bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purified Human Somatomedin A and Rat Multiplication Stimulating Activity
Authors: RECHLER Matthew M. , FRYKLUND Linda , NISSLEY S. Peter , HALL Kerstin , PODSKALNY Judith M. , SKOTTNER Anna , MOSES Alan C. 
Published: March 2005

We have compared the mitogenic activity and reactivity in receptor binding assays of somatomedin A and multiplication‐stimulating activity (MSA), two acid‐soluble polypeptides of molecular weight 7–10 000 that possess weak intrinsic insulin‐like metabolic activity. Somatomedin A was purified to homogeneity from human plasma, and MSA was purified to homogeneity from the conditioned culture media of a cell line, BRL 3A, derived from rat liver. The two peptides stimulated DNA synthesis in chick embryo fibroblasts 3–4‐fold and have superimposable dose‐response curves. Addition of either peptide to chick embryo fibroblasts plated in serum‐free medium increased the cell number by 150% in 4 days, compared with an increase of 25% in control cultures receiving no additions. The two peptides also stimulated DNA synthesis in human fibroblasts in culture 3–4‐fold.Specific receptors for MSA and/or somatomedin A could be demonstrated in intact cells or membranes from chick embryo fibroblasts, human fibroblasts, human placenta, rat liver, and the BRL 3A2 cell line, a subclone of the line that produces MSA. Unlabeled MSA and somatomedin A inhibited the binding of 125I‐labeled MSA and 125I‐labeled somatomedin A to each of these receptors with comparable potency. In chick embryo fibroblasts, human fibroblasts, and human placental membranes, the binding of both radioactive ligands also was inhibited by insulin, consistent with the interpretation that 125I‐labeled MSA and 125I‐labeled somatomedin A were binding to the same receptor. By contrast, in the BRL 3A2 cell line, insulin inhibited the binding of 125I‐labeled somatomedin A, but not the binding of 125I‐labeled MSA, suggesting that the two labeled peptides were binding to different receptors in this cell line. Moreover, 125I‐labeled MSA, but not 125I‐labeled somatomedin A, bound specifically to rat liver plasma membranes. These results indicate that human somatomedin A and rat MSA are closely related, but not identical, peptides. They have similar mitogenic activity for cultured fibroblasts, and cross‐react with specific cell receptors with comparable potency. The two radioactively labeled peptides, however, can be distinguished by certain cell surface receptors for somatomedin‐like peptides.
Binding, Uptake and Degradation of the Toxic Proteins Abrin and Ricin by Toxin‐Resistant Cell Variants
Authors: SANDVIG Kirsten , OLSNES Sjur , PIHL Alexander 
Published: March 2005

Ricin‐resistant variants derived from HeLa cells and baby hamster kidney (BHK) cells had a lower number of toxin‐binding sites than the sensitive parent lines, while the apparent association constants between the toxins and the surface receptors were almost the same. In the ricin‐resistant cell lines a much greater number of bound toxin molecules was required to inhibit protein synthesis by 50% after 3 h than in the parent lines, indicating that in addition to a reduced number of toxin‐binding sites other factors are involved in the reduced sensitivity of the variants to the toxins. Apparently, the toxin resistance involves an impairment of the transfer of the toxin A‐chain from the cell surface to the target, the ribosomes.Neuraminidase treatment of the various cell lines increased the number of binding sites for abrin and ricin, particularly in the resistant variants having originally a reduced number of binding sites and increased amount of sialic acid. The sensitivity of the neuraminidase‐treated cells to protein synthesis inhibition by abrin and ricin increased concomitantly with the increase in the number of binding sites. In most of the cell lines the number of toxin molecules bound to the cells when protein synthesis was decreased by 50% was about the same as before the neuraminidase treatment. The results indicate that in these cells the new binding sites appearing after neuraminidase treatment are as efficient as the normally occurring ones in mediating the uptake of the toxins.Most of the toxin bound to the cells may be washed off with 0.1 M lactose. However, after incubation at 37°C a certain fraction could not be released by this treatment. Subsequent treatment of these cells with 125I‐labelled antibodies to the toxins showed that the lactose‐resistant toxins were not accessible to the antibody and probably were internalized.Studies with 125I‐labelled abrin and ricin showed that the cell‐bound toxins are slowly degraded by the cells and that they are partly excreted in an apparently intact form into the medium. No evidence was obtained that the degradation of toxin was greater in resistant than in sensitive cell lines.The data indicate that the uptake of toxins by pinocytosis in two ricin‐resistant variants occurs at the same rate as the parent HeLa cells. In a ricin‐resistant variant of BHK cells it was reduced to about half of the control value. The possible role of pinocytosis in the intoxication process is discussed.
Control of Globin Synthesis by the Haemin‐Controlled Translational Repressor in a Fractionated Cell‐Free System from Rabbit Reticulocytes
Authors: SARRE Thomas , HILSE Kurt 
Published: March 2005

The control of protein synthesis by haemin in rabbit reticulocyte lysates is mediated by a translational repressor protein. We have developed a fractionated globin‐synthesizing system from rabbit reticulocyte lysates that is free of endogenous repressor and therefore well suited to study the action of purified translational repressor and its antagonism to haemin.The major components of this system are: (a) rabbit reticulocyte polysomes, (b) a mouse liver pH‐5 enzyme as the source of all components for peptide chain elongation, and (c) a fraction pelleted from the rabbit reticulocyte postpolysomal supernatant by ultracentrifugation (named 200 000 ×g fraction).Crude rabbit reticulocyte lysates, when stimulated to extensive globin synthesis by haemin, can be inhibited by preparationes of purified translational repressor in its haemin‐irreversible form. In contrast, globin synthesis directed by isolated polysomes is only slightly affected by the addition of purified repressor. Addition of the 200 000 ×g fraction to polysomes produces a strong stimulation of globin synthesis that is severely inhibited by the translational repressor. If the haemin‐reversible form of the repressor is used, this inhibition can be prevented by the addition of haemin. Thus, the fractionated system shows the same effects observed with crude rabbit reticulocyte lysates and, moreover, supplies a convenient tool for the purification of the translational repressor in its haemin‐reversible form.Addition of the 200 000 ×g fraction to a protein‐synthesizing system from Ehrlich ascites tumour cells reveals the existence of α‐globin mRNA within this fraction. As determined by sucrose gradient analysis, the 200 000 ×g fraction contains two major components sedimenting at 50 S and 80 S, respectively. In view of the results presented here, the nature of these two components and their relevance to the observed features of the 200 000 ×g fraction is discussed.
Riboflavin Binding in Egg‐White Flavoprotein: the Role of Tryptophan and Tyrosine
Authors: BLANKENHORN Gunter 
Published: March 2005

In egg‐white apoflavoprotein six of the nine tyrosines and three of the nine tryptophans have been shown to be accessible to ethylene glycol using solvent perturbation difference spectroscopy. For egg‐yolk flavoprotein the same ratio of exposed tyrosines to tryptophans was found, but the native protein structure proved to be more stable against denaturation by solvent.A single tryptophan and a single tyrosine have been demonstrated to be essential for binding of riboflavin by a combination of chemical modification and affinity chromatography techniques. Two of the exposed tyrosines and two of the exposed tryptophans could be modified without loss of binding capacity. Only the essential tyrosine was found to be protected by bound riboflavin against nitration with tetranitromethane, whereas the essential tryptophan was found not to be protected against alkylation by dimethyl‐(2‐hydroxy‐5‐nitrobenzyl)‐sulfonium bromide.Indocyanine green, a hydrophobic probe, binds to egg‐white flavoprotein, with a Kd of 10−6 M, most probably at the cofactor binding site. An intramolecular dihydroflavin · nicotinamide complex is bound to the apoprotein (Kd∼ 10−4 M) in a geometry still allowing charge transfer interaction between dihydro‐flavin and nicotinamide.These data lead us to propose that egg‐white flavoprotein binds riboflavin at a hydrophobic site near to or at the suface of the protein. Tyrosine is probably involved in a stacking interaction with riboflavin and tryptophan is also essential for binding. The dimethylbenzene part of the flavin nucleus appears to be buried in egg‐white flavoprotein in contrast to flavodoxins where it has been shown to be exposed.
Sequence at the Site of Attachment of an Affinity‐Label Derivative of Puromycin on 23‐S Ribosomal RNA of Escherichia coli Ribosomes
Authors: ECKERMANN David J. , SYMONS Robert H. 
Published: March 2005

The puromycin analogue, 5′O‐(N‐bromoacetyl‐p‐aminophenylphosphoryl)‐3′‐N‐L‐phenylalanyl puromycin aminonucleoside, acts as an affinity label in covalently binding to 23‐S RNA in the active centre of peptidyl transferase of Escherichia coli ribosomes [Greenwell, P., Harris, R. J. & Symons, R. H. (1974) Eur. J. Biochem. 49, 539–554]. The sequence of a pentanucleotide fragment of 23‐S RNA to which the affinity label is attached has now been determined.In an attempt to determine the nucleotide residue to which the affinity label was attached, a 32P‐labelled affinity label was reacted with ribosomal RNA, poly(A), poly(C), poly(G) and poly(U) under the conditions used for affinity labelling of ribosomes. However, no specific reaction occurred, confirming our earlier observation that this required the functional integrity of the ribosome. An alternative approach of affinity labelling ribosomes labelled by incubating E. coli in the presence of [3H]adenosine, [3H]cytidine, [3H]guanosine or [3H]uridine, showed that the affinity label was attached to a CMP residue.Affinity‐labelled oligonucleotide fragments were purified for sequence analysis as follows. The free α‐amino group of the affinity‐label molecule attached to 23‐S ribosomal [32P]RNA was coupled to biotin by reaction with the N‐hydroxysuccinimide ester of biotin. After digestion of the RNA with either ribonuclease A or T1, the basic protein avidin was added to form the avidin · biotin – affinity‐label‐oligonucleotide fragment. Stepwise chromatography on phosphocellulose, followed (after dissociation of the avidin) by electrophoresis on DEAE‐cellulose at pH 1.9 gave substantial purification of the biotin–affinity‐label‐oligonucleotide. From the nucleotide composition of the purified ribonuclease A and T1 fragments, the pentanucleotide sequence of G‐U‐C*‐C‐Gp was obtained, where C* represents C modified by the affinity label.
Subcellular Distribution of a Factor Inactivating Tyrosine Aminotransferase
Authors: BENEKING Margrit , SCHMIDT Helga , WEISS Gerhard 
Published: March 2005

The subcellular distribution of a tyrosine aminotransferase inactivating factor in rat liver has been investigated. Most of its activity is associated with plasma membranes, with minor amounts in mitochondria and endoplasmatic reticulum.The factor is also found in kidney and inactivates the enzyme reversibly in presence of cysteine, most likely by modification of −SH groups.ATP counteracts this inactivation only, when crude enzyme extracts are inactivated by purified subcellular fractions or when the purified enzyme is inactivated in presence of liver or kidney cortex homogenates.The relationship of this inactivation to reported different forms of the enzyme has been investigated. Form I of three different forms, that can be obtained by hydroxyl‐apatite chromatography, is readily inactivated, form III can be partly converted to form I by incubation in presence of purified plasma membranes.The relationship of these findings to a possible multistep mechanism in the turnover of the enzyme is discussed.
Induction and ‘Superinduction’ of Sialylation of Membrane‐Bound γ‐Glutamyltransferase during Liver Regeneration
Authors: KÖTTGEN Eckart , REUTTER Werner , GEROK Wolfgang 
Published: March 2005

The present paper shows that in the regenerating rat liver the membrane‐bound γ‐glutamyltransferase exists in two molecular forms. Depending on the state of proliferation, a sialic‐acid‐rich enzyme (in the fetal or regenerating liver) or a sialic‐acid‐poor enzyme (in the adult or quiescent liver) could be detected. In regenerating liver (24 h after 2/3 resection) only the sialic‐acid‐rich or fetal enzyme could be found. Since total enzyme activity (adult + fetal type) remained unchanged, it is assumed that the adult type of γ‐glutamyltransferase was modified by sialylation during the initial phase of liver regeneration.This process of sialylation was prevented by inhibitors of RNA or protein synthesis such as D‐galactosamine, actinomycin D or cycloheximide, provided that the inhibitor (D‐galactosamine) was given within the first 8 h after partial hepatectomy. Sialylation was not impaired by inhibitors of DNA synthesis, e.g. hydroxyurea or cytosine arabinoside.Administration of actinomycin D during a defined phase of proliferation (24 to 48 h after partial hepatectomy) stimulated the transfer of sialic acid to γ‐glutamyltransferase, a finding which describes for the first time the so‐called ‘superinduction’ of a sialylation process.
Effect of Polyamines on Isoleucyl‐tRNA Formation by Rat‐Liver Isoleucyl‐tRNA Synthetase
Authors: IGARASHI Kazuei , EGUCHI Kuniko , TANAKA Makoto , HIROSE Seiyu 
Published: March 2005

The effect of polyamines on rat‐liver isoleucyl‐tRNA formation was studied using isoleucyl‐tRNA synthetase purified by column chromatography successively on Sephadex G‐200, DEAE‐Sephadex A‐25, and tRNA‐Sepharose 4B. In the presence of 50 mM K+, isoleucyl‐tRNA formation was inhibited markedly by 1.5 mM or higher concentrations of Mg2+. However, the addition of spermine to the reaction mixture prevented the inhibitory effect of Mg2+. In the presence of 200 mM K+, the addition of spermine to the reaction mixture stimulated isoleucyl‐tRNA formation in the presence of Mg2+ concentrations from 0 to 5 mM. Although the effective concentration was different, spermidine exhibited a similar stimulative effect. The effective concentration of spermine required for stimulation was higher when larger amounts of tRNA were used.The stimulatory effect of isoleucyl‐tRNA formation by polyamines was shown to reflect on polypeptide synthesis. When formaldehyde‐treated poly (A, U) was used as messenger RNA, polypeptide synthesis from amino acids was stimulated by polyamines, but that from aminoacyl‐tRNAs was not stimulated by polyamines.
Fatty Acid, 3‐β‐Hydroxysterol, and Ketone Synthesis in the Perfused Rat Liver
Authors: BRUNENGRABER Henri , BOUTRY Mireille , LOWENSTEIN John M. 
Published: March 2005

The effects of oleate and hydroxycitrate on the rate of long‐chain fatty acid and 3‐β‐hydroxysterol synthesis were measured in perfused rat livers. Metabolite measurements show that in livers from fed animals inhibition of fatty acid synthesis by oleate or hydroxycitrate is associated with an increase in the tissue content of glucose 6‐phosphate and fructose 6‐phosphate, and a diminution in glycolytic intermediates from fructose diphosphate to phosphoenolpyruvate. Oleate also causes an increase in the tissue content of long‐chain fatty acyl‐CoA and citrate. The increase in long‐chain fatty acyl‐CoA is larger in livers from starved as compared to fed rats, while the increase in citrate is larger in livers from fed as compared to starved rats. However, the increase in the citrate content of livers from fed rats occurs in a range where it causes no further activation of acetyl‐CoA carboxylase in vitro.Ketogenesis by livers from fed rats perfused without free fatty acids is strongly inhibited by hydroxycitrate. However, ketogenesis is not inhibited by hydroxycitrate when livers from starved rats are perfused with oleate, and ketogenesis is increased somewhat by hydroxycitrate when livers from fed rats are perfused with oleate. These results are interpreted in terms of an extramitochondrial pathway of ketogenesis which operates in carbohydrate‐fed animals. The intramitochondrial pathway predominates in starved animals, or when the concentration of fatty acids is high, or both. Other interpretations, which cannot be ruled out at present, are also considered.
Biosynthesis of Stizolobinic Acid and Stizolobic Acid in Higher Plants
Authors: SAITO Koshi , KOMAMINE Atsushi 
Published: March 2005

Two new enzymes, which catalyze the synthesis of stizolobinic acid and stizolobic acid through oxidative ring cleavage of 3,4‐dihydroxyphenylalanine, subsequent recyclization and dehydrogenation of the resultant possible products were isolated from the soluble protein extract of Stizolobium hassjoo seedlings and were purified approximately 100‐fold by ammonium sulfate fractionation followed by column chromatography on Sephadex G‐100, DEAE‐cellulose and hydroxyapatite. The apparent molecular weights of both enzymes as determined by Sephadex G‐100 column chromatography were 45 000. The pH optima were 7.6 and 7.4, apparent Km values for 3,4‐dihydroxyphenylalanine as substrate were 1.67 mM and 1.39 mM for the enzymes to synthesize stizolobinic acid and stizolobic acid, respectively. Both enzymes required NADP+ for the activities. They were activated by Zn2+ and inhibited by sulfhydryl binding and zinc chelating agents. Both exhibited a strict specificity for 3,4‐dihydroxyphenylalanine as substrate and showed no affinities for other o‐dihydric phenols tested. The enzymes were very labile with a half‐life of 3–5 h at 30°C but they could be stored at −20°C for about 3 months under nitrogen gas with 0.1 mM dithiothreitol without appreciable loss of the catalytic enzyme activities.These enzymes have never been reported in any biological material. The trivial names, stizolobinic acid synthase and stizolobic acid synthase, are proposed.
A Characterization of Alfalfa‐Mosaic‐Virus Protein Polymerization in the Presence of Nucleic Acid
Authors: DRIEDONKS René A. , KRIJGSMAN Peter C. J. , MELLEMA Jan E. 
Published: March 2005

The polymerization of alfalfa mosaic virus (AMV) protein in the presence of homologous nucleic acids and a number of other natural and synthetic nucleic acids was studied. The conditions for optimal assembly were found to be pH 6.0 and low ionic strength (I= 0.1 M) at room temperature, irrespective of the type of nucleic acid. The resulting nucleoprotein particles exhibited the same structural characteristics as the virus. This information emerged from optical diffraction and computer filtering of electron micrographs from the reconstituted particles.Irrespective of the type of nucleic acid present the polymerization of the protein resulting in a nucleoprotein particle is a cooperative process. Evidence for this was obtained by nitrocellulose filter binding assay, sodium dodecylsulphate/polyacrylamide gel electrophoresis, sedimentation velocity and electron microscopy of the reaction mixtures. The rates and efficiencies of reconstitution were of the same order of magnitude for a number of ribonucleic acids. Sedimentation data derived from AMV protein and AMV RNA mixtures suggested the existence of a specific nucleation product in the first stage of assembly. The results are discussed in terms of a tentative model of the assembly, in which at least two different steps (nucleation and elongation) can be distinguished, each characterized by an association constant.
Protease II from Escherichia coli
Authors: PACAUD Michele 
Published: March 2005

Protease II, a cytoplasmic endopeptidase from Escherichia coli, has been further characterized. In agreement with previous evidence of a trypsin‐like specificity, this enzyme cleaves essentially the carboxymethylated B chain of insulin at the Arg22‐Gly23 bond, but after prolonged periods of incubation it is also able to cleave the Tyr16‐Leu17 bond. Protease II and pancreatic trypsin are inhibited in a similar fashion by diisopropylphosphofluoridate and tosyl lysine chloromethyl ketone suggesting that seryl and histidyl groups are commonly involved in the active site of the two enzymes.The analysis of the pH dependence of the steady‐state kinetic parameters of two related substrates. N‐benzoyl‐L‐arginine ethyl ester and N‐benzoyl‐L‐arginine p‐nitroanilide, indicates that two ionizing groups are present in the enzyme's active site, one has a pK(app) of 6.7–7.5, the other has a pK(app) of 8.7–9.6. These two enzyme groups control the binding of substrates, but the group with a pK around 9 does not appear to be essential for the catalysis. The assignment of these pK values is discussed in connection with the known features of the reaction mechanism of serine proteases. Attempts to obtain an insight into the catalytic mechanism from the kinetic analysis of the effects of added nucleophiles were unsuccessful.No ‘burst’ release of p‐nitrophenol is observed from the hydrolysis of p‐nitrophenyl‐p′‐guanidobenzoate. Deuterium isotope effects for kcat/Km range from 1 for N‐benzoyl‐L‐arginine ethyl ester to 1.5 for N‐benzoyl‐L‐arginine amide. Furthermore, the kinetics of the protease‐II‐catalyzed hydrolysis of various synthetic substrates are characterized by relatively weak variations in the values of Km(app), but significant differences in the values of kcat are observed with closely related amide and ester substrates. This implies that Km may be related to Ks, and that the step immediately following formation of the Michaelis enzyme‐substrate complex in the catalytic process, is rate‐determining.These findings do not agree with the acyl‐enzyme mechanism established for pancreatic serine hydrolases and α‐lytic protease.
Alterations in tRNAs Containing 2‐Methylthio‐N6‐(Δ2‐isopentenyl)‐adenosine during Growth of Enteropathogenic Escherichia coli in the Presence of Iron‐Binding Proteins
Authors: GRIFFITHS Elwyn , HUMPHREYS Janice 
Published: March 2005

Escherichia coli grown in chemically produced iron‐deficient media have well characterized alterations in the chromatographic properties of tRNAs containing the modified nucleoside 2‐methylthio‐N6‐(Δ2‐isophentenyl) adenosine. The present report shows that similar tRNA alterations occur in enteropathogenic E. coli inhibited by human milk and bovine colostrum, the inhibited bacterial containing 10% or less of the normal tRNA species. Adding sufficient iron to saturate the ironbinding capacity of the lactoferrin present in milk and colostrum reversed these changes which are probably due to a failure to methylthiolate the isopentenyladenosine.Although adding iron led to a rapid replacement of abnormal tRNA by the chromatographically normal species, and to a resumption of multiplication, the tRNA alterations are not directly related to the inhibition of growth. Strains of E. coli which grew normally in milk, colostrum and in defined media containing the iron‐binding protein transferrin or ovotransferrin also contained about 90% of the abnormal species. Rapid conversion of abnormal tRNA to normal tRNA occurred on adding iron and in the absence of RNA synthesis.The tRNA changes are discussed in relation to their possible connection with both the adaptation of E. coli to growth under the iron‐restricted conditions imposed by iron‐binding proteins in tissue fluids and with bacterial pathogenicity.