002

We have recently shown the presence of αB‐crystallin in non‐ocular tissues of diverse embryological origins such as the heart, brain, spinal cord, kidney, retina, etc. Using an αB‐crystallin‐specific antiserum and immunofluorescence, immunoblotting, immunoprecipitation and peptide mapping with Staphylococcus aureus protease, we demonstrate differential expression of αB‐crystallin in epithelial and fibroblast cell lines. αB‐Crystallin was detectable only in epithelial cell lines such as MDBK, MDCK, LLCPK1 and JTC‐12, and was not observed in two kidney fibroblast cell lines, one skin fibroblast cell line, and one corneal fibroblast cell line. Differential expression of the αB‐crystallin gene was also confirmed by Northern blot analysis of the RNAs isolated from these cell lines. These data suggest a cell‐type‐specific role for αB.
Ubisemiquinone in the NADH‐ubiquinone reductase region of the mitochondrial respiratory chain
Authors: Burbaev D.Sh. , Moroz I.A. , Kotlyar A.B. , Sled V.D. , Vinogradov A.D.
Published: July 2002

Coupled bovine heart submitochondrial particles exhibit a rotenone‐sensitive g = 2.00 low‐temperature EPR signal attributable to ubisemiquinone which is observed during steady‐state electron transfer from NADH to oxygen or from succinate to NAD+ in Δµ̃H+‐dependent reverse electron transfer. Quantitation of the signal under optimal conditions gives a value for maximal semiquinone of approx. 0.5 spins per spin of the fully reduced NADH dehydrogenase iron‐sulfur center N‐2. The intensity of the signal is drastically reduced when electron transfer from NADH to oxygen is blocked by cyanide or in the case of the reverse electron transfer from succinate to NAD+ being prevented by anaerobiosis. Only those particles which contain ‘turnover‐preconditioned” NADH‐ubiquinone reductase demonstrate the ubisemiquinone signal together with N‐1, N‐2, N‐3 and N‐4 iron‐sulfur centers. The spin relaxation characteristics of the rotenone‐sensitive ubisemiquinone signal point to its interaction with one of the rapidly relaxing (4Fe‐4S) centers, most probably N‐2.
High‐affinity Ca2+‐binding site inhibiting Ca2+ release from sarcoplasmic reticulum
Authors: Argaman Anat , Shoshan-Barmatz Varda
Published: July 2002

Ca2+ release from sarcoplasmic reticulum membranes, activated by alkaline pH occurs only when EGTA is present in the release medium. Addition of very low concentrations of Ca2+ to the medium inhibits Ca2+ release. The concentration of free Ca2+ required for 50% inhibition ranges from between 5 and 20 nM in different experiments and/or membrane preparations, irrespective of whether the free Ca2+ concentration is controlled by EGTA or CDTA. Other divalent cations such as Mn2+, Ba2+, Cu2+, Cd2+ and Mg2+ also exert an inhibitory effect on Ca2+ release, with higher or lower potency than that of Ca2+. The inactivation of Ca2+ release by Ca2+ is reversible. We suggest the involvement of high‐affinity Ca2+‐binding sites in the control of Ca2+ release.
The minor antenna complexes of an oxygen evolving photosystem II preparation: purification and stoichiometry
Authors: Barbato Roberto , Rigoni Fernanda , Giardi Maria Teresa , Giacometti Giorgio Mario
Published: July 2002

A photosystem II complex obtained by partial solubilization of spinach PS II membranes has been analyzed in terms of chlorophyll‐protein complexes. The complex was found to contain the minor antennas CP29 and CP26 and a very small amount of LHC II (less than 3%). No trace of CP24 was found to be associated with the PS II complex. This chlorophyll a/b‐protein was instead found in the fraction containing the main light‐harvesting complex LHC II. The stoichiometry of the minor antennas indicates the values 3:3:1 for the ratios CP29:CP26:P680.
TRH‐extended peptides in the olfactory lobe are formed by incomplete cleavage at pairs of arginine residues in the TRH prohormone
Authors: Cockle Sheena M.
Published: July 2002

High concentrations of thyrotrophin‐releasing hormone (TRH) are known to be present in the olfactory lobe, and the processing of the TRH prohormone in this region of the brain has been examined in this study. TRH‐extended peptides have been detected in the rat olfactory lobe: these peptides accounted for approximately 11% of the total TRH immunoreactivity present in the tissue and contained the sequence pGlu‐His‐Pro‐Gly‐Arg exclusively at their N‐termini. Extended peptides containing pGlu‐His‐Pro‐Gly‐Lys at their N‐termini were not detected suggesting that incomplete cleavage occurs only at Arg‐Arg residues in the TRH‐prohonnone. In view of the highly specific processing of the prohormone, it is likely that the TRH‐extended peptides play important physiological roles.
RGD peptides may only temporarily inhibit cell adhesion to fibronectin
Authors: Ylänne Jari
Published: July 2002

When human fibroblasts were cultured on fibronectin for 4 h in the presence of 0.5 of the GRGDSP peptide derived from the fibronectin cell‐binding site, they adhered and spread normally and organized talin and integrin α5 and β1 subunits into focal adhesions. When the adherent cells were quantitated as a function of time, submaximal peptide concentrations were found to delay cell adhesion on fibronectin, but they had no effect on the maximum. When the cells were plated on vitronectin, however, even relatively low peptide concentrations lowered the maximal amount of cells adhering and abolished cell spreading. The results suggest a different mechanism for cell adhesion on fibronectin and vitronectin.
Isolation and characterization of human reg protein produced in Saccharomyces cerevisiae
Authors: Itoh Takako , Tsuzuki Hiroshige , Katoh Takaaki , Teraoka Hiroshi , Matsumoto Kohichi , Yoshida Nobuo , Terazono Kimio , Watanabe Takuo , Yonekura Hideto , Yamamoto Hiroshi , Okamoto Hiroshi
Published: July 2002

reg was originally identified a a gene expressed during the regeneration of insulin‐producing pancreatic β‐cells of the rat. We built an expression vector containing human reg cDNA to drive Saccharomyces cerevisiae to synthesize the reg protein, and purified it from the culture medium. The 144‐amino acid sequence of the recombinant protein was consistent with that deduced from the cDNA and genomic DNA sequence except that the signal sequence of 22 amino acids was eliminated, and the amino‐terminal residue of the protein was pyroglutamic acid. The secondary structure of the reg protein was predicted by determination of the intramolecular cystine linkage and of α‐helix and β‐sheet contents.
Expression of spinach plastocyanin in E. coli
Authors: Nordling Margareta , Olausson Torbjörn , Lundberg Lennart G.
Published: July 2002

An expression vector designed for overexpression of plastocyanin in the periplasmic space of E. coli has been developed. The vector contains the signal peptide sequence of Pseudomonas aeruginosa azurin and the mature sequence of spinach plastocyanin. The precursor is efficiently translocated to the periplasmic space and correctly processed to mature plastocyanin. No detectable amount of plastocyanin was present in the cytoplasmic or in the membrane fraction. A large scale preparation of the recombinant plastocyanin in a 20 litre fennentor yielded approximately 30 mg of pure plastocyanin. The recombinant protein obtained from E. coli shows CD, EPR and optical properties identical to plastocyanin isolated from spinach.
Studies on the structural requirement for ligand binding to the neuropeptide Y (NPY) receptor from rat cerebral cortex
Authors: Baeza Claudio Rivera , Undén Anders
Published: July 2002

Analogues of the NPY model peptide NPY 1‐4‐Aca‐25‐36 were synthesized by solid phase peptide synthesis using the tea‐bag method for parallel peptide synthesis. The affinity of the peptides as ligands to the NPY receptor in rat cerebral cortex was investigated. The model compound NPY 1‐4‐Aca‐25‐36 was a relatively poor ligand to the NPY receptor in rat brain (IC50 = 0.40 μM). Arg35 and Arg33 were both important for ligand recognition by the NPY receptor. Substitutions in several positions in the region corresponding to the C‐terminal part of NPY resulted in analogues with only minor reduction of the affinity for the NPY receptor.
Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3/‐cyclic nucleotide phosphohydrolase revealed by amino acid squence comparison
Authors: Koonin Eugene V. , Gorbalenya Alexander E.
Published: July 2002

Related domains containing the purine NTP‐binding sequence pattern have been revealed in two enzymes involved in tRNA processing, yeast tRNA ligase and phage T4 polynucleotide kinase, and in one of the major proteins of mammalian nerve myelin sheath, 2',3'‐cyclic nucleotide 3'‐phospho‐hydrolase (CNPase). It is suggested that, similarly to the tRNA processing enzymes, CNPase possesses polynucleotide kinase activity, in addition to the phosphohydrolase one. It is speculated that CNPase may be an authentic mammalian polynucleotide kinase recruited as a structural component of the myelin sheath, analogously to the eye lens crystallins. Significant sequence similarity was revealed also between the N‐terminal regions of yeast tRNA ligase and phage T4 RNA ligase. A tentative scheme of the domainal organizations for the three complex enzymes is proposed. According to this model, tRNA ligase contains at least three functional domains, in the order: N‐ligase‐kinase‐phosphohydrolase‐C, whereas poly‐nucleotide kinase and CNPase encompass only the two C‐terminal domains in the same order.
Depurination of plant ribosomes by pokeweed antiviral protein
Authors: Taylor Blair E. , Irvin James D.
Published: July 2002

Mammalian ribosomes have been shown to be enzymatically modified by ribosomal inactivating protein (RIPs) via specific depurination of rRNA. Here we report that ribosomes isolated from wheat germ contain intact and undepurinated rRNA and are depurinated by pokeweed antiviral protein (PAP). Pokeweed ribosomes isolated under the same conditions are depurinated. Total RNA isolated from pokeweed in the presence of strong denaturants was found to pbe partially depurinated. We conclude that wheat germ ribosomes are resistant to the endogenous RIP, tritin, but are sensitive to PAP and that pokeweed ribosomes can be depurinated by the N‐glycosidase activity of endogenous PAP during isolation.
EXAFS of the type‐1 copper site of rusticyanin
Authors: Holt Steven D. , Piggott Brian , Ingledew W.John , Feiters Martinus C. , Diakun Gregory P.
Published: July 2002

Extended X‐ray absorption fine structure (EXAFS) spectra at the Cu K‐edge have been recorded of the oxidized and reduced form at pH 3.5 of rusticyanin, the type‐1 or ‘blue’‐copper protein from Thiobacillus ferrooxidans. The EXAFS of oxidized rusticyanin is well simulated with models assuming a ligand set of 2 N(His) and 1 S(Cys) at 1.99 and 2.16 Å, respectively. Upon reduction, the average Cu‐N ligand distance increases by approx. 0.08Å. For both redox states studied, the fit by the simulation is significantly improved by including a contribution of an additional sulfur ligand at approx. 2.8 Å. From comparison with structural data of other blue‐copper proteins, it is concluded that the copper coordination environment is relatively rigid, which may be a clue to its high redox potential.
Inhibition of cysteine proteinases by a protein inhibitor from potato
Authors: Rowan Andrew D. , Brzin Joze , Buttle David J. , Barrett Alan J.
Published: July 2002

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg‐white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.
Magnetic resonance studies of murine macrophages
Authors: King Nicholas J.C. , Ward Mary H. , Holmes Kerry T.
Published: July 2002

Proton magnetic resonance spectroscopy (1H‐MRS) was used to investigate the membranes of macrophages activated by γ‐interferon in vitro and by Listeria monocytogenes in vivo. We report the appearance with activation, of a high resolution spectrum indistinguishable from that found in activated T and B cells and embryonic and malignant cell types previously studied. We furthermore show that proliferation is not a prerequisite for the appearance of this activated spectrum. This supports the idea that membrane ‘activation’ in all cells, irrespective of origin, may be accompanied by similar architectural changes, and suggests that a common pathway exists for the activation of cell membranes of the immune system, possibly important in the acquisition of increased motility. The use of 1H‐MRS as a non‐invasive tool for analysis of activation is discussed.
The potency of amide protons for assignments of NMR spectra of carbohydrate chains of glycoproteins, recorded in 1H2 O solutions
Authors: Hård Karl , Spronk Bertina A. , Hokke Cornelis H. , Kamerling Johannis P. , Vliegenthart Johannes F.G.
Published: July 2002

Three glycoprotein N‐glycans, namely, a disialylated diantennary carbohydrate chain linked to Asn. a monosialylated, fucosylated diantennary glycopeptide with bisecting N‐acetylglucosamine, and a tetrasialylated, fucosylated tetra‐antennary oligosaccharide. have been investigated by two‐dimensional NOE and HOHAHA spectroscopy in 1H2O as solvent. The amide protons of all N‐acetylglucosamine and sialic acid residues could readily be assigned. The large chemical‐shift dispersion of the amide resonances of the N‐acetylglucosamine residues, allowed the unambiguous assignment of every N‐acetyl methyl signal, via strong NOEs. Subspectra could be obtained of all N‐acetylglucosamine residues in HOHAHA spectra. These results have as main implication that several biologically important large glycans, will not become amenable for conformational studies by multidimensional NMR in 1H2O solution.
Expression of rat liver vitamin D3 25‐hydroxylase cDNA in Saccharomyces cerevisiae
Authors: Akiyoshi-Shibata Megumi , Usui Emiko , Sakaki Toshiyuki , Yabusaki Yoshiyasu , Noshiro Mitsuhidel , Okuda Kyuichiro , Ohkawa Hideo
Published: July 2002

The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D3 25‐hydroxylase, cytochrome P450LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells. The transformed years cells produced a P450LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme. The expression level of the P450LMT25 hemoprotein was about 5 × 104 molecules per cell as determined by reduced CO‐difference spectra. The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25‐hydroxylase activity toward 1α‐hydroxyvitamin D3 and 27‐hydroxylase activity toward 5β‐cholestane‐3α, 7α, 12α‐triol in a reconstituted system containing bovine adrenodoxin and NADPH‐adrenodoxin reductase.
Calcium oscillation associated with reduced protein kinase C activities in ras‐transformed NIH3T3 cells
Authors: Fu Tao , Sugimoto Yoshikazu , Oki Tohru , Murakami Shigeko , Okano Yukio , Nozawa Yoshinori
Published: July 2002

We show here novel intracellular Ca2+ oscillation in ν‐K‐ras‐transformed NIH3T3 cells induced by mitogenic peptide hormones, bradykinin and bombesin, as well as fetal calf serum. Induction of the Ca2+ oscillation is strongly correlated with the malignant properties and inversely with PKC activities in vitro and in vivo. These results suggest that the mitogen‐induced Ca2+ oscillation is negatively regulated by PKC, which modulates Ca2+ influx in ν‐K‐ras‐transformed NIH3T3 cells.
Subsite specificity of the proteinase from myeloblastosis associated virus
Authors: Konvalinka Jan , Blaha Ivo , Skrabana Rostislav , Sedlacek Juraj , Pichova Iva , Kapralek Frantisek , Kostka Vladimir , Strop Petr
Published: July 2002

The subsite requirements of the aspartic proteinase from the mycloblastosis‐associated virus (MAV) for the cleavage of peptide substrates were studied with a series of synthetic peptides of general structure Ala‐Thr‐P4‐P3‐P2‐P1 ★ Nph‐Val‐Arg‐Lys‐Ala. The residues in positions P4, P3, P2 and P1 were varied and the kinetic parameters for the cleavage of substrates in 2.0 M NaCl were spectrophotometrically determined at pH 6.0 and 37°C. The acceptance of amino acid residues in particular subsites is similar to that observed with the human immunodeficiency virus type 1 (HIV‐1) proteinase in our earlier studies on the same substrate series: hydrophobic or aromatic residues are preferable in P1 position, a broad variety of residues are acceptable in P3 whereas the residues occupying P2 plays the decisive role in the substrate cleavage as evidenced by its dramatic influence on both k
cat and K
m values. The most remarkable difference between the two enzymes was found in P3 and P4 subsites. In P3, the introduction of negatively charged glutamate increases the substrate binding by the MAV proteinase 12‐fold and decreases binding by the HIV‐1 proteinase. In P4, Pro in this series is a favourable residue for the MAV proteinase and is strongly inacceptable for HIV‐1 the proteinase. The pH profile of the cleavage was studied with a chromogenic substrate and differences between HIV‐1 and MAV proteinases are discussed.
Protein kinase C subtypes in endothelial cells
Authors: Mattila Pirkko
Published: July 2002

Activation of protein kinase C (PKC) has been linked to the regulation of class II expression on endothelial cells by interferon‐γ (IFN‐γ). PKC subtypes in endothelial cells were analyzed using three different approaches, the immunoperoxidase staining of native and IFN‐γ stimulated cells cultured on chamber slides as well as immuno‐ and Northern blotting. All approaches revealed that of the conventional subtypes. α is the predominant form of PKC in endothelial cells. Even though IFN‐γ is able to induce PKC translocation to particulate fractions, no translocation was detected in histological stainings. Western blot studies as well as mRNA studies revealed that IFN‐γ is unable to increase the total amount of PKC in endothelial cells.
Identification of potential amino acid residues supporting anticodon recognition in yeast methionyl‐tRNA synthetase
Authors: Despons Laurence , Walter Philippe , Senger Bruno , Ebel Jean-Pierre , Fasiolo Franco
Published: July 2002

Sequence comparisons among methionyl‐tRNA synthetases from different organisms reveal only one block of homology beyond the lasts β strand of the mononucleotide fold. We have introduced a series of semi‐conservative amino acid replacements in the conserved motif of yeast methionyl‐tRNA synthetase. The results indicate that replacements of two polar residues (Asn584 and Arg588) affected specifically the aminoacylation reaction. The location of these residues in the tertiary structure of the enzyme is compatible with a direct interaction of the amino acid side‐chains with the tRNA anticodon.
A possible role for cysteine residues in the fidelity of DNA synthesis exhibited by the reverse transcriptases of human immunodeficiency viruses type 1 and type 2
Authors: Bakhanashvili Mary , Hizi Amnon
Published: July 2002

HIV reverse transcriptases (RTs) have few cysteine residues relative to other RTs and retain their DNA polymerization functions following chemical modification by thiol‐specific reagents. The functional role of the cysteines in the fidelity of the DNA‐dependent DNA synthesis of HIV RTs has been addressed by chemical modification of the wild‐type enzymes in combination with the analysis of an enzymatically active mutant HIV‐1 RT in which all cysteines were modified to serines. We have observed an increase in 3′‐terminal mispair extension efficiency exhibited by chemically modified HIV‐1 and HIV‐2 RTs. The possible involvement of cysteine residues was further substantiated using the cysteine‐free mutant HIV‐1 RT that displays an increased efficiency of mispair extension. These results provide evidence for a possible role of cysteine residues in the fidelity of DNA synthesis catalyzed by HIV RTs.