bluuebunny/crossref_metadata_2025 huggingface dataset:
The regulatory and basal phosphorylation sites of hormone‐sensitive lipase are dephosphorylated by protein phosphatase‐1, 2A and 2C but not by protein phosphatase‐2B
Authors: OLSSON Håkan , BELFRAGE Per
Published: March 2005

The activity of hormone‐sensitive lipase, the rate‐limiting enzyme in adipose tissue lipolysis, is controlled by cAMP‐mediated phosphorylation at a specific regulatory phosphorylation site. The lipase is also phosphorylated at a site, termed basal, without any effects on its activity [Strålfors et al. (1984) Proc. Natl Acad. Sci. USA 81, 3317–3321]. The capacity of protein phosphatase‐1, 2A, 2B and 2C to dephosphorylate the lipase, selectively phosphorylated by glycogen synthase kinase‐4 and cAMP‐dependent protein kinase at the basal and regulatory phosphorylation sites, was compared with that towards glycogen phosphorylase and phosphorylase kinase (α subunit). Protein phosphatase‐1, 2A and 2C were found to dephosphorylate both phosphorylation sites of hormone‐sensitive lipase, while protein phosphatase‐2B had no measureable activity towards any of the sites. When the activities of protein phosphatase‐1, 2A and 2C were normalized with respect to the reference substrates, they were found to dephosphorylate the lipase regulatory site in the approximate relations of 1:4:3 and the basal site in the approximate relations of 1:6:4.

Protein phosphatase‐1 showed 20% higher and protein phosphatase‐2A and 2C 80% higher activity towards the basal site compared to the regulatory site. The two phosphorylation sites of the lipase were comparable to good substrates for protein phosphatase‐2A and 2C, but relatively poor substrates for protein phosphatase‐1. Protein phosphatase‐2C activity towards the lipase was completely dependent on Mg2+ with a half‐maximal effect at 3 mM. Protamine increased the lipase dephosphorylation by protein phosphatase‐1 3–5‐fold with half‐maximal effect at 0.6 μg/ml, and by protein phosphatase‐2A about 2‐fold with half‐maximal effect at 3–5 μg/ml, thus illustrating the potential for control of these lipase phosphatase activities.
A monoclonal antibody against 2,2,7‐trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7‐methylguanosine‐capped RNAs
Authors: BOCHNIG Peter , REUTER Rolf , BRINGMANN Peter , LÜHRMANN Reinhard
Published: March 2005

A hybridoma secreting a monoclonal antibody (H‐20) that recognizes the 2,2,7‐trimethylguanosine(m3G)‐containing cap structure of U snRNAs was derived from a mouse which was immunized with a m3G‐containing human serum albumin conjugate. The antibody specifically reacts with intact small nuclear ribonucleoprotein particles, U snRNPs, and allows the snRNPs U1 to U6 to be isolated in one step from nuclear extracts of eucaryotic cells by affinity chromatography on a preparative scale. Antibody‐bound snRNPs are desorbed from the affinity column by elution with excess of the cross‐reactive nucleoside 7‐methylguanosine (m7G), which guarantees maintenance of their native structure. The 20 affinity column also allows the snRNPs U1, U2 and U5 to be separated from U4/U6 RNPs by sequential elution of the particles with m7G under differential salt concentrations. As determined by competitive radioimmunoassay and protein‐A‐Sepharose immunoprecipitation, mAb H‐20 crossreacts with intact m7G cap structures. In particular we could show that non‐denatured m7G‐capped SP6/β‐globin RNA was precipitated efficiently by the antibody while GpppG‐capped or non‐capped RNAs did not react. Thus the monoclonal antibody H‐20 should have a wide application, not only for studying the molecular biology and immunology of the U snRNPs from diverse organisms, but also for the characterization and isolation of m7G‐capped transcripts.
Isolation and characterization of ornitho‐kininogen
Authors: KIMURA Michio , SUEYOSHI Tatsuya , TAKADA Katsumi , TANAKA Kosaku , MORITA Takashi , IWANAGA Sadaaki
Published: March 2005

Ornitho‐kininogen was purified from chicken blood plasma by a two‐stage method using chromatography on columns of S‐alkylated papain‐Cellulofine and DEAE‐5PW. The yield was 1.7 mg from 44 ml plasma. The isolated preparation gave a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) with or without 2‐mercaptoethanol and on disc/polyacrylamide gel electrophoresis. The relative molecular mass, Mr, of ornitho‐kininogen was estimated as 74000 on SDS‐PAGE using the Ferguson plot method. Ornitho‐kininogen was found to have the similar properties to those of mammalian high‐Mr kininogen, in terms of the amino acid composition, molecular mass, and susceptibility to plasma kallikrein. No kininogen corresponding to mammalian low‐Mr kininogen and rat T‐kininogen could be detected in chicken plasma. In fact, ornitho‐kininogen was degraded rapidly by bovine plasma kallikrein, liberating a kinin. This kinin was isolated from the digest by reversed‐phase HPLC. The primary structure of the isolated kinin was determined as Arg1‐Pro2‐Pro3‐Gly4‐Phe5‐Thr6‐Pro7‐Leu8‐Arg9. The sequence of this peptide, named ornitho‐kinin, was similar to that of bradykinin except for the substitution of Thr6 and Leu8 for Ser6 and Phe8. The isolated ornitho‐kinin induced a contraction of chicken smooth muscle and had a strong hypotensive effect in the chicken. However, it did not contract the isolated rat uterus. It is suggested that this specificity difference is due to the replacement of Phe8 by Leu8. The sequence of residues 1–30 of ornitho‐kininogen exhibited 43% identity with that of bovine kininogen.
Mouse UDP glucuronosyltransferase
Authors: KIMURA Teruyuki , OWENS Ida S.
Published: March 2005

A cDNA clone (UDPGTm‐1) encoding a mouse UDP glucuronosyltransferase (transferase) was isolated from pBR322 and λgt11 libraries by hybridization to a rat transferase clone. This cDNA is 1860 bp long and 65–87% similar at both the nucleotide and the deduced amino acid sequence levels to three different rat transferase clones [Mackenzie, P. I. et al. (1984) J. Biol. Chem. 259, 12153–12160; Mackenzie, P. I. (1986) J. Biol. Chem. 261, 6119–6125]. UDPGTm‐1, like the rat transferase clones already described, contains an open reading frame of 1590 bp encoding 530 amino acids (unmodified Mr= 60856), an N‐terminus membrane‐insertion signal sequence, a carboxy‐terminus hydrophobic putative membrane‐spanning region, and potential asparagine‐linked glycosylation sites (residues 316 and 483). The cDNA contains two poly(A) addition consensus sequences at positions 1695–1837. UDPGTm‐1 is complementary to a 2200‐base mRNA and also cross‐hybridizes to a 2000‐base mRNA species due to sequence homology in the 5′ region of the clone. Both the 2200‐base and the 2000‐base mRNA are induced approximately 2.5‐fold by the hypolipidemic agent clofibrate, and also by phenobarbital and benzo[a]pyrene. A new and more potent hypolipidemic agent, perfluorooctancic acid, is also shown to induce both mRNA species. Each of these compounds induces bilirubin transferase activity in a manner parallel to the effects on mRNA, i. e. perfluorooctanoic acid being the most effective, followed by phenobarbital, benzo[a]pyrene, and clofibrate. Southern blot hybridization of UDPGTm‐1 to mouse genomic DNA showed sequence homology to a total DNA size of 40–50 kb. These data indicate that UDPGTm‐1 is a member of a new subfamily of transferases in mouse with patterns of regulation of their mRNAs similar to that seen for bilirubin transferase activity.
Completion of the amino acid sequence of the α1 chain of human basement membrane collagen (type IV) reveals 21 non‐triplet interruptions located within the collagenous domain
Authors: BRAZEL Dieter , OBERBÄUMER Ilse , DIERINGER Hans , BABEL Wilfried , GLANVILLE Robert W. , DEUTZMANN Rainer , KÜHN Klaus
Published: March 2005

The cDNA and protein sequences of the N‐terminal half of human basement membrane collagen (type IV) have been determined. Overlapping cDNA clones were constructed by repeated primer extension with synthetic oligonucleotides. They cover 2953 bp, beginning at the 5′ end of the corresponding mRNA. At the protein level, the sequence of the cyanogen bromide peptide CB6 adjacent to the 7S domain has been additionally elucidated. The data presented here complete the protein sequence and nearly the entire cDNA sequence of the human α1(IV) chain. The amino‐terminal half of the α1(IV) chain contains 8 cysteine residues involved in intramolecular and intermolecular cross‐links. The entire triple‐helical domain of α1(IV) is interrupted by 21 non‐triplet regions.
Purification and characterization of human fibroblast‐derived hybridoma growth factor identical to T‐cell‐derived B‐cell stimulatory factor‐2 (interleukin‐6)
Authors: VAN DAMME Jo , CAYPHAS Sylvie , VAN SNICK Jacques , CONINGS René , PUT Willy , LENAERTS Jean‐Pierre , SIMPSON Richard J. , BILLIAU Alfons
Published: March 2005

Human fibroblast cultures, when stimulated with interleukin‐1 (IL‐1) produce a growth factor for B‐cell hybridoma and plasmocytoma cell lines. The availability of both a fast‐growing and high‐producer cell line (MG‐63 osteosarcoma cells) and of a highly sensitive and specific assay system for this hybridoma growth factor (HGF) allowed us to obtain analytically pure preparations. Crude HGF from MG‐63 cells was processed through a five‐step concentration and purification schedule. Sequential adsorption to controlled pore glass (CPG) beads, antibody affinity chromatography and gel filtration resulted in a 10000‐fold purification to a specific activity of 109 units/mg HGF. Electrophoretically pure HGF was obtained after additional purification by cation‐exchange chromatography and reversed‐phase HPLC. The purification procedure revealed two distinct biologically active HGF components. The amino‐terminal sequence of one of the two components was determined and found to correspond to that already predicted from cDNA clones of a protein alternatively called 26‐kDa protein, interferon‐β2 (IFN‐β2) or B‐cell stimulating factor‐2 (BSF‐2). The first two designations (26‐kDa protein and IFN‐β2) refer to a postulated fibroblast secretory protein with so far no unambiguously defined function; the latter designation (BSF‐2) refers to a T‐cell product possessing differentiation stimulatory effect on B‐cell lines. The reported results firmly establish that the protein is secreted by fibroblasts and reveal that it possesses B‐cell growth stimulatory activity. The new designation interleukin‐6 (IL‐6) is, proposed to resolve prescribing nomenclature confusion.
Biochemical characterization of polypeptide components involved in neurite fasciculation and elongation
Authors: WOLFF J. Michael , RATHJEN Fritz G. , FRANK Rainer , ROTH Siegfried
Published: March 2005

Polypeptide components and carbohydrate linkage types of F11 antigen and G4 antigen, two chick cell‐surface glycoproteins implicated in neurite fasciculation and elongation [Rathjen, F.G., Wolff, J.M., Bonhoeffer, F. and Rutishauser, U. (1987) J. Cell Biol. 104, 343–353], have been studied in comparison to mouse L1 antigen. Tryptic fingerprint analysis does not reveal any relation of the 130‐kDa components of G4 or F11 antigens to each other or to neural cell‐adhesion molecules. The 180/190‐kDa component of G4 antigen comprises parts of the 130‐kDa and 80/65‐kDa components and shares a sequence corresponding to the amino terminus of the G4 130‐kDa component as shown serologically with anti‐peptide sera. This closely parallels the relationship found for mouse L1 antigen components. In contrast, the F11 170‐kDa component is different from the F11 130‐kDa component, as shown serologically and by fingerprint analysis. A combination of chemical and enzymatic deglycosylation methods reveals that while O‐glycosylation cannot be detected F11 130‐kDa, G4 130‐kDa and L1 140‐kDa components contain N‐linked carbohydrates. Endoglycosidase H treatment shows that the oligosaccharides present in the G4 130‐kDa component and mouse L1 are mostly of the complex type, while the F11 130‐kDa component consists of two populations, one containing mainly complex‐type carbohydrates and a second containing high‐mannose/hybrid‐type carbohydrates.
Characterisation of oligosaccharides released from human‐blood‐group O erythrocyte glycopeptides by the endo‐β‐galactosidase of Bacteroides fragilis
Authors: SCUDDER Peter , LAWSON Alexander M. , HOUNSELL Elizabeth F. , CARRUTHERS Robert A. , CHILDS Robert A. , FEIZI Ten
Published: March 2005

Desialylated human blood group O erythrocyte glycopeptides were digested with the endo‐β‐galactosidase of Bacteroides fragilis and the enzyme‐released products reduced with NaBH4 and purified by Bio‐Gel P‐4 chromatography. Three linear and six branched oligosaccharides of poly(N‐acetyllactosamine) type, which together accounted for 90% of the oligosaccharide alditols, were characterised by fast‐atom‐bombardment mass spectrometry and gas‐liquid chromatography/mass spectrometry. Linkage and composition data were obtained for the remaining material. The salient findings were (a) the branched oligosaccharide alditols each contained the sequence:

image

and (b) there was no evidence for the terminal branch‐point sequence:

image

Together these observations indicate that, as with erythrocyte glycolipids described previously [Scudder, P., Hanfland, P., Uemura, K. & Feizi T. (1984) J. Biol. Chem. 259, 6586–6592], the endo‐β‐galactosidase of Bacteroides fragilis cannot hydrolyse branch‐point β‐galactosidic linkages on erythrocyte membrane glycopeptides.
Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder
Authors: CONLON J. Michael , DAVIS Michael S. , FALKMER Sture , THIM Lars
Published: March 2005

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast‐atom bombardment mass spectrometry. The peptides show strong homology to residues (49–60), (63–96) and (98–125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61–Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residuc processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin‐14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.
Comparative studies on phosphorylation of synthetic peptide analogue of ribosomal protein S6 and 40‐S ribosomal subunits between Ca2 +/phospholipid‐dependent protein kinase and its protease‐activated form
Authors: SAKANOUE Youichirou , HASHIMOTO Eikichi , MIZUTA Keiko , KONDO Hiroki , YAMAMURA Hirohei
Published: March 2005

Ca2 +/phospholipid‐dependent protein kinase (protein kinase C) and trypsin‐activated protein kinase C (protein kinase M) phosphorylated the synthetic peptide R1– A13 (Arg‐Arg‐Leu‐Ser‐Ser‐Leu‐Arg‐Ala‐Ser‐Thr‐Ser‐Lys‐Ala) which contains both cAMP‐ and insulin‐regulated phosphorylation sites in rat liver ribosomal protein S6 [Wettenhall, R. E. H. & Morgan, F. J. (1984) J. Biol. Chem. 259, 2084–2091]. Both enzymes showed essentially the same kinetic properties; V and apparent Km were determined to be 0.16 μmol min−1 mg−1 and 30 μM, respectively. At first, tryptic phosphopeptides were prepared at the early stage of phosphorylation and purified by high‐performance liquid chromatography (HPLC). Through these analyses, four radioactive peptides were isolated. When protein kinase C was employed, phosphorylation was observed on all four peptides in a Ca2 +/phospholipid‐dependent manner. Irrespective of the protein kinase employed, phosphate incorporation into these peptides increased linearly with time; the peptide concentration did not affect the ratio of phosphate distribution into these four peptides.

Analysis of amino acid composition and phosphoamino acid of radioactive peptides obtained after extensive phosphorylation showed that phosphates were incorporated into Ser‐4, Ser‐5, Ser‐9 and Ser‐11. The latter three serine residues were major phosphorylated sites. When rat liver 40‐S ribosomal subunits were employed as substrate for protein kinases C and M, a radioactive protein with Mr,app= 31 000, which corresponded to S6 protein, was detected on an autoradiogram of a sodium dodecyl sulfate/polyacrylamide slab gel. The rate of phosphorylation with protein kinase M was twice as fast as that with protein kinase C. The elution profile of radioactive tryptic peptides in HPLC suggest that phosphorylation occurred on the sites in S6 protein corresponding to Ser‐5, Ser‐9 and Ser‐11 as major sites and Ser‐4 as the minor one. These results indicate that protein kinase C has an ability to recognize at least four sites derived from hormone‐dependent phosphorylation sites in ribosomal protein S6 irrespective of the mode of activation of this enzyme.
Cyclic nucleotide binding to cAMP receptor protein from Escherichia coli
Authors: DONOSO‐PARDO Josefa L. , TURNER Peter C. , KING Rodney W.
Published: March 2005

cAMP receptor protein from Escherichia coli has been purified on a large scale. Analogues of cAMP modified on the 6‐NH2 group of the adenosine ring, the ribose 2′OH group or the cyclic phosphate are able to displace cAMP from its binding site with dissociation constants of similar magnitude to that of cAMP. More extensive modification produces weaker binding. Ultraviolet/visible difference spectroscopy and fluorescence spectroscopy show that the environment of the bound adenosine moiety is considerably less polar than that in aqueous solvent, while an anthraniloyl group substituted on the 2′OH position remains accessible to solvent. The 2‐NH2 group of cGMP appears to be protonated in the bound form, while no change in the charge state of cAMP is apparent.
Human protein binding to DNA sequences surrounding the human T‐cell lymphotropic virus type‐I long terminal repeat polyadenylation site
Authors: LEVINGER Louis F. , LAUTENBERGER James A.
Published: March 2005

The long terminal repeats (LTRs) of RNA tumor viruses, including human T‐cell lymphotropic virus type I (HTLV‐I), contain the control elements for expression of viral genes. Sequence‐specific LTR‐DNA‐binding proteins could regulate viral functions. To search for such proteins we have used an in vitro non‐denaturing polyacrylamide gel assay, with restriction fragments of the HTLV‐I LTR and nuclear protein extracts from several HTLV‐I‐infected cell lines and an uninfected T‐cell line, H9.

Four DNA‐binding activities were observed, including non‐specific DNA‐binding activity and at least two activities (forms I and II) which bind specifically to a HinfI restriction fragment from nucleotides + 181 to + 334 relative to the transcription start site. DNA‐binding activities I and II were partially by ion‐exchange chromatography and mapped by protection experiments to two 10–20‐bp blocks surrounding the polyadenylation site at + 221. Of the cell lines tested, form II was abundantly found in C10/MJ, and forms I and IV were also found in C91/PL, C81‐66‐45, MT2 and H9 cells.
NAD(P)+‐independent aldehyde dehydrogenase from Pseudomonas testosteroni
Authors: POELS Paulus A. , GROEN Barend W. , DUINE Johannis A.
Published: March 2005

Aldehyde dehydrogenase from Pseudomonas testosteroni was purified to homogeneity. The enzyme has a pH optimum of 8.2, uses a wide range of aldehydes as substrates and cationic dyes (Wurster's blue, phenazine methosulphate and thionine), but not anionic dyes (ferricyanide and 2.6‐dichloroindophenol), NAD(P)+ or O2, as electron acceptors. Haem c and pyrroloquinoline quinone appeared to be absent but the common cofactors of molybdenum hydroxylases were present. Xanthine was not a substrate and allopurinol was not an inhibitor. Alcohols were inhibitors only when turnover of the enzyme occurred in aldehyde conversion. The enzyme has a relative molecular mass of 186000, consists of two subunits of equal size (Mr 92000), and 1 enzyme molecule contains 1 FAD, 1 molybdopterin cofactor, 4 Fe and 4 S. It is a novel type of NAD(P)+‐independent aldehyde dehydrogenase since its catalytic and physicochemical properties are quite different from those reported for already known aldehyde‐converting enzymes like haemoprotein aldehyde dehydrogenase (EC 1.2.99.3), quinoprotein alcohol dehydrogenases (EC 1.1.99.8) and molybdenum hydroxylases.
Inhibition of Na+–Ca2+ exchange in heart sarcolemmal vesicles by phosphatidylethanolamine N‐methylation
Authors: PANAGIA Vincenzo , MAKINO Naoki , GANGULY Pallab K. , DHALLA Naranjan S.
Published: March 2005

The effect of phosphatidylethanolamine N‐methylation on Na+‐Ca2+ exchange was studied in sarcolemmal vesicles isolated from rat heart. Phosphatidylethanolamine N‐methylation following incubation of membranes with S‐adenosyl‐l‐methionine, a methyl donor for the enzymatic N‐methylation, inhibited Na+i‐dependent Ca2+ uptake by about 50%. The N‐methylation reaction did not alter the passive permeability of the sarcolemmal vesicles to Na+ and i‐dependent Ca2+ uptake by about 50%. The N‐methylation reaction did not alter the passive permeability of the sarcolemmal vesicles to Na+ and Ca2+ and did not modify the electrogenic characteristics of the exchanger. The depressant effect of phosphatidylethanolamine N‐methylation on Na+i‐dependent Ca2+ uptake was prevented by S‐adenosyl‐l‐homocysteine, an inhibitor of the N‐methylation. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino‐group‐blocking agent, also prevented methylation‐induced inhibition of Ca2+ uptake. In the presence of exogenous phospholipid substrate, the phospholipid N‐methylation process in methyl‐acetimidate‐presence of exogenous phospholipid substrate, the phospholipid N‐methylation process in methyl‐acetimidate‐treated sarcolemmal vesicles was restored and the inhibitory effect on Ca2+ uptake was evident. These results suggest that phosphatidylethanolamine N‐methylation influences the heart sarcolemmal Na+‐Ca2+ exchange system.
Partial purification and specificity studies of the d‐glutamate‐adding and d‐alanyl‐d‐alanine‐adding enzymes from Escherichia coli K12
Authors: MICHAUD Catherine , BLANOT Didier , FLOURET Bernard , VAN HEIJENOORT Jean
Published: March 2005

The d‐glutamate‐adding and d‐alanyl‐d‐alanine‐adding enzymes from Escherichia coli were partially purified by fast protein liquid chromatography on an anion exchanger. Their relative molecular masses, determined by gel filtration on Superose 12, were 54000 ± 2000 and 51000 ± 2000, respectively. In order to investigate the specificity of these ligases, several compounds derived from their respective nucleotide substrates were prepared. In the case of the d‐Glu‐adding enzyme, DDP‐MurNAc‐l‐Ala (DDP = dihydrouridine 5′‐diphosphate) and P1‐MurNAc‐l‐Ala were substrates of the reaction. In the case of the d‐Ala‐d‐Ala‐adding enzyme, only DDP‐MurNAc‐l‐Ala‐d‐Glu(‐meso‐A2pm) was a subsrate; P1‐MurNAc‐l‐Ala‐d‐Glu(‐meso‐A2pm) was neither a substrate nor an inhibitor. Concerning the amino acid site of the d‐Glu‐adding enzyme, even closely related analogues of d‐glutamate hardly inhibited the reaction.
Effects of arsenate on the Ca2+ ATPase of sarcoplasmic reticulum
Authors: ALVES Elias Walter , dE MEIS Leopoldo
Published: March 2005

The effect of arsenate on the partial reactions of the catalytic cycle of the Ca2+ ATPase of skeletal muscle of sarcoplasmic reticulum was studied. With the use of native vesicles it was found that arsenate accelerates the rate of ITP hydrolysis and inhibits both Ca2+ or Sr2+ uptake. These effects were not observed when ATP was used as substrate or, with the use of ITP, when leaky vesicles were assayed. Activation of ITP hydrolysis is related to an increase of the enzyme's apparent affinity for ITP. Arsenate increases the steady‐state level of the phosphoenzyme formed from ITP. This depends on the concentration of both Pi and Ca2+, in the medium. Ca2+ and Sr2+ efflux were accelerated by arsenate. The fast Ca2+ efflux promoted by arsenate is impaired by external Ca2+. Arsenate competes with Pi for the phosphorylating site of the enzyme.
The heme biosynthetic pathway in the regenerating rat liver
Authors: SCHOENFELD Nili , MAMET Rivka , EPSTEIN Orit , LAHAV Meir , LURIE Yoav , ATSMON Abraham
Published: March 2005

Enzymes of heme synthesis, porphyrins and heme content of regenerating rat livers were examined. During the first three days of regeneration the weights of livers of one‐third and two‐third hepatectomized rats increased 1.5‐fold and 2.7‐fold and the activity of porphobilinogen deaminase increased 2‐fold and 4‐fold and was inversely correlated with ferrochelatase activity. δ‐Aminolevulinic acid synthase and δ‐aminolevulinic acid dehydratase activities were reduced. Concomitantly an increase in the concentration of porphyrins and a decrease in that of heme were observed.

The changes in the biosynthetic pathway of heme during rapid growth of the liver are discussed.
Isolation and characterization of neurokinin A, neurokinin A(3–10) and neurokinin A(4–10) from a neutral water extract of a metastatic ileal carcinoid tumour
Authors: THEODORSSON‐NORHEIM Elvar , ANDERSSON Monica , JÖRNVALL Hans , NORHEIM Ingrid , ÖBERG Kjell , JACOBSSON Göran
Published: March 2005

A metastasis to the right liver lobe of an argyrophil/argentaffin midgut carcinoid tumour in a patient with the classical carcinoid syndrome was examined for the presence of tachykinins other than substance P, using a specific antiserum. The extract was initially purified using SepPak cartridges, and subsequently subjected to cation‐exchange chromatography on SP Sephadex C‐25 which separated the immunoreactive material into two main components (components I and II). Both were further purified by anion‐exchange chromatography on DEAE‐Sephadex A‐25, and by reverse‐phase fast protein liquid chromatography. Component II was identified as neurokinin A by its immunochemical and chromatographic properties and amino acid sequence analysis. Component I consisted of two molecular forms which were identified as neurokinin A(3–10) and neurokinin A(4–10) by amino acid sequence analysis.

The tumour tissue contained only small amounts of the eledoisin‐like peptide that has earlier been demonstrated in mammalian tissues. Although this component behaved like the nonmammalian peptide eledoisin on reverse‐phase HPLC and on reverse‐phase ion‐pair chromatography, eledoisin‐specific antiserum E2 indicated that eledoisin‐like peptide is not identical to eledoisin.

Neurokinin A in carcinoid tumours has an N‐terminal heterogeneity; this multiplicity constitutes a further support for the hypothesis that carcinoid tumours produce a number of tachykinins which may be present in different relative amounts in individual patients and may contribute to the individual differences in symptomatology.
Identification of two isoenzymes of protein phosphatase 2C in both rabbit skeletal muscle and liver
Authors: McGOWAN Clare H. , COHEN Philip
Published: March 2005

Protein phosphatase 2C was isolated from rabbit skeletal muscle by a procedure that involved chromatography on DEAE‐cellulose, precipitation with ammonium sulphate, gel‐filtration on Sephadex G‐100, affinity chromatography on thiophosphorylated myosin‐P‐light‐chain–Sepharose and chromatography on Mono Q. The enzyme was purified about 35000‐fold and 0.3–0.4 mg was isolated from 2500 g skeletal muscle within 5 days. The final step resolved the activity into two peaks, termed protein phosphatases 2C1 and 2C2, that possessed identical substrate specificities and enzymatic properties. About 2.5‐fold more protein phosphatase 2C2 was isolated than protein phosphatase 2C1. Protein phosphatases 2C1 and 2C2 migrated as single bands on SDS/polyacrylamide gels yielding apparent molecular masses of 44 kDa and 42 kDa, respectively, and the native proteins were both monomeric at pH 7.5 as judged by their elution from Sephadex G‐100 and Sephacryl S200. Peptide maps of protein phosphatases 2C1 and 2C2, obtained after separate digestions with four different proteinases, were different, indicating that they are isoenzymes.

Protein phosphatases 2C1 and 2C2 were purified from rabbit liver by the same procedure, and 0.2 mg (2C1+ 2C2) was isolated from 120 g hepatic tissue. Hepatic protein phosphatases 2C1 and 2C2 were also isolated in a molar ratio of about 1:2.5, and their enzymatic properties and apparent molecular masses in the presence and absence of SDS were identical to the skeletal muscle enzymes. Protein phosphatases 2C4 from muscle and liver displayed identical peptide maps, as did protein phosphatases 2C2 from these two tissues. It is concluded that the same two isoenzymes of protein phosphatase 2C are present in skeletal muscle and liver.
Isolation of a cDNA for adrenodoxin reductase (ferredoxin –NADP+ reductase)
Authors: HANUKOGLU Israel , GUTFINGER Tamar , HANIU Mitsuru , SHIVELY John E.
Published: March 2005

Using specific antibodies against adrenodoxin reductase (AR), we screened λ gt11 cDNA expression libraries constructed from bovine adrenal cortex mRNA, and isolated several putative clones coding for this enzyme. Concurrently we determined the amino acid sequences of fragments from it. A deoxyinosine‐containing oligonucleotide probe, generated for one of the sequences, reacted specifically with one of the cloned cDNAs of about 1600 base pairs. The codon sequence of this cDNA matched the peptide sequences, further confirming its identity as a copy of AR mRNA. RNA blot analysis indicates that in the adrenal cortex and corpus luteum there is only one major mRNA (∼ 2000 bp) for AR. The levels of this mRNA are at least 40‐fold lower in the liver and kidney which are also known to contain in homologue of AR. As compared to adrenodoxin and cytochrome P‐450scc mRNAs, AR mRNA levels in the adrenal cortex appear to be about 10‐fold lower. Southern blot analysis of bovine and human genomic DNAs reveals that in both of these species there is only one gene for AR. These results indicate that only a single reductase serves the different mitochondrial P‐450 systems in steroidogenic tissues.
Quantitative EPR of an S= 7/2 system in thionine‐oxidized MoFe proteins of nitrogenase
Authors: HAGEN Wilfred R. , WASSINK Hans , EADY Robert R. , SMITH Barry E. , HAAKER Huub
Published: March 2005

Thionine‐oxidized nitrogenase MoFe proteins from Azotobacter vinelandii. Azotobacter chroococcum and Klebsiella pneumoniae exhibit excited‐state EPR signals with g= 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20‐ 50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g= 10.4 from 12 K to 86 K, are consistent with an S= 7/2 system with spin Hamiltonian parameters D=−3.7 ± 0.7 cm−1, [E] = 0.16 ± 0.01 cm−1 and g= 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g∼ 10, ∼ 2.5 and ∼ 1.8. The S= 7/2 EPR is ascribed to the multi‐iron exchange‐coupled entities known as the P clusters. Quantification relative to the S= 3/2 EPR signal from dithionite‐reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one‐electron oxidation, the P clusters factorize into two populations, Pa and Pb, only one of which is further oxidized with thionine resulting in the S= 7/2 system. Both models require eight‐electron oxidation of tetrameric MoFe protein to reach the S= 7/2 state.
Geminate ligand recombination as a probe of the R, T equilibrium in hemoglobin
Authors: MARDEN Michael C. , HAZARD E. Starr , KIMBLE Cindy , GIBSON Quentin H.
Published: March 2005

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand.

The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb‐CO was probed with a weaker time‐delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected.

The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model‐dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states.
Hepatocyte heterogeneity in response to extracellular ATP
Authors: HÄUSSINGER Dieter , STEHLE Thomas , GEROK Wolfgang , TRAN‐THI Thuy‐Anh , DECKER Karl
Published: March 2005

The metabolic and hemodynamic effects of extracellular ATP in perfused rat liver were compared during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion. ATP in concentrations up to 100 μM was completely hydrolyzed during a single liver passage regardless of the perfusion direction.

The ATP (20 μM)‐induced increases of glucose output, perfusion pressure and ammonium ion release seen during antegrade perfusions were diminished by 85–95% when the perfusion was in the retrograde direction, whereas the amount of Ca2+ mobilized from the liver was decreased by only 60%. The maximal rate of initial K+ uptake following ATP was dependent on the amount of Ca2+ mobilized regardless of the direction of perfusion. In the presence of UMP (1 mM), an inhibitor of ATP hydrolysis by membrane‐bound nucleotide pyrophosphatase, the effect of the direction of perfusion on the glycogenolytic response to ATP (20 μM) was largely diminished.

For a maximal response of glucose output, Ca2+ release and perfusion pressure to extracellular ATP, concentrations of about 20 μM, 50 μM and 100 μM were required during antegrade perfusion, respectively. These maximal responses could also be obtained during retrograde perfusion, but higher ATP concentrations were required (120 μM, 80 μM, above 200 μM, respectively).

14CO2 production from [1‐14C]glutamate which occurs predominantly in the perivenous hepatocytes capable of glutamine synthesis was stimulated by extracellular ATP (20 μM); it was only slightly affected by the direction of perfusion. In antegrade perfusions, ATP (20 μM) increased 14CO2 production from 88 to 162 nmol g−1 min−1, compared to an increase from 91 to 148 nmol g−1 min−1 in retrograde perfusion.

The data are interpreted to suggest that (a) extracellular ATP is predominantly hydrolyzed by a small hepatocyte population located at the perivenous outflow of the acinus; (b) glycogenolysis to glucose is predominantly localized in the periportal area; (c) contractile elements (sphincters) exist near the inflow of the sinusoidal bed; (d) a considerable portion of the Ca2+ mobilized by ATP is derived from liver cells that do not contribute to hepatic glucose output.
Expression of interleukins in L cells transfected with human DNA
Authors: CORBO Laura , PIZZANO Rosa , SCALA Giuseppe , VENUTA Salvatore
Published: March 2005

We have obtained, by transfection of mouse L cells with total human DNA, clones that constitutively secreted human interleukins IL1, IL2 and B‐cell grown‐factor activities, as assessed by specific biological assays.

Southern analysis with IL2 and IL1β cDNAs confirmed the integration of the corresponding human genes in the genome of recipient mouse cells and showed their amplification and rearrangement. All the four IL2‐secreting clones integrated in the mouse genoma the human IL2 gene. Three out of the sixteen IL1‐producing clones contained the IL1β gene. The IL1 activity secreted by the remaining clones exhibited a molecular mass of 17 kDa similar to that reported for mature human macrophage IL1.

Our results demonstrate that DNA‐mediated gene transfer may represent a suitable tool for the production of human growth and differentiation factors and the cloning of their genes.
Stimulation of the erythrocyte Ca2+‐ATPase and of bovine brain cyclic nucleotide phosphodiesterases by chemically modified calmodulin
Authors: GUERINI Danilo , KREBS Joachim , CARAFOLI Ernesto
Published: March 2005

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+‐ATPase whereas the stimulation of the bovine brain cyclic‐nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic‐nucleotide phosphodiesterase. Modification of arginine residues by 1,2‐cyclohexanedione had no effect on the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N‐chlorsuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C‐terminal half of calmodulin are more important for the interaction with the Ca2+‐ATPase.
Molecular cloning and sequence determination of the tuf gene coding for the elongation factor Tu of Thermus thermophilus HB8
Authors: KUSHIRO Akira , SHIMIZU Masato , TOMITA Ken‐ichi
Published: March 2005

The tuf gene, which encodes the elongation factor Tu (EF‐Tu) of Thermus thermophilus HB8, and its flanking regions were cloned and sequenced. The gene encoding EF‐G was found upstream of the 5′ end of the tuf gene. The tuf gene of T. thermophilus HB8 had a very high G + C content and 84.5% of the third base in codon usage was either G or C. The deduced primary structure of the EF‐Tu was composed of 405 amino acid residues with a Mr= 44658. A comparison of the amino acid sequence of EF‐Tu from T. thermophilus HB8 with those of Escherichia coli and Saccharomyces cerevisiae mitochondria showed a very high sequence homology (65–70%). Two Cys residues out of the three found in E. coli EF‐Tu had been replaced with Val in T. thermophilus HB8 EF‐Tu. An extra amino acid sequence of ten residues, consisting predominantly of basic amino acids (Met‐182–Gly‐191), which does not occur in EF‐Tu of E. coli, was found in T. thermophilus HB8.
Rabbit apolipoprotein A‐I mRNA and gene
Authors: PAN Te‐cheng , HAO Qian‐lin , YAMIN Ting‐Ting , DAI Pei‐Hua , CHEN Bao‐sheng , CHEN Shiow‐ling , KROON Paulus A. , CHAO Yu‐sheng
Published: March 2005

In order to study the tissue‐specific expression of rabbit apolipoprotein (apo) A‐I, a 923‐base‐pair clone, pRBA‐502, complementary to rabbit apo A‐I mRNA was identified from a rabbit intestinal cDNA library by hybrid‐select translation and immunoprecipitation methods. Northern blot and dot‐blot hybridization, utilizing 32P‐labeled pRBA‐502, revealed that the rabbit apo A‐I gene is expressed in the intestine, not in the liver and that rabbit apo A‐I mRNA is about 950 nucleotides in length. The entire nucleotide sequence of pRBA‐502 has been determined and the complete amino acid sequence of the corresponding apo A‐I has been deduced. The mRNA codes for a protein comprising 265 amino acids. Amino acids 1–18 and 19–24 of the primary translation product represent the presegment and prosegment, respectively, of apo A‐I. Matured rabbit apo A‐I contains 241 amino acids and has a molecular mass of 27612 Da. Using pRBA‐502 as a probe, a 15.5‐kb genomic fragment, which contains the entire apo A‐I gene, was isolated from a rabbit liver genomic library. Sequence analysis of the gene shows that the 200 base pairs of the 5′ upstream flanking region of the rabbit and human apo A‐I genes showed 78% sequence homology. Like the human apo A‐I gene, the rabbit apo A‐I gene is interrupted by three intervening sequences. Except for two nucleotides in the fourth exon, the coding sequence of the rabbit liver apo A‐I gene is identical to that of pRBA‐502. Our data showed that the lack of expression of apo A‐I gene in rabbit liver is not due to the alternation of rabbit liver apo A‐I gene sequence and suggest that the expression of apo A‐I gene in rabbit liver is regulated by a trans‐acting regulating element(s).
Isolation, characterization and amino‐acid sequence of γ‐seminoprotein, a glycoprotein from human seminal plasma
Authors: SCHALLER Johann , AKIYAMA Kazuko , TSUDA Ryouichi , HARA Mitsuwo , MARTI Thomas , RICKLI Egon E.
Published: March 2005

γ‐Seminoprotein (γ‐SM), a glycoprotein from human seminal plasma, was isolated in highly purified form by ion‐exchange chromatography on a Mono Q column. The main form, fraction M, was homogeneous by PAGE at pH 8.3 and by SDS‐PAGE. The complete amino acid sequence of γ‐SM was determined with the aid of fragments generated by cleavages with cyanogen bromide, clostripain, chymotrypsin and Staphylococcus aureus V8 protease. The fragments were aligned with overlapping sequences. The single polypeptide chain of γ‐SM contains 237 amino acids with a calculated Mr of 26079. A single N‐linked carbohydrate side chain is attached to Asn45. The complex structure of this oligosaccharide has been determined recently [van Halbeek H. et al. (1985) Biochem. Biophys. Res. Commun. 131, 507–514]. Sequence comparison with serine proteases shows a high degree of homology, especially with the kallikrein family. The residues in the vicinity of the active site of serine proteases are also highly conserved in γ‐SM, indicating the participation of His41, Asp96 and Ser189 in its active site.

γ‐SM hydrolyzed M‐casein with a pH optimum at 8.0, but failed to hydrolyze any of the synthetic substrates tested. This proteolytic activity could be inhibited with diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, Zn2+ or Hg2+ ions.
Heterogeneity of insulin‐like growth factor binding proteins and relationships between structure and affinity
Authors: HARDOUIN Sylvie , HOSSENLOPP Paul , SEGOVIA Berta , SEURIN Danielle , PORTOLAN Ghyslaine , LASSARRE Claudine , BINOUX Michel
Published: March 2005

Circulating insulin‐like growth factors (IGFs) are bound to specific, high‐affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150000 (‘large’ complex) and 40000 (‘small’ complex). The large complex appears to be under growth‐hormone control and its proportions vary with those of the IGFs.

Molecular heterogeneity among the binding proteins was revealed by polydcrylamide gel electrophoresis (SDS‐PAGE) of serum in which they were cross‐linked to 125I‐labelled IGF I or II. Out of the six specific bands observed, of 150000, 120000, 49000, 46000, 40000 and 37000 Mr, the last three appeared in both complexes, whereas the first three were visible only in the large complex. Some or all of the 49000–37000‐Mr species may constitute the subunits of 150000‐Mr and/or 120000‐Mr IGF‐BP complexes.

With electrophoresis followed by transfer onto nitrocellulose and incubation with either 125I‐labelled IGF I or II (western blot), the different binding proteins were identified per se. There were five molecular forms with Mr of 41 500, 38 500, 34000, 30000 and 24000. In normal serum the 41 500 and 38 500‐Mr forms were the major binding proteins. They appeared in both complexes, but were predominant in the large complex where they constitute the elementary binding units. These two proteins therefore bind to IGFs to form both ‘monomeric’ IGF‐BP and ‘oligomeric’ (IGF‐BP)n complexes. The 34000, 30000 and 24000‐Mr forms, by contrast, were visible only in the small complex. Different mechanisms appear to regulate the different binding proteins: in acromegalic serum the 41 500 and 38 500‐Mr forms were augmented and the 34000‐Mr form diminished, whereas in hypopituitary serum the reverse was true and, in addition, the 30000‐Mr form was augmented.

With chromatofocusing, the 34000, 30000 and 24000‐Mr forms eluted in three peaks between pH 6.0 and 4.0, whereas the 41 500 and 38 500‐Mr forms eluted throughout the gradient, principally at pH 7.5 and 7.0.

Competitive binding studies, done on binding proteins separated either by chromatofocusing or by SDS‐PAGE and transfer onto nitrocellulose, revealed different affinities for the IGFs among the different molecular forms. The 41 500 and 24 000‐Mr binding proteins preferentially bound IGF I and the 38 500, 34 000 and 30 000‐Mr proteins preferentially bound IGF II.

Our findings demonstrate the molecular heterogeneity of the binding proteins and the existence of a relationship between their structure and their affinities for the IGFs. They also suggest that, apart from their function as IGF carriers, the binding proteins also play a modulating role in the interaction between IGFs and their target cells.
Semi‐preparative HPLC purification of ribosomal proteins from Bacillus stearothermophilus and sequence determination of the highly conserved protein S19
Authors: HIRANO Hisashi , ECKART Klaus , KIMURA Makoto , WITTMANN‐LIEBOLD Brigitte
Published: March 2005

Several proteins from the Bacillus stearothermophilus 30S ribosomal subunit which could not be isolated by conventional open‐column chromatography were purified by high‐performance liquid chromatography using a semi‐preparative reverse‐phase C4 column. Protein S19 was purified by this technique and the complete amino acid sequence determined. Protein S19 was fragmented and the peptides isolated in picomole quantities were sequenced by an improved manual 4‐N,N‐dimethylaminoazobenzene‐4′‐isothiocyanate (DABITC) technique; the presence of five consecutive C‐terminal lysines in the S19 sequence was confirmed by gas‐phase sequencing and fast‐atom‐bombardment (FAB) mass spectrometry.

Protein S19 is composed of 91 amino acid residues which correspond to a molecular mass of 10428 Da. 71% of the B. stearothermophilus S19 sequence was found to be identical with the corresponding ribosomal protein from Escherichia coli [Yaguchi and Wittmann (1978), FEBS Lett. 88, 227] and both sequences can be aligned without gaps. Among the known 26 amino acid sequences of the B. stearothermophilus and E. coli ribosome such a high degree of conservation has only been observed for a few proteins, all of which are known to be involved in the protein biosynthesis process. Although a clear function has not yet been assigned to protein S19, its high sequence conservation in these two eubacteria clearly indicates an important role of this protein for the function of the ribosome.