bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
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.
Purification of a new acidic glutathione S‐transferase, GST‐Yn1 Yn1, with a high leukotriene‐C4 synthase activity from rat brain
Authors: TSUCHIDA Shigeki , IZUMI Takashi , SHIMIZU Takao , ISHIKAWA Takashi , HATAYAMA Ichiro , SATOH Kimihiko , SATO Kiyomi 
Published: March 2005

A new acidic form of glutathione S‐transferase (GST, pI 6.2) was purified from rat brain by S‐hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20–25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6‐6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2‐dichloro‐4‐nitrobenzene and immunological properties. On two‐dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST‐MT. Therefore, the brain acidic form is a homodimer, and named GST‐Yn1 Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene‐C4 synthase. This form (GST‐Yn1Yn1) showed the highest leukotriene‐C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 μM and 3.5 mM respectively.A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST‐P (7–7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST‐P also showed the significant leukotriene‐C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 μM, fourfold higher than that of GST‐Yn1 Yn1. These results suggest that mainly GST‐Yn1 Yn1 may be involved in leukotriene‐C4 synthesis in rat brain.
Microquantitative determination of intra‐acinar distribution profiles of low‐Km and high‐Km aldehyde dehydrogenase activity in rat liver
Authors: MALY Ireneusz Piotr , SASSE Dieter 
Published: March 2005

Microquantitative measurements of total and of low‐Km aldehyde dehydrogenase (ALDH) activity with millimolar and micromolar concentrations of acetaldehyde and propionaldehyde were carried out on the livers of male and female rats. Lyophilized cryostat sections of liver parenchyma were microdissected along the entire sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ALDH activity was measured in a microbiochemical assay using the oil‐well technique with luminometric determination of NADH. On the basis of single measurements, mean values of total, low‐Km and high‐Km ALDH activity could be calculated and the specific distribution patterns graphically demonstrated.The two substrates acetaldehyde and propionaldehyde yielded similar values of ALDH activity, the intra‐acinar distribution profiles of which showed characteristic sex differences. In the liver of the male rat high‐Km ALDH activity has two flat peaks in the periportal and the perivenous area, while low‐Km ALDH activity is almost evenly distributed throughout the acinus. In the livers of female rats, both high‐Km and low‐Km ALDH activity shows a continuous gradient which decreases from the periportal to the perivenous zone (pp/pv = 1.4:1). It was therefore possible to demonstrate that the maxima of alcohol dehydrogenase activity and of low‐Km ALDH activity are localized in opposite parts of the liver acinus of the female rat. This heterotopy should have consequences with respect to hepatotoxicity after alcohol ingestion.