bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Structural Studies on Yeast 3‐Phosphoglycerate Kinase
Authors: FATTOUM Abdellatif , ROUSTAN Claude , FEINBERG Jeanne , DESVAGES Gisèle , PRADEL Louise‐Anne
Published: March 2005

Cyanogen bromide cleavage of yeast 3‐phosphoglycerate kinase yielded four fragments which account for the amino acid composition of the entire molecule. These results are consistent with a single polypeptide chain of molecular weight 42000. Affinity chromatography on Sepharosemercurial followed by gel filtration on Sephadex was used with success for separation of peptides. The carboxyl and N‐terminal fragments were characterized. The N‐terminal fragment contained the single cysteinly residue of the protein. After cyanylation and subsequent cleavage, this cysteinyl residue was located near position 100.
Optical Spectroscopic Study of the ADP‐Myosin Interaction
Authors: MARSH David J. , d'ALBIS Anne , GRATZER Walter
Published: March 2005

The binding of ADP to heavy meromyosin, and the separated subfragment 1 components S‐1 (A1) and S‐1 (A2), has been observed by ultraviolet spectrophotometry. The results are compatible with the presence of spectroscopically equivalent and independent sites, one per head, at both 10°C and 25°C. We do not observe the heterogeneity of binding and of the spectroscopic response that has been reported. The binding has also been followed by other methods sensitive to the effect of ligand on the aromatic residues of the protein, viz. intrinsic fluorescence of heavy meromyosin and changes in the near‐ultraviolet Cotton effects of myosin, and its active fragment. Within the limits of our experimental precision, the binding profiles, based on concentration of myosin heads, are the same for myosin as for subfragment 1. A perturbation in the circular dichroism is also generated by pyrophosphate, which compets with ADP. The spectra suggest that subsites for the purine ring and the diphosphate can be recognised. The sensitivity of binding profiles obtained by methods of the kind used here to cooperative or antagonistic interactions between the binding sites has been analysed. It is clear that sizeable effects of this nature could be concealed by the binding curves, even for high experimental precision.
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.
Separation and Characterization of Two Long‐Chain Acyl‐CoA Synthetases from Candida lipolytica
Authors: MISHINA Masayoshi , KAMIRYO Tatsuyuki , TASHIRO Shin‐Ichi , NUMA Shosaku
Published: March 2005

Two activities of long‐chain acyl‐CoA synthetase have been demonstrated in the particulate fraction derived from cells of Candida lipolytica. One is observed both in oleic‐acid‐grown and in glucose‐grown cells and is independent of phosphatidylcholine. The other is found only in oleicacid‐grown cells and is dependent on phosphatidylcholine. The phosphatidylcholine‐independent activity is more resistant to heat or Triton X‐100 than the phosphatidylcholine‐dependent activity. Moreover, the phosphatidylcholine‐independent activity is readily resolved from the particulate fraction by Triton X‐100 treatment, whereas the phosphatidylcholine‐dependent activity remains in the particulate fraction after this treatment. Thus, the two activities have been separated from each other. These results indicate that C. lipolytica cells grown on·oleic acid possess two distinct long‐chain acyl‐CoA synthetases. The phosphatidylcholine‐independent enzyme is designated as acyl‐CoA synthetase I, and the phosphatidylcholine‐dependent enzyme as acyl‐CoA synthetase II. The presence of two distinct long‐chain acyl‐CoA synthetases is further supported by the occurrence of mutant strains of C. lipolytica that are defective in acyl‐CoA synthetase I but possess acyl‐CoA synthetase II. Acyl‐CoA synthetase II exhibits a broader substrate specificity with respect to fatty acid than acyl‐CoA synthetase I. This, together with the inducible nature of acyl‐CoA synthetase II, supports the concept, based on the phenotype of the mutants mentioned above, that acyl‐CoA synthetase I is responsible for the production of acyl‐CoA to be utilized for the synthesis of cellular lipids, while acyl‐CoA synthetase II provides acyl‐CoA which is degraded to yield acetyl‐CoA [Kamiryo, T., Mishina, M., Tashiro, S. & Numa, S. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 4947–4950].
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.
Transfer of Proteins across Membranes
Authors: SCHEELE George , DOBBERSTEIN Bernhard , BLOBEL Günter
Published: March 2005

Canine pancreas was fractionated into free ribosomes and rough microsomes. Detached ribosomes were prepared by treatment of rough microsomes with detergent. Poly(A)‐containing mRNA was extracted from rough microsomes. The biosynthesis of canine pancreatic secretory proteins was studied by comparing proteins synthesized in vitro by translation of mRNA or by completion of nascent chains present in free ribosomes, rough microsomes, and detached ribosomes with proteins synthesized in tissue slices using polyacrylamide gel electrophoresis in sodium dodecyl sulfate and subsequent autoradiography.

The banding pattern of authentic secreted proteins synthesized in tissue slices was largely congruent with that obtained from the translation products of rough microsomes indicating that the bulk of the mRNA engaged with rough microsomes codes for secretory proteins.

The banding pattern of translation products from mRNA in the absence of microsomal membranes was not congruent with that of authentic secretory proteins. Primary translation products for trypsinogen and the other serine protease zymogens using mRNA appeared to be larger in molecular weight than authentic proteins by 1000–2000 and are thus designated ‘presecretory’ proteins.

The banding pattern from the translation products of free ribosomes, which are essentially devoid of membranes, was similar to that of ‘presecretory’ proteins.

Translation of mRNA in the presence of microsomal membranes yielded a banding pattern for serine protease zymogens congruent with that of the translation products of rough microsomes, and these products were resistant to posttranslational proteolysis, indicating that segregation and processing of these polypeptide chains had taken place during translation in vitro.
Kinetics of Synthesis of Cytoplasmic Messenger‐Like RNA not Associated with Ribosomes in HeLa Cells
Authors: MAURON Alex , SPOHR Georges
Published: March 2005

The turn‐over of cytoplasmic messenger‐like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin‐D‐chase experiments, cytoplasmic messenger‐like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients.

The half‐life of free mlRNA was found to be of 1–2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short‐lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non‐polyadenylated fractions showed that this short‐lived minor component of half‐life less than 1 h is non‐polyadenylated.
Histone Phosphorylation and Chromatin Structure during Mitosis in Chinese Hamster Cells
Authors: GURLEY Lawrence R. , D'ANNA Joseph A. , BARHAM Steven S. , DEAVEN Larry L. , TOBEY Robert A.
Published: March 2005

Histone phosphorylation and chromatin structure were examined in synchronized CHO Chinese hamster cells during progression through mitosis. Cell population distribution in various phases of mitosis was determined by electron microscopy. Entry into mitosis was seen to occur in two stages: (1) the gathering of chromatin into aggregates of dense chromatin clumps during preprophase, followed by (2) the condensation of these aggregates into chromosome structures during prophase. Exit from mitosis was observed essentially as the reverse process, chromosomes first being disorganized into dense chromatin clumps during telophase, followed by dispersion of these aggregates in early G1. Correlating these structural changes with histone phosphorylation revealed that interphase‐type histone H1 phosphorylation (H1I) involving 1–3 phosphates per molecule existed in interphase and during the chromatin aggregation stages of mitosis (preprophase and telophase). Also, no histone H3 phosphorylation occurred during these periods of the cell cycle. It is proposed that H1I phosphorylation may be involved with the submicroscopic changes in chromatin organization observed during interphase using molecular probes of chromatin structure. However, during mitosis, histone phosphorylation was correlated with microscopic chromatin structural changes. During the second stage of mitosis (prophase, metaphase, and anaphase), when chromosome structures were fully condensed, virtually all histone H1 existed as superphosphorylated molecules (H1M) containing 3–6 phosphates, and all histone H3 molecules were phosphorylated. Exit of cells from anaphase correlated closely with the dephosphorylation of H3 to unphosphorylated H3 and with the dephosphorylation of H1M to subphosphorylated H1 containing 0–3 phosphates. Further dephosphorylation of subphosphorylated H1I to unphosphorylated H1 occurred as these cells left telophase and entered G1. These experiments demonstrated that H1M superphosphorylation and H3 phosphorylation are strictly mitotic events which occur only when chromosomes are fully condensed. The absence of Colcemid in some of these experiments eliminates the possibility that H1M and H3 phosphorylations are artifacts of the Colcemid treatment. It is proposed that histones H1 and H3 may impose a restriction on chromatin structure which prevents chromosome condensation during interphase and that the H1M and H3 phosphorylations remove this restriction during mitosis.
Purification, Biochemical and Immunological Characterisation of Hexosaminidase A from Variant AB of Infantile GM2 Gangliosidosis
Authors: CONZELMANN Ernst , SANDHOFF Konrad , NEHRKORN Heidi , GEIGER Benjamin , ARNON Ruth
Published: March 2005

Variant AB of infantile GM2 gangliosidosis is a fatal disease leading invariably to death within the first few years of life, due to the excessive storage of the glycolipids GM2 and GA2 which occurs in the nervous tissue of the patient. Unlike other variants of this hereditary disease, where a deficiency of hexosaminidase A, the ganglioside‐GM2‐degrading enzyme, could be demonstrated, the variant AB is characterized by a normal or even elevated level of this enzyme.

To examine the possibility of a mutant hexosaminidase A, well capable of hydrolyzing the fluorogenic synthetic substrates but unable to attack the ganglioside, the enzyme was isolated from a patient's tissue and characterized biochemically and immunologically in comparison with an enzyme preparation from normal control tissue. No differences between hexosaminidase A from normal and variant AB tissue could be detected indicating that the defect involved in this disease is not at the genetic level of production of either α or β chains of hexosaminidase A.
Enzymatic Synthesis of Blood‐Group Lewis‐Specific Glycolipids
Authors: PROHASKA Rainer , SCHENKEL‐BRUNNER Helmut , TUPPY Hans
Published: March 2005

A blood‐group Lewis precursor glycolipid was isolated from the plasma of a Lewis‐negative individual [Le(a–b–)] and treated with fucosyltransferases from human gastric mucosa and GDP‐fucose. Subsequently the glycolipid was adsorbed onto Le(a–b–) erythrocytes and the presence of blood‐group Lewis antigens was assessed by passive hemagglutination with anti‐Lewis sera. It was shown that the precursor glycolipid was enzymatically transformed to blood‐group Lewisa (Lea) and Lewisb (Leb) specific glycolipids. Leb‐glycolipid was also synthesized by fucosylation of an isolated Lea‐glycolipid. Moreover Le(a–b–) erythrocytes were shown to develop Lea and Leb activities when subjected to enzymatic fucosylation, thus showing that Lewis‐negative cells carry blood‐group Lewis precursor glycolipid on the surface of their membrane. Le(a + b‐) erythrocytes, upon enzymatic fucosylation, acquired Leb activity.
Conformation of Methylated Sequences in HeLa Cell 18‐S Ribosomal RNA: Nuclease S1 as a Probe
Authors: KHAN M. Shah N. , MADEN B. Edward H.
Published: March 2005

18‐S rRNA from HeLa cells was digested with nuclease S1. Under the conditions employed 15% of the total nucleotides and some 50% of the methylated nucleotides were released as low‐molecular‐weight products. The material which was precipitable by 70% ethanol after nuclease S1 digestion was subjected to further digestion by combined T1 plus pancreatic riboncleases or by T1 ribonuclease alone, and fingerprints were prepared. It was found that the four sites which are modified late during ribosome maturation, and which contain base modifications, were all accessible to nuclease S1. By contrast fewer than one‐half of the sites which are modified early during ribosome maturation, and which contain 2′‐O‐methyl groups, were accessible to nuclease S1; the remainder were protected, presumably by secondary or tertiary interactions within 18‐S rRNA.
Conversion of the Active‐Site Cysteine Residue of Papain into a Dehydro‐serine, a Serine and a Glycine Residue
Authors: CLARK Peter I. , LOWE Gordon
Published: March 2005

Photolysis of papain which had been inhibited with 2‐bromo‐2′,4′‐dimethoxyacetophenone regenerated papain, but also formed [δSer25]papain (i.e. papain in which the active‐site cysteine residue 25 was replaced by dehydroserine) via the intermediate dehydrocysteine analogue, [δCys25]‐papain. Reduction with sodium borohydride gave [Ser25]papain. Both [Ser25]papain and [δSer25]‐papain had binding properties similar to those of papain, but were devoid of enzymic activity. Their fluorescence properties were also investigated. Incubation of [δSer25]papain at pH 9.0 gave [Gly25]papain.
Membranes Rich in Acetylcholine Receptor: Characterization and Reconstitution to Excitable Membranes from Exogenous Lipids
Authors: SCHIEBLER Werner , HUCHO Ferdinand
Published: March 2005

Characterization of acetylcholine‐receptor‐enriched membranes from Torpedo californica electric tissue by negative‐staining electron‐microscopy and by lipid analysis is described. The protein/lipid ratio is 70%/30%. The lipids consist of 70% phospholipids (46% phosphatidylcholine, 31% phosphatidylethanolamine, 14% phosphatidylserine, 7% sphingomyelin, 2% phosphatidylinositol of the phospholipids determined) and 20% cholesterol. The acetylcholinesterase‐enriched membranes show a similar composition. The only differences are a lower protein/lipid ration (45%/55%) and a lower phosphatidylcholine/sphingomyelin ratio of 39%/14% as compared to 46%/7% for the receptor‐enriched membranes. A method of preparing single‐walled phosphatidylcholine vesicles by gel filtration on Sephadex G50 according to Brunner et al. (Biochem. Biophys. Acta, 455, 322–331, 1976) is used to recombine the lipid‐depleted receptor complex with artificial lipid vesicles. Starting from a lipid mixture of 46% phosphatidylcholine, 31% phosphatidylethanolamine, 14% phosphatidylserine, 7% sphingomyelin, 2% phosphatidylinositol and 15% cholesterol we obtained vesicles associated with the acetylcholine receptor complex. These receptor vesicles are chemically excitable by 10 μM carbamoylcholine as measured by efflux of 22Na+ from the vesicles. The excitability is blocked by preincubation with 0.5 mM α‐toxin from Naja naja siamensis venom and by reduction with 5 mM dithioerythritol.
The Aminoacyladenylate Mechanism in the Aminoacylation Reaction of Yeast Phenylalanyl‐tRNA Synthetase
Authors: FASIOLO Franco , FERSHT Alan R.
Published: March 2005

It is shown from a combination of rapid quenching and steady‐state kinetics that the phenylalanyl‐tRNA synthetase from yeast catalyses the formation of phenylalanyl‐tRNA by the aminoacyladenylate pathway at pH 7.8 and 25°C. The rate‐determining step at saturating reagent concentrations is not the dissociation of the charged tRNA from the enzyme.
The Action of Lysozyme on Partially Deacetylated Chitin
Authors: AMANO Ken‐ichi , ITO Eiji
Published: March 2005

The effect of N‐unsubstitution of chitin and chitin oligosaccharides on their hydrolysis by lysozyme was studied.

Lysozyme digests of chitin deacetylated at about 70% of its glucosamine residues were fractionated by gel chromatography, paper chromatography and paper electrophoresis, giving six oligosaccharides with N‐unsubstituted glucosamine residues, C1–C6, together with the monomer to tetramer of N‐acetylglucosamine. Oligosaccharides C1, C2 and C3, which accounted individually for about 4–7% of the total glucosamine residues in the isolated saccharides, were identified as GlcNAc‐GlcNAc‐GlcNAc‐GlcN, GlcNAc‐GlcN‐GlcNAc‐GlcNAc and GlcNAc‐GlcN‐GlcNAc‐GlcN, respectively. Oligosaccharide C4, present in a smaller amount, and oligosaccharide C5, present in a very small amount, were identified as GlcNAc‐GlcNAc‐GlcN and GlcN‐GlcNAc‐GlcNAc, respectively. Oligosaccharide C6, accounting for about 7% of the glucosamine residues recovered, was identified as GlcNAc‐GlcN.

On further digestion with excess lysozyme, tetrasaccharide C1 yielded small amounts of N‐acetylglucosamine and GlcNAc‐GlcNAc‐GlcN in addition to major products, (GlcNAc)2 and GlcNAc‐GlcN. Tetrasaccharide C2 was hydrolyzed by lysozyme into GlcNAc‐GlcN and (GlcNAc)2; tetrasaccharide C3 into GlcNAc‐GlcN alone; trisaccharide C4 into N‐acetylglucosamine and GlcNAc‐GlcN. In contrast, trisaccharide C5 was shown to be completely resistant to lysozyme.

The results of digestion of the isolated oligosaccharides with lysozyme, together with the structural feature of these saccharides, were accounted for by the importance of the N‐acetyl groups of the N‐acetylglucosamine residues located on subsites C and E in the enzyme · substrate complexes.
Glycosylation in vitro of Semliki‐Forest‐Virus and Influenza‐Virus Glycoproteins and Its Suppression by Nucleotide‐2‐deoxy‐hexose
Authors: SCHWARZ Ralph T. , SCHMIDT Michael F. G. , LEHLE Ludwig
Published: March 2005

Cell‐free enzyme preparations from cultured fibroblasts infected with Semliki forest virus or fowl plague virus (an influenza A virus) incorporate [14C]mannose from GDP‐[14C]mannose into dolichol‐phosphate‐mannose, lipid‐linked oligosaccharides and into endogenous virus‐specific glycoproteins.

When GDP‐2‐deoxy‐d‐[14C]glucose serves as substrate 2‐deoxy‐d‐[14C]glucose is transferred to dolichol phosphate yielding dolichol‐monophosphate‐2‐deoxy‐d‐[14C]glucose. UDP‐2‐deoxy‐d‐[14C]‐glucose gives rise also to a lipid which, however, is not a polyprenol derivative.

The transfer of [14C]mannose to lipid‐extractable fractions and glycoproteins in vitro is blocked by GDP‐2‐deoxy‐d‐glucose. It can be restored by exogenous dolichol monophosphate only with regard to the formation of dolichol‐monophosphate‐[14C]mannose. GDP‐2‐deoxy‐d‐glucose, however, does not inhibit the transfer of [14C]mannose‐labelled oligosaccharides into glycoproteins. UDP‐2‐deoxy‐d‐glucose has no inhibitory effect on transfer reactions of [14C]mannose from GDP‐[14C]mannose into various lipid fractions or into glycoprotein. It is concluded therefore, that the inhibition of glycosylation brought about by 2‐deoxyglucose in vivo is caused by an interference of its GDP derivative with the formation of a correct lipid‐oligosaccharide.
Effect of Choline Deficiency on the Enzymes that Synthesize Phosphatidylcholine and Phosphatidylethanolamine in Rat Liver
Authors: SCHNEIDER Wolfgang Johann , VANCE Dennis E.
Published: March 2005

Activities have been determined in subcellular fractions of livers from choline‐deficient and normals rats for the enzymes that convert choline and ethanolamine to phosphatidylcholine and phosphatidylethanolamine respectively, that methylate phosphatidylethanolamine to yield phosphatidylcholine, and that oxidize choline to betaine. The activities of ethanolamine kinase, phosphoethanolamine cytidylyltransferase, and CDP‐ethanolamine: 1,2‐diacylglycerol phosphoethanol‐aminetransferase are not changed in the livers from choline‐deficient rats for at least 18 days. Similarly, the activities of choline kinase and CDP‐choline: 1,2‐diacylglycerol phosphocholine transferase were unaffected by choline depletion. A decrease of 30–41% was observed, however, in the mitochondrial oxidation of choline to betaine. Also, the activity of the phosphocholine cytidylyltransferase was reduced in the choline‐deficient livers to 60% of the control values. The only observed increase in enzyme activity was a 62% elevation of the phosphatidylethanolamine‐S‐adenosylmethionine methyltransferase activity after 2 days of choline deficiency. This increased activity was maintained for at least 18 days of choline deprivation. The results suggest a lack of adaptive change in the levels of these phospholipid biosynthetic enzymes as a result of choline deficiency.
Active‐Site Catalytic Efficiency of Acetylcholinesterase Molecular Forms in Electrophorus, Torpedo, Rat and Chicken
Authors: VIGNY Marc , BON Suzanne , MASSOULIÉ Jean , LETERRIER François
Published: March 2005

The active sites of acetylcholinesterase multiple forms from four widely different zoological species (Electrophorus, Torpedo, rat and chicken) were titrated using a stable, irreversible phosphorylating inhibitor (O‐ethyl‐S2‐diisopropylaminoethyl methyl‐phosphonothionate). In all cases, we found that within a given species, the molecular forms we examined were equivalent in their catalytic activity per active site. As pure preparations of the molecular forms of Electrophorus acetylcholin‐esterase were available, we were able to establish that one inhibitor molecule binds per monomer unit for each of them. This had already been shown by several authors for the tetrameric globular form, but not for the tailed molecules.

Analysis of the phosphorylation reaction showed that they are equally reactive. Under our experimental conditions, their turnover number per site was 4.4 × 107 mol of acetylthiocholine hydrolysed · h−1 at 28 °C, pH 7.0. The corresponding value was less than half for Torpedo (1.64 × 107 mol · h−1), and again lower for rat (1.32 × 107 mol · h−1).

In the case of rat acetylcholinesterase, the activity per active site of solubilized (with or without Triton X‐100) and membrane‐bound enzyme were identical.

We discuss the implications of these findings with respect to the quaternary structure of acetylcholinesterase, and to the physico‐chemical state and physiological properties of its molecular forms.
Affinity Chromatography of Phenylalanine Hydroxylase
Authors: COTTON Richard G. H. , JENNINGS Ian G.
Published: March 2005

Four independent methods have established that the structure of a previously reported pteridine affinity adsorbent, 6,7‐dimethyl‐5,6,7,8‐tetrahydropterin–CH‐Sepharose, is 5(CH‐Sepharosyl)‐6,7‐dimethyl‐5,6,7,8‐tetrahydropterin.

A novel reaction, the carbodiimide‐promoted coupling of a carboxyl group to N‐5 of a tetrahydropterin, is described.

Two novel adsorbents, 5‐formyl‐tetrahydrofolate–AH‐Sepharose and 5‐methyl‐tetrahydrofolate–AH‐Sepharose, are described which may be useful not only in the study of phenylalanine hydroxylase but also in the study of folate‐metabolizing enzymes.
Carbamoylphosphate Synthetase I of Rat‐Liver Mitochondria
Authors: LUSTY Carol J.
Published: March 2005

An improved purification of carbamoylphosphate synthetase I from rat liver mitochondria is described. The enzyme is essentially homogeneous with enhanced specific activity and stability. The enzyme is stable at alkaline pH in concentrated solution of salts. Kinetic studies indicate two type of inactivation, reversible and irreversible, depending on pH. The monomeric unit of carbamoylphosphate synthetase I consists of one polypeptide chain. The protein migrates in dodecylsulfate/polyaacrylamide gel as a single component corresponding to a molecular weight of about 155000. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M guanidine hydrochloride containing dithioerythritol is 158000 ± 5000. The weight‐average molecular weight of the native enzyme, 188000, suggests that the monomeric form is the predominant form of the enzyme, under the conditions described. Glutamine is inactive as a substrate. With ammonium, carbamoyl phosphate synthesis is optimal in the pH range 6.8–7.6. Mg2+ and Mn2+ are equally effective metal ions in the direction of carbamoyl phosphate synthesis, but excess Mn2+ is inhibitory. At less than saturating concentrations of substrate, considerable activation of the enzyme is observed when metal ions are in excess of ATP; when MgATP2− concentration is held constant at 1 mM and 10 mM, plots of v versus free Mg2+ concentration are hyperbolic, and extrapolate to zero. These results suggests that free divalent metal ion is required in the forward reaction. The apparent Km of the substrates is similar to other preparations of the enzyme. The determination of the maximal activity of carbamoylphosphate synthetase in rat liver mitochondria is reported, providing a measure of the total enzyme concentration that is about 1–1.5 mM.

The purification procedure described also provides an easy method to obtain ornithine transcarbamylase in partially purified form.
Alteration of the Intracellular Concentration of Aminoacyl‐tRNA Synthetases and Isoaccepting tRNAs during Amino‐Acid‐Limited Growth in Escherichia coli
Authors: THOMALE Jürgen , NASS Gisela
Published: March 2005

Growth of Escherichia coli AB 2271 under threonine or isoleucine deficiency leads to a derepression of the threonyl‐tRNA synthetase and isoleucyl‐tRNA synthetase respectively. During this amino‐acid‐limited growth the concentrations of isoaccepting fractions of the cognate tRNA species were changed, as demonstrated by their altered reversed‐phase‐5 chromatograms. But, in addition, the profiles of the isoacceptors of all other tRNA species investigated, i.e. of tRNAsLeu, tRNAsSer and tRNAsArg were also altered. This means that, if there is a correlation between regulation of the level of an aminoacyl‐tRNA synthetase and its cognate isoaccepting tRNAs, it is superimposed by the effect of amino acid limitation upon the concentration of all isoaccepting tRNAs.

So far drastic changes in profiles of isoaccepting tRNAs have only been observed under unbalanced growth in relaxed cells or during treatment with antibotis. Here we demonstrate that similar heavy alterations in patterns of isoaccepting tRNAs occur in a proven stringent E. coli strain growing exponentially under amino acid limitation signal a meaningful biological function of those newly or increasingly occuring isoaccepting tRNAs.

During the growth under amino acid limitation the total acceptor activity of eight investigated tRNA species, however, stayed unchanged, except that under threonine‐limited growth the total amount of tRNAIle was reduced to about half and that of tRNAGlu increased; both tRNA species of these isoacceptors are known [30,31] as spacers between ribosomal RNAs.