bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemiosmotic coupling in cytochrome oxidase
Authors: Mitchell Peter , Mitchell Roy , John Moody A. , West Ian C. , Baum Harold , Wrigglesworth John M.
Published: July 2002

Using the principle of specific vectorial ligand conduction, we outline directly coupled protonmotive O loop and O cycle mechanisms of cytochrome oxidase action that are analogous to protonmotive Q loop and Q cycle mechanisms of QH2 dehydrogenase action. We discuss these directly coupled mechanisms in the light of available experimental knowledge, and suggest that they may stimulate useful new research initiatives designed to elucidate the osmochemistry of protonmotive oxygen reduction in cytochrome oxidase.
Characterization of monoclonal antibodies against human protein C specific for the calcium ion‐induced conformation or for the activation peptide region
Authors: Laurell Martin , Ikeda Kyoichi , Lindgren Stefan , Stenflo Johan
Published: July 2002

Three monoclonal antibodies have been produced that are specific for the activation peptide region m human protein C. These antibodies inhibited the activation of protein C by thrombin and by the thrombin‐thrombomodulin complex. A fourth monoclonal antibody specifically recognized the Ca2+‐stabilized conformation in protein C. This antibody bound both intact protein C and protein C from which the γ‐carboxy‐glutamic acid‐containing region had been removed by limited proteolysis. These results indicate that this antibody recognizes the conformation in protein C stabilized by Ca2+ bound to the single binding site that is independent of γ‐carboxyglutamic acid.
On the 6‐phosphofructo‐1‐kinase phosphatase activity of protein phosphatase 2C and its dimeric nature
Authors: Mieskes Gottfried , Söling Hans-Dieter
Published: July 2002

A recently described 6‐phosphofructo‐1‐kinase phosphatase (PFK‐phospatase [1]) shared several properties with protein phosphatase 2C [2,3], but exhibited differences with respect to molecular mass and substrate specificity. Chromatography on histone‐Sepharose, gel filtration experiments on Sephacryl S‐200 and Sephadex G‐100 as well as sucrose density gradient centrifugation [4] show that both enzyme preparations behave identically under all experimental conditions used. The low activity of PFK‐phosphatase phosphorylated histone H2B [1] had resulted from an inhibition of the enzyme by high concentrations of this substrate. The apparent molecular mass of protein phosphatase 2C as calculated from Sephacryl chromatography and sedimentation analysis is about 90 kDa, the molecular mass obtained by SDS gel electrophoresis about 45 kDa. The native enzyme therefore seems to be a dimer consisting probably of 2 identical subunits. Accordingly, the previously described PFK‐phosphatase is protein phosphatase 2C.
A new function of kininogens as thiol‐proteinase inhibitors: inhibition of papain and cathepsins B, H and L by bovine, rat and human plasma kininogens
Authors: Sueyoshi Tatsuya , Enjyoji Keiichi , Shimada Toshio , Kato Hisao , Iwanaga Sadaaki , Bando Yoshiaki , Kominami Eiki , Katunuma Nobuhiko
Published: July 2002

Kininogen
Thiol‐proteinase inhibitor
Papain
Cathepsin B
Cathepsin H
Cathepsin L
Solubilization of C18‐Co A and C20‐CoA elongases from Allium porrum L. epidermal cell microsomes
Authors: Lessire René , Bessoule Jean-Jacques , Cassagne Claude
Published: July 2002

The effects of n‐octyl‐β‐D‐glucopyranoside, Triton X‐100 and deoxycholate on acyl‐CoA elongation by Allium porrum L. epidermal cell microsomes showed that the Triton X‐100 specifically stimulated the synthesis of C22–C26 acids using C18‐CoA as primer, whereas the fatty acid elongation products of C20‐CoA remained essentially unchanged. n‐Octyl‐β‐D‐glucopyranoside increased the C20 and C22 fatty acid syntheses to the same extent and deoxycholate inhibited C18‐CoA and C20‐CoA elongation. The presence of two different elongation systems, as suggested by these results, has been demonstrated. After solubilization by Triton X‐100, the C18‐CoA and C20‐CoA elongases were separated by sucrose density centrifugation. The fractions corresponding to sucrose concentrations of 0.51 and 0.62 M presented the maximal activities for C18‐CoA and C20‐CoA elongases, respectively. In addition, by gel filtration on a Sephacryl S‐300 column, the C20‐CoA and the C18‐CoA elongases have estimated apparent molecular masses under detergent conditions of 600 and 350 kDa, respectively.
The properties of the complex between ribosomal protein L2 and tRNA
Authors: Remme Jaanus , Metspalu Ene , Maimets Toivo , Villems Richard
Published: July 2002

Escherichia coli ribosomal protein L2 interacts with fMet‐tRNAMet and NacPhe‐tRNAPhe in solution, protecting their 3'‐ends from enzymatic degradation. At the same time L2 enhances the rate of spontaneous hydrolysis of the ester bonds between terminal riboses and amino acyl moieties of these two peptidyl‐tRNA analogues. L2 has, however, only a slight effect on the rate of spontaneous deacylation of aminoacyltRNAs. We suggest that the role of L2 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its P‐site, and speculate that this protein is directly involved in the peptidyl transferase (PT) reaction. Peptidyl transferase Protein L2 tRNA‐protein complex
Purification and biochemical characterization of hepatic ferredoxin (hepatoredoxin) from bovine liver mitochondria
Authors: Waki Noriko , Hiwatashi Atsuo , Ichikawa Yoshiyuki
Published: July 2002

Hepatic ferredoxin (hepatoredoxin) was purified from bovine liver mitochondria. The monomeric molecular mass of the hepatoredoxin was larger than that of adrenocortical ferredoxin (adrenodoxin) from bovine adrenocortical mitochondria at 14 kDa. We studied the amino acid residues and NH2‐terminal sequence of this protein. The hepatoredoxin was organ‐specific protein. The optical absorption spectrum of oxidized hepatoredoxin had two peaks, at 414 and 455 nm in the visible region. Hepatoredoxin formed an immunoprecipitin lime against anti‐adrenodoxin immunoglobulin in Ouchterlony double diffusion, and an immunochemical staining band in Western blotting.
Structural and immunological differences between human platelet and endothelial thrombospondins
Authors: Clezardin Philippe , Hunter Nikolas R. , McGregor John L. , Pepper Duncan S. , Dawes Joan
Published: July 2002

The structural and immunological properties of human thrombospondins isolated from platelets and from endothelial cells were compared. Both thrombospondins were digested with either trypsin or thermolysin, in the presence or absence of calcium, then injected onto a Superose 12 gel filtration column. The isolated thermolysin‐generated fragments of thrombospondins were identified by radioimmunoassays using either different monoclonal antibodies or a polyclonal antibody directed against platelet thrombospondin. The results show that platelet and endothelial thrombospondins are both partially protected from trypsin digestion in the presence of calcium but have different trypsin and thermolysin fragmentation patterns. The thermolysin‐generated fragments from platelet and endothelial thrombospondins are recognized differently by a monoclonal antibody whereas all of them are identified by a polyclonal antibody.
Binding of concanavalin A to the outer membrane of potato tuber mitochondria detected by flow cytometry
Authors: Petit P. , Diolez P. , Muller P. , Brown S.C.
Published: July 2002

Purified and intact mitochondria isolated from potato tuber show a specific concanavalin A (Con A) binding as deduced from fluorescence labelling studies in the presence of the specific sugar α‐D‐methylmannose. This specific binding also occurs in mitoplasts, indicating the presence of Con A‐binding sites on the inner mitochondrial membrane. In contrast, the binding of wheat germ agglutinin appears non‐specific. Flow cytometry with purified mitochondria enables assessment of the binding by individual mitochondria. Thus, three mitochondrial subpopulations were identified by their reactivity towards fluorescent Con A.
Binding of seminalplasmin to the plasma and acrosomal membranes of bovine spermatozoa
Authors: Shivaji Sisinthy
Published: July 2002

The binding of seminalplasmin, a protein secreted by the accessory sex glands of bull, to the plasma and outer acrosomal membrane of bovine spermatozoa was studied using three different fluorescent probes. 8‐Aniline‐1‐naphthalenesulfonate fluorescence, pyrene excimer fluorescence and diphenylhexatriene fluorescence polarisation studies indicate that seminalplasmin binds to the spermatozoal membranes, and leads to an increase in the fluidity of both the plasma and the acrosomal membranes. Calcium was found to have no influence on the interaction of seminalplasmin with the spermatozoal membranes. These results suggest that protein(s) present in the seminal plasma could interact with spermatozoal membranes and increase their fluidity.
Presence of cytochrome b−245 in NADPH oxidase preparations from human neutrophils
Authors: Bellavite Paolo , Cassatella Marco A. , Papini Emanuele , Megyeri Pál , Rossi Filippo
Published: July 2002

The composition of NADPH oxidase purified by Red Sepharose chromatography of extracts from human neutrophil membranes was investigated. In contrast to that was recently reported by others, the enzyme isolated according to this procedure contained a high concentration of cytochrome b
−245 and little FAD. The results reinforce the belief that cytochrome b
−245 is a major component of the NADPH oxidase and plays a fundamental role in the formation of O2
− by neutrophils.
Thermal stability of myosin subfragment‐1 decreases upon tryptic digestion in the presence of nucleotides
Authors: Pintér Katalin , Lu Renne Chen , Szilágyi László
Published: July 2002

Myosin subfragment‐1 (S‐1), digested with trypsin in the presence of ATP, rapidly loses its ATPase activity upon mild heat treatment even if ATP or ADP is present. The heat‐treated molecule is very sensitive to further tryptic digestion. Undigested S‐1 and S‐1 digested in the absence of ATP are protected by nucleotides. The loss of the protective effect of nucleotides correlates with the tryptic splitting of the 25 kDa aminoterminal fragment between Arg 23 and Ile 24.
A shift in chloroplast energy coupling by KCl from localized to bulk phase delocalized proton gradients
Authors: Beard William A. , Dilley Richard A.
Published: July 2002

Spinach thylakoids were resuspended in a buffer including either 200 mM sucrose or 100 mM KCl. singleturnover flash excitation and the luciferin‐luciferase system were used to energize and follow ATP formation. The effect of the permeable buffer pyridine was measured on the ATP formation onset lag and on the post‐illumination ATP yield. Consistent with a bulk phase delocalized proton gradient coupling model, thylakoids stored in 100 mM KCl exhibited an increase in these two parameters in the presence of pyridine. Thylakoids isolated in the absence of KCl showed no effect of pyridine on the two parameters indicating a localized energy coupling mode.
Construction, aminoacylation and 80 S ribosomal complex formation with a yeast initiator tRNA having an arginine CCU anticodon
Authors: Beauchemin Nicole , Grosjean Henri , Cedergren Robert
Published: July 2002

The digestion of yeast initiator methionine tRNA with mung bean nuclease and U2 ribonuclease yielded 5'‐ and 3'‐fragments, respectively. These two fragments together represent the entire tRNA sequence except for A35, the central nucleotide of the anticodon, and the CCA terminus. Using RNA ligase, a cytosine was added and the anticodon loop having a C35 was reformed. Subsequent treatment of this product with CCA‐transferase yielded a full‐length methionine tRNA having an arginine CCU anticodon. This recombinant tRNAMet (CCU) was charged with methionine by the yeast tRNA synthetase. Aminoacylation of the recombinant was however less extensive than in the case of native tRNAMet (CAU). After aminoacylation the recombinant tRNA formed an 80 S ribosomal complex.
Phosphorylation of myosin in non‐muscle and smooth muscle cells
Authors: Kuźnicki Jacek
Published: July 2002

Reversible phosphorylation of myosin subunits is observed in almost all eukaryotic cells. The data concerning sites and effects of phosphorylation on actin‐activated ATPase activity of myosin and on its filament formation are described. These observations are discussed in terms of possible evolutionary trends and rules which may govern the process of myosin phosphorylation.
Evolutionary conservation of the substrate‐binding cleft of phosphoglycerate kinases
Authors: Mori Nozomu , Singer-Sam Judith , Riggs Arthur D.
Published: July 2002

The primary structures of six phosphoglycerate kinases (PGKs) are known: three from mammals, one from yeast, and two from trypanosomes. Comparison of the amino acid sequence of these enzymes reveals 154 invariant positions out of 392 positions in the aligned sequences. Most of the conserved positions fall into the twelve β‐sheets and adjacent peptide regions that form the inner loops surrounding the ATP and 3‐phosphoglycerate‐binding cleft. The homology between mammalian and yeast PGKs is greater than 94% for the inner‐loop region, even though the overall homology is less than 65%. Trypanosome PGK has only 44% overall homology with the mammalian enzyme, but shows 74% homology in the inner‐loop region. Trypanosome PGK contains a polypeptide segment in its N‐terminal domain that is transposed in comparison with the other species.
DNA sequence analysis of diuron‐resistant mutations in the mitochondrial cytochrome b gene of Saccharomyces cerevisiae
Authors: di Rago J.-P. , Perea X. , Colson A.-M.
Published: July 2002

Diuron (3‐[34‐dichlorophenyl]‐1,1‐dimethylurea), an inhibitor of mitochondrial respiration blocks the yeast respiratory chain between cytochrome b and c
1. Diuron‐resistant mutants of Saccharomyces cerevisiae have been selected and several mutations localized to the mitochondrial cytochrome b gene. The present paper identifies specific DNA base changes within the cytochrome b gene conferring diuron‐resistance. DNA sequence analysis was done utilizing primer extension of crude mitochondrial RNA preparations in the presence of reverse transcriptase. Five independent diuron‐resistant mutations have been sequenced.
Altered protein kinase activities of lymphoid cells transformed by Abelson and Moloney leukemia viruses
Authors: Brunati Anna Maria , Saggioro Daniela , Chieco-Bianci Luigi , Pinna Lorenzo A.
Published: July 2002

Five different types of protein kinase acivities have been evaluated in cell lines from murine lymphomas induced by Abelson leukemia virus (A‐MuLV), whose oncogene codes for a tyrosine protein kinase. Such activities were compared with those of normal cells and of cells transformed by Moloney leukamia virus (M‐MuLV), lacking oncogene sequences in its genome. While cAMP‐dependent protein kinase and casein kinase‐1 do not undergo significant changes, casein kinase‐2 rises in both A‐MuLV and M‐MuLV infected lymphocytes, becoming largely associated with the particulate fraction of transformed cells. Protein kinase‐C on the other hand is unchanged in M‐MuLV transformed cells but it undergoes a 2–3‐fold increment in both soluble and particulate fractions of A‐MuLV transformed lymphocytes, which also display high tyrosine protein kinase activity.
The L‐form of N‐7‐mercaptoheptanoyl‐O‐phosphothreonine is the enantiomer active as component B in methyl‐CoM reduction to methane
Authors: Kobelt A. , Pfaltz A. , Ankel-Fuchs D. , Thauer R.K.
Published: July 2002

The reduction of methyl‐CoM to methane in methanogenic bacteria is dependent on a low‐M
r, heat‐stable compound designated component B, the structure of which has recently been assigned as N‐7‐mercaptoheptanoyl‐O‐phosphothreonine. We report here that only the enantiomer derived from O‐phospho‐L‐threonine has cofactor activity. N‐7‐Mercaptoheptanoyl‐O‐phospho‐D‐threonine was neither active nor inhibitory.
A point mutation in the chloroplast 16 S rRNA gene of a streptomycin resistant Nicotiana tabacum
Authors: Etzold Thure , Fritz Christian C. , Schell Jozef , Schreier Peter H.
Published: July 2002

We sequenced a 82 bp fragment in the chloroplast 16 S rRNA gene of the streptomycin resistant mutant SR1 of Nicotiana tabacum. The sequence contains a transition from cytosine to adenine in position 860 of the 16 S rRNA. We suggest that this mutation is sufficient for the streptomycin resistance phenotype in the SR1 mutant.
Catalytic and noncatalytic nucleotide binding sites of the Escherichia coli F1 ATPase Amino acid sequences of β‐subunit tryptic peptides labeled with 2‐azido‐ATP
Authors: Wise John G. , Hicke Brian J. , Boyer Paul D.
Published: July 2002

Under appropriate conditions tight, noncovalent binding of 2‐azido‐adenine nucleotides to either catalytic or noncatalytic binding sites on the E. coli F1‐ATPase occurs. After removal of unbound ligands, UV‐irradiation results primarily in the covalent incorporation of nucleotide moieties into the β‐subunit in both catalytic and noncatalytic site labeling experiments. Minor labeling of the α‐subunit was also observed. After trypsin digestion and purification of the labeled peptides, microsequencing studies identified two adjacent β‐subunit tryptic peptides labeled by 2‐azido‐ADP or ‐ATP. These β‐subunit peptides were labeled on tyrosine‐331 (catalytic sites) and tyrosine‐354 (noncatalytic sites) in homology with the labeling patterns of the mitochondrial and chloroplast enzymes.
Primary structures of three highly acidic ribosomal proteins S6, S12 and S15 from the archaebacterium Halobacterium marismortui
Authors: Kimura Junko , Arndt Evelyn , Kimura Makoto
Published: July 2002

The amino acid sequences of three extremely acidic ribosomal proteins, S6, S12, and S15, from Halobacterium marismortui have been determined. The sequences were obtained by the sequence analysis of peptides derived by enzymatic digestion with trypsin. Stapylococcus aureus protease and chymotrypsin, as well as by cleavage with dilute HCl. The proteins, S6, S12 and S15, consist of 116, 147 and 102 amino acid residues, and have molecular masses of 12251, 16440 and 11747 Da, respectively. Comparison of the amino acid sequences of these proteins with ribosomal protein sequences of other organisms revealed that halobacterial protein S12 has homology with the eukaryotic protein S16A from Saccharomyces cerevisiae, while S15 is significantly related to the Xenopus laevis S19 protein. No homology was found between these halobacterial proteins and any eubacterial ribosomal proteins.
Absorption mode two‐dimensional NOE spectroscopy of exchangeable protons in oligonucleotides
Authors: Sklenár̆ Vladimír , Brooks Bernard R. , Zon Gerald , Bax Ad
Published: July 2002

A new NMR method is described for the generation of absorption mode two‐dimensional NOE spectra of oligonucleotides in H2O solution. The method yields spectra that are free of baseline distortions with excellent suppression of the intense H2O resonance. The method is demonstrated for a sample of the dodecamer d(CGCGAATTCGCG)2. All exchangeable base protons are identified and a number of new types of NOE connectivities are observed.
The 3′‐orf protein of human immunodeficiency virus shows structural homology with the phosphorylation domain of human interleukin‐2 receptor and the ATP‐binding site of the protein kinase family
Authors: Samuel Kenneth P. , Seth Arun , Konopka Andrzej , Lautenberger James A. , Papas Takis S.
Published: July 2002

The primary amino acid sequence within a stretch of 25 residues (positions 91–116) of the middle portion of the 3‐′orf protein (p273′‐orf) of the human immunodeficiency virus (HIV) shares structural homology with a highly charged region within the intracytoplasmic phosphorylation domain of human interleukin‐2 receptor (IL‐2R) and the ATP‐binding site of the catalytic subunit of cAMP‐dependent protein kinase (cAMP‐PK) and other members of the protein kinase family. Comparison of the predicted secondary structure within this region of p273′‐orf with the phosphorylation domain of human IL‐2R and the ATP‐binding region of the phospho‐kinase family of protein suggests that the 3′‐orf protein could serve homologous function(s).
Fructose 2,6‐bisphosphate in isolated foetal hepatocytes
Authors: Martín-Sanz Paloma , Cascales María , Boscá Lisardo
Published: July 2002

Fru 2,6‐P2 was present in isolated foetal hepatocytes at a concentration of 1.6 nmol per g cells. When foetal hepatocytes were exposed to glucagon no changes were observed either in the concentration of Fru 2,6‐P2 and lactate release or in the activities of 6‐phosphofructo‐2‐kinase and pyruvate kinase. Incubation of purified 6‐phosphofructo‐2‐kinase with the catalytic subunit of protein kinase did not change the enzyme activity. The inhibition by sn‐glycerol 3‐phosphate was much lower for the foetal than for adult enzyme. These results suggest that an isoenzyme of 6‐phosphofructo‐2‐kinase in foetal hepatocytes different from that of adult hepatocytes may be present.
Lipocortin inhibition of extracellular and intracellular phospholipases A2 is substrate concentration dependent
Authors: Aarsman A.J. , Mynbeek G. , van den Bosch H. , Rothhut B. , Prieur B. , Comera C. , Jordan L. , Russo-Marie F.
Published: July 2002

Hydrolysis of Escherichia coli membrane phospholipids by pancreatic phospholipase A2 was inhibited by lipocortin from human monocytes in a substrate dependent manner. Inhibition was completely overcome at substrate concentrations above 250 μM. Lipocortin also inhibited partially purified preparations of two intracellular phospholipases A2, isolated from rat liver mitochondria and rat platelets when these enzymes were assayed at low micromolar concentrations of phosphatidylethanolamine. Inhibition gradually decreased with increasing substrate concentrations both for pancreatic and platelet phospholipase A2 and became completely abolished above 15 and 50 μM phosphatidylethanolamine, respectively.
Isolation of protein kinase C subspecies from a preparation of human T lymphocytes
Authors: Shearman Mark S. , Berry Nicola , Oda Tomiichiro , Ase Katsuhiko , Kikkawa Ushio , Nishizuka Yasutomi
Published: July 2002

Using a preparation of purified human T lymphocytes, we were able to resolve a partially purified protein kinase C (PKC) enzyme fraction into two distinct subspecies, of approximately equal activity. Biochemical and immunocytochemical analysis revealed that these fractions closely resembled the type II(β) and type III(α) PKC subspecies previously identified and characterised from brain tissue. These results provide valuable information for further studies on the role of individual PKC subspecies in T lymphocyte proliferation.
Mobile sequences in the pyruvate dehydrogenase complex, the E2 component, the catalytic domain and the 2‐oxoglutarate dehydrogenase complex of Azotobacter vinelandii, as detected by 600 MHz 1H‐NMR spectroscopy
Authors: Hanemaaijer Roeland , Vervoort Jacques , Westphal Adrie H. , de Kok Arie , Veeger Cees
Published: July 2002

600 MHz 1H‐NMR spectroscopy demonstrates that the pyruvate dehydrogenase complex of Azotobacter vinelandii contains regions of the polypeptide chain with intramolecular mobility. This mobility is located in the E2 component and can probably be ascribed to alanine‐proline‐rich regions that link the lipoyl subdomains to each other as well as to the E1 and E3 binding domain. In the catalytic domain of E2, which is thought to form a compact, rigid core, also conformational flexibility is observed. It is conceivable that the N‐terminal region of the catalytic domain, which contains many alanine residues, is responsible for the observed mobility. In the low‐field region of the 1H‐NMR spectrum of E2 specific resonances are found, which can be ascribed to mobile phenylalanine, histidine and/or tyrosine residues which are located in the E1 and E3 binding domain that links the lipoyl domain to the catalytic domain. In the 1H‐NMR spectrum of the intact complex, these resonances cannot be observed, indicating a decreased mobility of the E1 and E3 binding domain.
Casein kinase 2 activity increases in the prereplicative phase of liver regeneration
Authors: Pérez Mercè , Grande Jorge , Itarte Emilio
Published: July 2002

Cytosolic casein kinase activity increased up to 2‐fold in the first 6 h after partial hepatectomy and then decreased to control values. This increase was due mainly to casein kinase 2, which reached maximal values at 6–8 h of liver regeneration. In contrast, casein kinase 1 showed a smaller increase at 4 h and then started to decrease reaching values of about 70% of control at 16 h. The increase in total casein kinase 2 was accompanied with an activation of the enzyme, as determined by the low/high β‐casein activity ratio assay. Administration of an acute dose of glucagon to control rats also increased the activity ratio but failed to cause any rise in total casein kinase 2 activity.
Two‐chain structure of the interleukin 1 receptor
Authors: Kroggel Roswitha , Martin M. , Pingoud Vera , Dayer J.M. , Resch K.
Published: July 2002

By crosslinking radioiodinated recombinant human IL1 α to mouse EL4 thymoma cells we have identified in addition to the known IL1‐binding proteins of 80 kDa, a second IL1‐binding protein of about 40 kDa. This second binding protein could be demonstrated most easily when crosslinking to higher protein complexes was inhibited. This finding suggests that the IL1 receptor, similar to the receptor for other cytokines such as interleukin 2, is composed of a heterodimer, of which both polypeptides contribute to ligand binding.
Expression of protein kinase C subspecies in human leukemia‐ lymphoma cell lines
Authors: Sawamura Satoru , Ase Katsuhiko , Berry Nicola , Kikkawa Ushio , McCaffrey Patricia G. , Minowada Jun , Nishizuka Yasutomi
Published: July 2002

Expression of protein kinase C (PKC) subspecies was studied in various human leukemia‐lymphoma cell lines. The PKC in most cell lines examined was resolved into two major fractions corresponding to type II (β‐sequence) and type III (α‐sequence) PKC of the rat brain. The amounts of these two subspecies greatly varied among the cell lines. Type I PKC (γ‐sequence) was expressed in none of the cell lines tested, but PKCs with undefined structures were frequently detected. The differential co‐expression of several PKC subspecies is presumably related to the state of cell differentiation.
The organisation of the mouse chromogranin B (secretogranin I) gene
Authors: Pohl Thomas M. , Phillips Elizabeth , Song Kyuyong , Gerdes Hans-Hermann , Huttner Wieland B. , Rüther Ulrich
Published: July 2002

A cosmid clone containing the gene for mouse chromogranin B (secretogranin I), a secretory protein found in secretory granules of most endocrine cells and neurons, was isolated and sequenced. The chromogranin B protein was found to be encoded by 5 exons which correspond to the cleaved signal peptide, the short N‐terminal sequence preceding the disulfide‐bonded loop structure, the disulfide‐bonded loop structure itself, the large, variable region comprising ~90% of the protein, and the conserved C‐terminal sequence. The promoter region of the chromogranin B gene is very GC‐rich and contains a CATAA motif, a cAMP‐responsive element and an Spl binding site.
A soluble form of the interleukin‐1 receptor produced by a human B cell line
Authors: Symons J.A. , Duff G.W.
Published: July 2002

A soluble protein that binds specifically to interleukin‐1 (IL‐1)β was released from a B cell line (Raji). The covalently cross‐linked binding protein/ [125I]IL‐1β migrated at 60 kDa by SDS‐PAGE. The IL‐1 receptor (IL‐1R) on Raji cells had the same ligand specificity. Stimulation of Raji with dexamethasone increased surface expression of the IL‐1R and the rate of release of soluble binding protein. A serine protease inhibitor prevented release of the binding protein and increased IL‐1R expression on the cells. These results suggest that the soluble IL‐1β binding protein is a proteolytically cleaved form of the novel B cell IL‐1R.
Internalization of the Hm1 muscarinic cholinergic receptor involves the third cytoplasmic loop
Authors: Maeda Sadaaki , Lameh Jelveh , Mallet William G. , Philip Mohan , Ramachandran J. , Sadée Wolfgang
Published: July 2002

The m1 muscarinic receptor was previously shown to stimulate phosphatidyl inositol (PI) turnover and to internalize rapidly upon agonist activation. Three receptor mutants with large deletions of the third cytoplasmic loop (i3) of human Hm1, leaving only 11 and 8 amino acids at the amino and carboxy terminal junctions of i3, respectively, retained full ability to stimulate PI turnover, when expressed in U293 cells, but receptor internalization was greatly reduced in two mutants with deletions reaching close to the NH2 terminal of i3. We propose that a receptor domain located toward the amino terminal junction ofi3 plays a role in Hm1 internalization.
A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties
Authors: Boyot Philippe , Pillet Laurence , Ducancel Frédéric , Boulain Jean-Claude , Tremeau Odile , Ménez André
Published: July 2002

We previously reported the production of a fused snake neurotoxin composed of protein A and erabutoxin a in E. coli [1]. The hybrid had much lower toxicity and affinity for the acetylcholine nicotinic receptor than natural erabutoxin. By treating the hybrid with cyanogen bromide we generated a toxin which was purified in a single step by RP‐HPLC. This compound, produced in a good yield, recovered all properties of native erabutoxin a, implying that the lower toxic activities of the hybrid were due to the bulky protein A and not to an incorrect folding of the toxin. This work serves as a basis for future studies of toxin‐receptor interactions using engineered toxin mutants.
The nirSTBM region coding for cytochrome cd1‐dependent nitrite respiration of Pseudomonas stutzeri consists of a cluster of mono‐, di‐, and tetraheme proteins
Authors: Jüngst Angelika , Wakabayashi Sadao , Matsubara Hiroshi , Zumft Walter G.
Published: July 2002

Genes for respiratory nitrite reduction (denitrification) of Pseudomonas stutzeri are clustered within 7 kbp. A 4.6‐kbp Hind III‐Kpn I fragment carrying nirS, the structural gene for cytochrome cd
1, was sequenced. An open reading frame immediately downstream of nirScodes for a 22.8‐kDa protein with four heme c‐binding motifs. Mutagenesis of this gene causes an apparent defect in electron donation to cytochrome cd
1. Following this ORF are the structural genes for cytochrome c
552, cytochrome c
551, and ORF5 that codes for a 11.9‐kDa monoheme protein. All cytochromes have a signal sequence for protein export.
Release of periplasmic proteins induced in E. coli by expression of an N‐terminal proximal segment of the phage fd gene 3 protein
Authors: Rampf Bettina , Bross Peter , Vocke Tobias , Rasched Ihab
Published: July 2002

We used the enzymes β‐lactamase and alkaline phosphatase to quantitatively evaluate the release of periplasmic proteins from E. coli cells transformed by plasmids harboring gene 3 of phage fd. Different deletion mutants of gene 3 released varying fractions of the enzymes. From these results we conclude that essentially the amino‐terminal proximal part, upstream of the first glycine‐rich region but not this region itself, is responsible for the excretion of periplasmic proteins in E. coli cells expressing the gene 3 protein of phage fd.
Proteolytic inactivation of human α1 antitrypsin by human stromelysin
Authors: Winyard Paul G. , Zhang Zhi , Chidwick Keith , Blake David R. , Carrell Robin W. , Murphy Gillian
Published: July 2002

α1Antitrypsin (α1AT) is the main physiological inhibitor of neutrophil elastase, a serine protease which has been implicated in tissue degradation at inflammatory sites. We report here that the connective tissue metalloproteinase, stromelysin, cleaved α1AT (54 kDa), producing fragments of approximately 50 kDa and 4 kDa, as shown by gel electrophoresis. The cleavage of α1AT was accompanied by inactivation of its elastase inhibitory capacity. Isolation of the 4 kDa fragment by reversed‐phase HPLC, followed by N‐terminal amino acid sequencing, demonstrated that the cleavage of α1AT occurred at the Pro357‐Met358 (P2–P1) peptide bond, one peptide bond to the N‐terminal side of the inhibitory site. We suggest that stromelysin may potentiate the activity of neutrophil elastase by proteolytically inactivating α1AT.
Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis
Authors: Togni Giuseppe , Sanglard Dominique , Falchetto Rocco , Monod Michel
Published: July 2002

The extracellular acid protease of Candida tropicalis was purified from the supernatant fraction of culture medium containing bovine serum albumin as nitrogen source and the NH2‐terminal amino acid (aa) sequence of the protein was determined. The gene for the acid protease (ACP) was isolated using a pool of synthetic oligonucleotides as a probe and a segment of the deduced aa sequence was found to be in agreement with the NH2‐terminal aa sequence of the protein. The deduced aa sequence of ACP is similar to the aa sequence of proteases of the pepsin family. The nucleotide sequence of the 5′ portion of this gene revealed a coding sequence for a 60 residue propeptide containing two Lys—Arg amino acid pairs that have been identified as sites for peptidase processing of several exported peptides and proteins. The final Lys—Arg site occurs at the junction with the mature extracellular form or the acid protease.
Binding sites in fibronectin for an enterotoxigenic strain of E. coli B342289c
Authors: Visai Livia , Bozzini Silvia , Petersen Torben E. , Speciale Livianna , Speziale Pietro
Published: July 2002

The binding of fibronectin and fibronectin fragments to the enterotoxigenic strain E. coli B34289c was studied. E. coli cells bound to two distinct sites of fibronectin, one being the N‐terminal domain, which also contains the binding sites for staphylococci and streptococci, and the other located within the central heparin binding region. In addition, the N‐terininal and the heparin binding domain mediated the attachment of bacteria in a solid phase binding assay. E. coli cells expressed two classes of receptors, the first, a 17 kDa protein, recognized by the N‐terminal fragment and the second, having a mol. mass of 55 kDa, which interacts with the internal heparin binding domain. Bacterial receptors, which bind the N‐terminal end of fibronectin, may be structurally related.
Isolation of mitotic p34cdc2 apoenzyme from human cells
Authors: Meikrantz William , Feldman Robert P. , Sladicka Melissa M. , Ho David , Krupnick Jason , Anderson Karen , Schlegel Robert A.
Published: July 2002

A simple procedure was devised for isolating from homogenates of mitotic cells the human homolog to the fission yeast
cdc2
gene product. The identity of the purified protein was established with anti‐p34
cdc2
antibodies and p13
suc1
, both specific ligands for p34
cdc2
. Active‐site labeling with oxidized [α32P]ATP showed the purified molecule to be an ATP‐binding protein. Its ability to phosphorylate casein but not histone, and its phosphorylation on tyrosine, detected by anti‐phosphotyrosine antibodies, indicates the form of p34
cdc2
purified is the inactive or apoenzyme form. Purified quantities of human p34
cdc2
should be of considerable value in establishing the mechanism of its activation at mitosis by phosphatases.
Flash‐photolysis studies of the electron transfer from genetically modified spinach plastocyanin to photosystem I
Authors: Nordling Margareta , Sigfridsson Kalle , Young Simon , Lundberg Lennart G. , Hansson Örjan
Published: July 2002

Plastocyanin (Pc) has been modified by site‐directed mutagenesis at two separate electron‐transfer (ET) sites: Leu‐12‐Glu at a hydrophobic patch, and Tyr‐83‐His at an acidic patch. The reduction potential at pH 7.5 is decreased by 26 mV in Pc(Leu‐12‐Glu) and increased by 35 mV in Pc(Tyr‐83‐His). The latter mutant shows a 2‐fold slower intracomplex ET to photosystem I (PSI) as expected from the decreased driving force. The affinity for PSI is unaffected for this mutant but is drastically decreased for Pc(Leu‐12‐Glu). It is concluded that the hydrophobic patch is more important for the ET to PSI.
Binding of an import protein to intact chloroplasts and to isolated chloroplast envelopes of Chlamydomonas reinhardii
Authors: Su Qing-iang , Niklaus Andreas , Rothen René , Boschetti Arminio
Published: July 2002

The binding affinity of the precursor of the small subunit of ribuloseo‐1,5‐bisphosphate carboxylase (pSS) to isolated, intact chloroplasts and to isolated chloroplast envelopes from the green alga Chlamydomonas reinhardii was studied under conditions where no import into chloroplasts occurred, pSS bound to both chloroplasts and envelopes with equally high affinity, The dissociation constants were 5.9 ± 2.1×10−9 M and 2.9 ± 1.4× 10−9 M, respectively. The number of binding sites per chloroplast was determined to be 8.1 ± 4.1×104. Binding of pSS to isolated envelopes or intact chloroplasts was specific with respect to the type of the membrane and the presence of the transit sequence.
The major Thiobacillus ferrooxidans outer membrane protein forms low conductance ion channels in planar lipid bilayers
Authors: Silva Macarena , Ferreira Arianna , Rodriguez Manuel , Wolff Daniel
Published: July 2002

A protein isolated and purified from the outer membrane of the acidophilic, chemolithotrophic bacterium, Thiobacillus ferrooxidans with an oligomeric molecular weight of 90 000 Da (p9O) was incorporated into phosphatidylethanolamine planar lipid bilayers. The protein formed slightly anionic channels in KCl solutions, with a conductance of 25 pS in 100 mM KCl. The current—voltage relationship was linear between ±6O mV, and the conductance was a saturating function of the salt concentration. These channels fluctuated from a single open to closed state at low potentials, but present flickering activity at higher potentials.
Homomeric and native α7 acetylcholine receptors exhibit remarkably similar but non‐identical pharmacological properties, suggesting that the native receptor is a heteromeric protein complex
Authors: Anand René , Peng Xiao , Lindstrom Jon
Published: July 2002

Sucrose gradient analysis of chick acetylcholine receptor (AChR) α7 subunits expressed in oocytes indicates that they form pharmacologically active homomers of the same size as native α7 AChRs, a size compatible with a complex of five α7 subunits. By immunoisolating the [35S]methionine‐labeled α7 subunits we also demonstrate that they do not appear to assemble with endogenous Xenopus AChR subunits. Pharmacological characterization of detergent‐solubilized brain α7 AChRs and α7 homomers reveals that they have similar but nonidentical properties. The pharmacological difference is most accentuated for cytisine (~50‐fold). Thus, at least in E18 chicken brain, most or all of the native α7 AChRs do not appear to be homomeric.
Yeast mitochondrial ATP‐dependent protease: purification and comparison with the homologous rat enzyme and the bacterial ATP‐dependent protease La
Authors: Kutejová Eva , DurčovÁ Gabriela , SurovkovÁ Eva , Kužela Štefan
Published: July 2002

Homogenous ATP‐dependent protease has been isolated for the first time from mitochondria of the yeast Saccharomyces cerevisiae. The enzyme molecule consists of six 120 kDa subunits. It is a serine protease with an absolute ATP requirement for its activity. Basic enzymatic characteristics of the yeast protease are similar to those of the corresponding rat mitochondrial enzyme and of the E. coli protease La. The yeast enzyme immunochemically cross‐reacts with the bacterial protease La.
ζ‐Crystallin versus other members of the alcohol dehydrogenase super‐family Variability as a functional characteristic
Authors: Jörnvall Hans , Persson Bengt , Du Bois Garrett C. , Lavers Gene C. , Chen John H. , Gonzalez Pedro , Rao P.Vasantha , Zigler J.Samuel
Published: July 2002

Species variability of the lens protein ζ‐crystallin was correlated with those of alcohol dehydrogenases of classes I and III and sorbitol dehydrogenase in the same protein family. The extent of overall variability, nature of residues conserved, and patterns of segment variability, all fall within the limits typical of the ‘variable’ group of medium‐chain alcohol dehydrogenases. This shows that ζ‐crystallin is subject to restrictions similar to those of classical liver alcohol dehydrogenase and therefore derived from a metabolically active enzyme like other enzyme crystalline. Special residues at the active site, however, differ substantially, including an apparent lack of a zinc‐binding site. This is compatible with altered functional properties and makes the spread within this medium‐chain dehydrogenase family resemble the wide spread within the short‐chain dehydrogenases. Schematic plotting is useful for illustrating the differences between ‘variable’ and ‘constant’ enzymes.
Induction of stathmin mRNA during liver regeneration
Authors: Okazaki Takashi , Himi Toshiyuki , Peterson Christine , Mori Nozomu
Published: July 2002

Stathmin is a 19 kDa phosphoprotein, and is proposed to play a role in signal transduction in response to various extracellular stimuli that promote cellular growth and/or differentiation. We examined stathmin mRNA expression during development and liver regeneration in mice. Stathmin mRNA expression declined during the post‐natal period and was undetected in adult liver. 36 h after partial hepatectomy, stathmin mRNA was rapidly induced and remained at elevated levels for at least 10 days. In situ hybridization experiments confirmed that stathmin mRNA expression occurred in hepatocytes. These results indicate that the stathmin gene expression appears to be repressed during the post‐natal liver development, and is de‐repressed by liver regeneration, which suggests that stathmin may be a good molecular marker of liver plasticity.
Accumulation of trehalose in Saccharomyces cerevisiae growing on maltose is dependent on the TPS1 gene encoding the UDPglucose‐linked trehalose synthase
Authors: Petit Thomas , François Jean
Published: July 2002

When yeast strains were cultivated on maltose, the synthesis of trehalose already started in the exponential phase of growth, well before exhaustion of the sugar from the medium. This active pattern of trehalose accumulation was also observed in a maltose constitutive mutant strain growing on glucose. However, this accumulation was completely prevented by deletion of the TPS1 gene coding for the catalytic subunit of the UDPglucose‐linked trehalose‐6‐phosphate synthase, indicating that no alternative pathway for trehalose synthesis exists in yeast. The active pattern of trehalose accumulation seems to be consistent with the finding that trehalose‐6‐phosphate synthase is more active in strains growing on maltose than on glucose.
Optimal and heuristic policies for lot sizing with learning in setups
Authors: Rachamadugu Ram , Schriber Thomas J.
Published: July 2002

We develop heuristic and optimal methods for determining lot sizes when setup cost reductions occur over time due to emphasis on continuous improvement, learning effects and incremental process changes. Our heuristic methods are intuitively appealing, easy to implement, require little information about setup costs, and have low computational burden. Computational studies show that choosing the appropriate heuristic yields nearly optimal solutions. Recommendations for choosing the appropriate heuristic are also provided. The optimal method developed in this paper is also useful to managers for evaluating investments in hardware and/or worker training for setup reduction. Concepts and methods are illustrated with numerical examples. Managerial implications of using our policies are discussed.
Structure of the cytosolic domain of TOM5, a mitochondrial import protein
Authors: Hammen Philip K , Weiner Henry
Published: July 2002

TOM5 is a small outer mitochondrial membrane protein in Saccharomyces cerevisiae and is part of a multi‐protein translocator complex, which mediates protein import into mitochondria. Presently, nothing is known about the conformational preferences of TOM5 or other mitochondrial import proteins. In this report, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy are used to determine the conformational preferences of the cytosolic domain of TOM5. The CD spectra show evidence of a helical structure that is invariant with pH. NOESY data revealed that TOM5 forms a stable helical core between E11 and R15 with a less structurally rigid helix extending to the C‐terminus.
The natural osmolyte trimethylamine N‐oxide (TMAO) restores the ability of mutant tau to promote microtubule assembly
Authors: Smith Michael J , Crowther R.Anthony , Goedert Michel
Published: July 2002

Coding region and intronic mutations in the gene for microtubule‐associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP‐17). Most coding region mutations effect a reduced ability of tau protein to interact with microtubules and lead to the formation of a filamentous pathology made of hyperphosphorylated tau. Here we show that trimethylamine N‐oxide (TMAO) restores the ability of tau with FTDP‐17 mutations to promote microtubule assembly. To mimic phosphorylation, serine and threonine residues in tau were singly or multiply mutated to glutamic acid, resulting in a reduced ability of tau to promote microtubule assembly. With the exception of the most heavily substituted protein (27 glutamic acid residues), TMAO increased the ability of mutant tau to promote microtubule assembly. However, it had no significant effect on heparin‐induced assembly of tau into filaments.
Diverse PAH transcripts in lymphocytes of PKU patients with putative nonsense (G272X, Y356X) and missense (P281L, R408Q) mutations
Authors: Ellingsen Ståle , Knappskog Per M , Apold Jaran , Eiken Hans Geir
Published: July 2002

The majority of mutations in the human phenylalanine hydroxylase (PAH) gene that lead to the recessive disease phenylketonuria (PKU) are believed to affect the activity or stability of the PAH enzyme. In this study we have performed in vivo analyses of lymphocyte PAH mRNA from PKU patients homozygous for the PKU missense mutations P281L and R408Q as well as the nonsense mutations G272X and Y356X. The mutations G272X, P281L and R408Q, which are located outside the consensus splice site sequence, result in transcripts with one or more exons skipped in addition to full‐length transcripts. The mutation Y356X results in transcripts with one or more exons skipped, but no full‐length transcripts. Our findings question the value of functional and structural predictions of mutations at the protein level without analyses of the corresponding transcript.
Posttranscriptional regulation of oligodendroglial thyroid hormone (T3) receptor β1 by T3
Authors: Baas Dominique , Puymirat Jack , Sarliève Louis L.
Published: July 2002

3,5,3′‐triiodo‐L‐thyronine interacts with the genome by binding and activating nuclear 3,5,3′‐triiodo‐L‐thyronine receptors. To determine how in secondary oligodendrocyte cultures, exogenous 3,5,3′‐triiodo‐L‐thyronine influences the expression of different 3,5,3′‐triiodo‐L‐thyronine receptor isoforms, we studied the regulation of α1, α2 and β1 3,5,3′‐triiodo‐L‐thyronine receptor mRNAs. In culture, we find that β1 3,5,3′‐triiodo‐L‐thyronine receptor mRNA, but not α1 and α2 3,5,3′‐triiodo‐L‐thyronine receptor mRNAs, is up‐regulated by 3,5,3′‐triiodo‐L‐thyronine in a time and dose dependent manner. In addition, we present evidence indicating that β1 3,5,3′‐triiodo‐L‐thyronine receptor expression is posttranscriptionally regulated by 3,5,3′‐triiodo‐L‐thyronine.

Previous studies from our laboratory and others have shown that in the rat oligodendrocyte lineage, 3,5,3′‐triiodo‐L‐thyronine receptors α1 and α2 were expressed in both early progenitor cells and mature oligodendrocytes. In contrast, β1 3,5,3′‐triiodo‐L‐thyronine receptor was found to be expressed only in mature oligodendrocytes. This suggests that thyroid hormone may influence oligodendrocyte differentiation and maturation via 3,5,3′‐triiodo‐L‐thyronine receptor β1, which is expressed only in oligodendrocytes and not in progenitor cells. We therefore show that this effect is indirect and is mediated by 3,5,3′‐triiodo‐L‐thyronine which acts posttranscriptionally on the 3,5,3′‐triiodo‐L‐thyronine receptor β1 gene.