bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Short‐Term Changes in Hepatic β‐Hydroxy‐β‐methylglutaryl‐CoA Reductase Activity
Authors: DUGAN Richard E. , BAKER Terry A. , PORTER John W.
Published: March 2005

Immunotitrations of rat liver hydroxymethylglutaryl‐CoA (HOMeGlt‐CoA) reductase activity were performed before and after short‐term changes in the nutritional or hormonal state of the animals. Changes in enzyme activity (increase or decrease) within 1 h following cholesterol feeding or glucagon or mevalonolactone administration to normal rats, or insulin administration to diabetic rats were accompanied by no change in the specific activity of the enzyme, as determined from the quantity of enzyme activity inactivated by a fixed quantity of antibody. These results support the conclusion that the loss in enzyme activity was due to conversion of the enzyme to immunounreactive products. In agreement with this conclusion the enzyme activity lost after these short‐term physiological changes was not restorable by phosphoprotein phosphatase action. On the other hand, incubation of rat liver microsomes with ATP and Mg2+ decreased the specific activity of HOMeGlt‐CoA reductase about tenfold, as determined by immunotitration. The low specific activity produced under these conditions was increased by phosphatase action to nearly the original level. The above evidence suggests that the changes in HOMeGlt‐CoA reductase activity that resulted from short‐term physiological changes in hormonal or nutritional states of an animal were brought about by a change in the quantity of enzyme, and not by reversible phosphorylation of preexisting enzyme.
Infrared Spectroscopy, a Tool for Probing Bacterial Peptidoglycan
Authors: NAUMANN Dieter , BARNICKEL Gerhard , BRADACZEK Hans , LABISCHINSKI Harald , GIESBRECHT Peter
Published: March 2005

Infrared spectroscopic measurements are used to obtain insights into the three‐dimensional architecture of peptidoglycan (murein), the rigid component of almost all bacterial cell walls. The infrared spectra of various types of peptidoglycans (including all chemotypes and examples of the so called A and B groups) were compared to each other and to those obtained from crystalline chitin.

All peptidoglycans investigated exhibited very similar infrared spectra. In particular the conformationally sensitive amide A, I and I1 absorption bands were found to be constantly centered around 3300 cm−1, 1657 cm−1 and 1534 cm−1 respectively; furthermore, the spectral region between 1200 cm−1 and 800 cm−1, characterized by several strong absorption bands connected to complex sugar ring modes, proved to be remarkably uniform. Additionally the infrared spectra remained significantly constant between −175 °C and +75 °C and turned out to be rather independent of sample preparation (solvent replacement, freeze‐drying and film producing).

An analysis of band half‐widths revealed no high crystalline state of order of peptidoglycan. On the basis of band positions and half‐widths of amide bands, regular conformations like α helices of β pleated sheets could be excluded.

Several distinctive, fingerprint‐like spectral features of the various murein samples permitted a facile identification of individual peptidoglycans. Moreover, infrared spectroscopy seems to be very promising as an analytical tool, e.g. for tracing variations of cell wall structure, detecting conformational changes and estimating crosslinking indices in a quick and simple way.

The comparative analysis of amide band positions and band half‐widths yielded substantial differences between infrared spectra of chitin and murein, thus rejecting previous models based on the assumption of a nearly crystalline chitin‐like structure of the glycan chains of murein.
Subcellular Localization of the Methylcitric‐Acid‐Cycle Enzymes in Propionate Metabolism of Yarrowia Zipolytica
Authors: UCHIYAMA Hiroo , ANDO Masayasu , TOYONAKA Yohko , TABUCHI Takeshi
Published: March 2005

The subcellular localization of the four characteristic enzymes of the methylcitric acid cycle was studied with glucose‐grown as well as n‐alkane‐grown cells of Yarrowia lipolytica. Microsomes and peroxisomes showed no cycle enzyme activities. The four cycle enzymes were constitutively localized in mitochondria, with the exception of the dual localization of the fourth enzyme, 2‐methylisocitrate lyase, in mitochondria and cytoplasm, where the lyase may function to supply pyruvate (the end‐product of the catabolism of the propionate residue) to various reactions.
Radioactive Human Lymphoblastoid Interferon
Authors: YONEHARA Shin
Published: March 2005

Human lymphoblastoid interferon (α type), labeled with [3H]leucine added to virus‐induced Namalwa cells, was purified quantitatively and in one step from the culture fluid by immune precipitation. The material showed, upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only four radioactive bands with molecular weights ranging from 17000 to 21000, which coincided well with interferon activity. They coincided also with the four interferon protein bands in the electropherogram of unlabeled interferon purified by a different method. The purity of the labeled interferon was ascertained also by polyacrylamide gel electrophoresis in the absence of dodecyl sulfate.

Pulse‐labeling of interferon with [3H]leucine for 1 h at various times after induction indicated that the cells always synthesized and secreted the four interferon species in parallel during the interferon production period.

Competitive radioimmunoassay for human interferon a was achieved by the use of purified radioactive inter‐ feron, anti‐(interferon α) serum, and bacterial adsorbent. The immune precipitation of the labeled interferon was inhibited by unlabeled interferon a, and 100 international reference units of interferon a could be measured in this way.
ATP‐Independent Proteolysis of Globin Cyanogen Bromide Peptides in Rabbit Reticulocyte Cell‐Free Extracts
Authors: McKAY Malcolm J. , HIPKISS Alan R.
Published: March 2005

The catabolism of two rabbit globin cyanogen bromide peptides in cell‐free extracts of ATP‐depleted rabbit reticulocytes has been studied. Proteolysis of the peptides (3533 and 5957 molecular weight) proceeded rapidly in the absence of ATP, had a pH optimum of approximately 7.8, and was inhibited by o‐phenanthroline, N‐ethylmaleimide, cystamine, zinc and cobalt ions, and puromycin peptide high‐molecular‐weight aggregates. Proteolysis of puromycin peptides was inhibited by the rabbit globin cyanogen bromide peptides. The ability of cell‐free extracts of degrade the globin cyanogen bromide peptides decreased with the reticulocyte maturity. Blocking the globin cyanogen bromide peptide amino groups by succinic and maleic anhydride treatment decreased susceptibility to degradation. It is suggested that the globin cyanogen bromide peptides might provide model substrates, replacing puromycin peptides, for the investigation of ATP‐independent proteolytic events.
Fluorogenic Substrates for Bacterial Aminopeptidase P and Its Analogs Detected in Human Serum and Calf Lung
Authors: FLEMINGER Gideon , CARMEL Amos , GOLDENBERG Dalia , YARON Arieh
Published: March 2005

A sensitive fluorimetric assay was developed for bacterial aminopeptidase P, based on intramolecularly quenched fluorogenic substrates. Two substrates were synthesized, Phe(NO2)‐Pro‐HN‐CH2‐CH2‐NH‐ABz(sub‐strate I) and Phe(NO2)‐Pro‐Pro‐HN‐CH2‐CH2‐NH‐ABz (substrate II), in which the Phe(NO2) group (p‐nitro‐l‐phenylalanyl) quenches the fluorescence of the ABz group (o‐aminobenzoyl). Both substrates were readily cleaved by aminopeptidase P from Escherichia coli, releasing p‐nitro‐l‐phenylalanine and causing a proportional increase in fluorescence. Complete hydrolysis of the two substrates resulted in a 7.5‐fold and 3.4‐fold fluorescence increase, respectively. Applying this fluorogenic assay, we were able to detect and measure quantitatively amino‐ peptidase P‐like activity in the human serum and calf‐lung extracts. Substrate I1 was shown to be specifically cleaved by aminopeptidase P in these preparations, while substrate I was apparently cleaved by other enzymes as well. In both preparations, the enzyme activity was independent of Co2+ ions, and Pro‐HN‐CH2‐CH2‐NH‐ ABz(Cbz) was inhibitory. The kinetic constant Km was determined as 0.35 mM and 0.28 mM for the human serum and the calf‐lung enzymes respectively. The enzyme activity was only slightly dependent on pH in the range 7.0 ‐ 8.4.
Classification of Trimethoprim‐Resistant Dihydrofolate Reductases Mediated by R‐Plasmids Using Isoelectric Focussing
Authors: BROAD David F. , SMITH John T.
Published: March 2005

An analytical isoelectric focussing technique has been developed to distinguish between different groups of R‐plasmid‐mediated trimethoprim‐resistant dihydrofolate reductases. Those mediated by the R‐plasmids R483 and R721, which are type I dihydrofolate reductases have isoelectric point (PI) values of 6.4 whereas those mediated by the R‐plasmids R67bis and R27, which are type I1 enzymes, have PI values of 5.5. There has been some confusion as to the classification of the dihydrofolate reductases mediated by R‐plasmids R388 and R751; however, our studies indicate that the one mediated by R388 is a type I1 enzyme, having a PI value of 5.5, whereas the one mediated by R751 is different from both the type I and type I1 enzymes having a PI value of 7.2. The differences in PI value of these three groups of trimethoprim‐resistant enzymes were confirmed by DEAE‐ cellulose chromatography at pH 8.5 where it was found that the type I enzymes were eluted with a different concentration of NaCl than were the type I1 enzymes including that mediated by R388, whereas the enzyme mediated by R751 did not bind to the DEAE‐cellulose at this pH.

A ‘scale‐down’ technique was developed for analytical isoelectric focussing and was used to study the dihydrofolate reductases mediated by ten R‐plasmids present in trimethoprim‐resistant Escherichia coli strains isolated from pigs. All these R‐plasmids coded for type I enzymes and all also coded for resistance to streptomycin, indicating that the trimethoprim‐resistance gene(s) were present on a transposon similar to Tn7.
Tuftsin Analogs for Probing Its Specific Receptor Site on Phagocytic Cells
Authors: GOTTLIEB Philip , BERETZ Alain , FRIDKIN Mati
Published: March 2005

Six new analogs of the phagocytosis‐stimulating peptide tuftsin were synthesized with the eventual aim of characterizing and isolating the tuftsin receptor. These analogs can be classified as follows : (a) photoaffinity label‐ ling analogs for the specific covalent attachment to the tuftsin receptor; (b) fluorescent analogs containing either rhodamine or dansyl fluorescent probes for microscopic visualization of the tuftsin receptor; (c) biotin analog for separation and purification of the receptor by affinity methods.

In this paper we describe the various synthetic pathways employed to introduce sensitive prosthetic groups into the tuftsin molecule while preserving its biological activity. Activities of the various analogs synthesized as compared to tuftsin in biological and receptor‐binding assays are described. All analogs are able to stimulate phagocytosis of the macrophage cell as well as compete specifically for tuftsin binding sites on these cells.
The Effect of Acceptor Oligoribonucleotide Sequence on the T4 RNA Ligase Reaction
Authors: McLAUGHLIN Larry W. , ROMANIUK Elena , ROMANIUK Paul J. , NEILSON Thomas
Published: March 2005

In order to make efficient use of T4 RNA ligase in a program involving chemical‐enzymatic oligoribonucleotide synthesis, the sequence effects of the acceptor oligomer have been refined. The reaction of the donor molecules pCp and pUpUpUpCp with a series of trinucleoside diphosphates ApApN, CpNpA and NpCpA (where N = U, C, A or G) was examined. High‐performance liquid chromatography was used for simultaneous analysis of all substrates, intermediates and products. For all trinucleoside bisphosphates tested a substantial amount of the intermediate A(5′)pp(5′)Cp or A(5′)pp(5′)UpUpUpCp was observed. This indicated that the extent of ligation was dependent upon sequence of the acceptor molecule and not upon adenylation of the donor. Con‐ versely, examination of the four nucleoside 3′,5′‐bisphosphate donors p u p, pCp, pAp and pGp with a common acceptor ApApU indicated that in the case of poor ligation little of the intermediate adenylated donor was formed.
Two Forms of Adrenodoxin Reductase from Mitochondria of Bovine Adrenal Cortex
Authors: SUHARA Katsuko , NAKAYAMA Kazuo , KATAGIRI Masayuki , TAKIKAWA Osamu
Published: March 2005

Two forms of adrenodoxin reductase, differing in isoelectric properties and thermal stability, have been highly purified from bovine adrenal cortex mitochondria. Both preparations appear to be homogeneous based upon polyacrylamide disc‐gel electrophoresis, isoelectric focusing, and ion‐exchange chromatography. Both are monomeric proteins containing one FAD per molecule (Mr 50000) and exhibit spectra typical of flavoproteins. The two preparations are indistinguishable by their amino acid compositions and N‐terminal (serine) and C‐ter‐ minal (leucine) amino acids, but reveal distinct sugar contents. Electrophoretic mapping of the protease digests of the two preparations indicates that the two have identical, or closely similar, primary structures.

Each form functions equally as an adrenodoxin‐dependent electron transport component from NADPH to the P‐450 cytochromes in the two different reconstituted steroid monooxygenase systems which catalyze cholesterol side‐chain cleavage reaction and steroid 11β‐(18‐)hydroxylase reaction, respectively. In all adrenodoxin reductase‐ containing fractions from adrenal cortex so far examined, i.e. the soluble fraction, and the sonic‐solubilized and detergent‐solubilized mitochondrial fractions, a constant ratio of about 2:5 between the two forms is found as judged by the DEAE‐cellulose chromatogram. No evidence has been obtained for mutual conversion of the two forms of the reductase, or for dissociation of the sugar moiety during the purification process and under various conditions examined.
Proton‐Magnetic‐Resonance Studies on the Coat Protein of Alfalfa Mosaic Virus
Authors: KAN Jaap H. , ANDREE Piet‐Jan , KOUIJZER Luuk C. , MELLEMA Jan E.
Published: March 2005

A model is postulated for the alfalfa mosaic virus (AlMV) coat protein (Mr 24250), consisting of a rigid core and a flexible N‐terminal part of approximately 36 amino acid residues. This model is based on a proton nuclear magnetic resonance study of the AlMV coat protein. The NMR spectrum of the coat protein shows several motionally narrowed resonances which strongly decrease in intensity when 25 N‐terminal amino acid residues are removed by a mild tryptic treatment. The motionally narrowed resonances, therefore, are assigned to the N‐terminal region. Specific assignments in the spectrum for tyrosine‐21, threonine‐15 and the N‐terminal N‐acetyl group can be made. The model is supported by photochemically induced dynamic nuclear polarization experiments on the intact protein and the trypsinated protein, which indicate that tyrosine‐21 is the only tyrosine which is easily accessible from the solvent. Furthermore, NMR spectra were obtained from a polymerisation product of the coat protein, the so‐called 12‐S product in which only the sharp resonances of the N‐terminal part are observed. Apparently, due to the large particle mass (about 400 kDa), the resonances of the core part of the protein are broadened to such an extent that they are no longer observable. The part of the spectrum which originates from the mobile region of the coat protein can be simulated by calculating a spectrum for a random coil peptide of about 36 N‐terminal amino acids. This indicates that the flexible region extends to about 36 amino acid residues from the N terminus and that it probably occurs in a random coil structure. Evidence is presented for a change in structure of the protein in solution when it assembles into the virus particle. This conclusion is based on an observed spectral change of histidine‐220.
Purification of l‐Glutamate‐Dependent Citrate Lyase from Clostridum sphenoides and Electron Microscopic Analysis of Citrate Lyase Isolated from Rhodopseudomonas gelatinosa, Streptococcus dacetilactis and C. sphenoides
Authors: ANTRANIKIAN Garabed , KLINNER Christina , KÜMMEL Angela , SCHWANITZ Diethard , ZIMMERMANN Thomas , MAYER Frank , GOTTSCHALK Gerhard
Published: March 2005

Citrate lyase from Clostridium sphenoides was purified 72‐fold with a yield of 11%. In contrast to citrate lyase from other sources the activity of this enzyme was strictly dependent on the presence of l‐glutamate. The purified enzyme was only stable in the presence of 150 mM l‐glutamate or 7 mM l‐glutamate plus glycerol, sucrose or bovine serum albumin. Changes of the l‐glutamate pool and of enzyme activity in growing cells of C. sphenoides indicated that citrate lyase activity in this organism was regulated by the intracellular l‐glutamate concentration.

Citrate lyase isolated from C. sphenoides, Rhodopseudomonas gelatinosa and Streptococcus diacetilactis was investigated by electron microscopy using the negative staining technique. Three different projections of enzyme molecules were observed: ‘star’ form, ‘ring’ form and ‘triangle’ form. In samples from R. gelatinosa and S. diacetilactis, star and ring forms occurred in a ratio of about 1:9. Using the enzyme from S. diacetilactis it was demonstrated that this ratio could be altered in favour of the star form by the addition of citrate or tricarballylate. The triangle form was observed in less than 1% of all evaluated molecules and may represent a transition form. In lyase samples from C. sphenoides there existed a correlation between enzyme activity and the proportion of stars and rings at varying concentrations of l‐glutamate.
Analysis of Structure‐Function Relationships in Citrate Lyase Isolated from Rhodopseudomonas gelatinosa as Revealed by Cross‐linking and Immunoelectron Microscopy
Authors: ZIMMERMANN Thomas , GIFFHORN Friedrich , SCHRAMM Hans J. , MAYER Frank
Published: March 2005

Citrate lyase (EC 4.1.3.6) isolated from Rhodopseudomonas gelatinosa was analyzed using cross‐linking experiments and immunoelectron microscopy. Different cross‐linking reagents and antibodies directed against citrate lyase and specifically against all three subunit types (L, M and S) were applied. A structure‐function model is proposed for citrate lyase from R. gelatinosa: the enzyme occurs in two configurations, ‘rings’ and ‘stars’. The ring contains two identical layers each consisting of three subunits L, with one subunit S as a polar cap sitting on each L, and three subunits M in alternating sequence (18 subunits altogether). In the star, the same 18 subunits are arranged in a different way. Whereas the subunits L are located at the periphery, the subunits M are concentrated in the center of the particle. The subunits S are positioned relative to L as in the ring; however, their location relative to the subunits M is changed. By transition from ring to star, areas on S are brought into contact with areas on M by rotation of structural units, consisting of one L, one M and one S subunit per layer, against each other, with S of one structural unit close to M of the neighbouring structural unit. This transition is assumed to work also in reversed direction. The observation of rings and stars as two distinct molecular forms is proposed to reflect the two states of citrate lyase, the ring being the form where substrate is bound by acyl exchange, and the star being the form where the substrate is consumed by cleavage, i.e. the catalyzed reaction is completed.
The Alkylating Agent, Dimethyl Sulphate, Stimulates ADP‐Ribosylation of Histone H1 and Other Proteins in Permeabilised Mouse Lymphoma (L1210) Cells
Authors: THI MAN Nguyen , SHALL Sydney
Published: March 2005

Exposure of mouse lymphoma L1210 cells to the alkylating agent, dimethyl sulphate at 500 μM for 1 h produced an eightfold increase in the activity of nuclear ADP‐ribose transferase as measured by [3H]NAD incorporation after permeabilisation of the cells. The radioactive product formed was shown to be (ADP‐ribose)n, (a) by identification of the snake venom phosphodiesterase products as 5′AMP and phosphoribosyl‐AMP,(b) by its hydrolysis by snake venom phosphodiesterase and poly(ADP‐ribose) glycohydrolase but not by DNase and RNase, and (c) by treatment with the ADP‐ribosyl transferase inhibitor, 3‐aminobenzamide, which almost completely prevented [3H]NAD incorporation.

Of this acid‐insoluble radioactivity, 4% was associated with the histone fraction while over 80% was extracted with the non‐histone chromatin protein fraction. After treatment with dimethyl sulphate, radioactivity in the histone fraction increased 20‐fold; the radioactivity in this histone fraction increased proportionately from 4% to 10% of the total radioactivity incorporated.

Analysis of the histones on acid/urea gels revealed that most of the [3H]NAD incorporated co‐migrated with histone H1, both with and without dimethyl sulphate treatment, and there was a large increase in radioactivity of histone H1 after this treatment. The average chain length of newly synthesised (ADP‐ribose)n on histones was between three and four ADP‐ribose units.

The nature of the ADP‐ribosyl–histone bond was investigated; neutral hydroxylamine liberated 56% of the (ADP‐ribose)n, a further 30% was released by alkaline hydrolysis and 14% was resistant to alkaline hydrolysis.

This stimulation of the ADP‐ribosylation of histones by an alkylating agent known to damage DNA provides further support for the participation of ADP‐ribosylation of chromatin proteins in DNA repair.
A New Acrosin Inhibitor from Boar Spermatozoa
Authors: TSCHESCHE Harald , WITTIG Bernd , DECKER Günther , MÜLLER‐ESTERL Werner , FRITZ Hans
Published: March 2005

A new proteinase inhibitor (Mr 7500) was isolated to apparent homogeneity from boar spermatozoa by repeated gel filtration on Sephadex G‐50 and affinity chromatography on concanavalin‐A‐Sepharose 4B. The inhibitor strongly inhibits boar acrosin in a competitive 1:1 stoichiometric reaction with a Kass= 7 × 1010l mol−1.

The inhibitor is a glycoprotein and represents a first member of a new class of proteinase inhibitor with a rather short polypeptide backbone of only 42 amino acid residues and a low cystine content. The basic protein (isoelectric point 9.4) contains a single disulfide loop, which is easily reducible by sodium borohydride. Upon reduction the inhibitory activity is lost, but rapidly regained after air reoxidation of the corresponding half‐cystine residues. The reactive site residue was established to be arginine by inhibition with 2,3‐butanedione. The inhibitor is rather specific for acrosin and inhibits bovine trypsin only to a limited extent. However, incubation with catalytic amounts of trypsin (or acrosin) at acid pH (pH 2–3) rapidly leads to a limited proteolysis at the reactive site with formation of 67% modified (reactive site hydrolysed), but still active inhibitor. The equilibrium constant was established to be Khyd= 2.0.
A Cytochrome b/c1 Complex with Ubiquinol–Cytochrome c2 Oxidoreductase Activity from Rhodopseudomonas sphaeroides GA
Authors: GABELLINI Nadia , BOWYER John R. , HURT Eduard , MELANDRI B. Andrea , HAUSKA Günter
Published: March 2005

A cytochrome b/c1 complex which catalyses the reduction of cytochrome c by ubiquinol has been isolated from Rhodopseudomonas sphaeroides GA. It contains two hemes b and substoichiometric amounts of ubiquinone‐10 and of the Rieske Fe‐S center per cytochrome c1, and is essentially free of reaction center and bacteriochlorophyll. The complex consists of three major polypeptides with apparent molecular masses of 40, 34 and 25 kDa. The 34‐kDa polypeptide carries heme. Cytochrome c1 has a midpoint potential of 285 mV. For cytochrome b two midpoint potentials, at 50 and –60 mV, at pH 7.4, can be derived if one assumes two components of equal amount. Ubiquinol–cytochrome c oxidoreductase activity is specific for ubiquinol and bacterial cytochromes c, and is inhibited by antimycin A and 5‐n‐undecyl‐6‐hydroxy‐4,7‐dioxobenzothiazole. The complex shows oxidant‐induced reduction of cytochrome b.
Complexity of Polysomal Polyadenylated RNAs in Vicia faba Meristematic Root Cells
Authors: BUFFARD Dominique , VAILLANT Victor , ESNAULT Robert
Published: March 2005

As a first step in quantifying gene expression during differentiation of the Vicia faba root cells we have analysed the mRNA population from meristematic cells. Using cDNA · mRNA hybridizations we have shown that this population can be divided into three abundance classes (abundant, intermediate and rate) representing 37%, 34% and 29% of the mRNAs and containing 26, 610 and 11700 sequences respectively. The total base‐sequence complexity of the mRNA population, as determined by cDNA · mRNA hybridization, was found to be 1.6 × 107 nucleotides. This estimate was confirmed by the determination of the amount of single‐copy genomic DNA hybridizing to the mRNA. Expressed as the number of structural gene transcripts, this complexity corresponds to about 13000 mRNA sequences.
Biosynthesis of Mammary Glycoproteins
Authors: VIJAY Inder K. , PERDEW Gary H.
Published: March 2005

The lipid‐linked glucosyloligosaccharide intermediates for the biosynthesis of asparagine‐linked glycoproteins in the lactating bovine mammary tissue were isolated from incubations of microsomes of this tissue with labeled sugar‐nucleotides. These were shown to have the general structure (Glc)1–3(Man)9(GlcNAc)2. A structural characterization indicated that glucose residues in these oligosaccharides are linked as Glcα1 → 3, Glcα1 → 3Glcα1 → 3, and Glc1 → 2Glcα1 → 3Glcα1 → 3, to the tetramannosyl branch of (Man)9(GlcNAc)2 portion previously characterized as Manα1 → 2Manα1 → 2Manα1 → 3(Manα1 → 2Manα1 → 6[Manα1 → 2Manα1 → 3]‐Manα1 → 6)Manβ1 → 4(3)GlcNAcβ1 → 4(3)GlcNAc [Vijay, I. K., Perdew, G. H., and Lewis, D. E. (1980) J. Biol. Chem. 255, 11210–11220]. The lipid‐linked (Man)9(GlcNAc)2 appears to be the initial acceptor of the first glucosyl residue. Even though incubations of the bovine mammary tissue in vitro synthesize multiple isomers of a number of mannosyloligosaccharides, the glucosyloligosaccharides are all monoisomeric.
Similarity of Acetylcholine Receptors of Denervated, Innervated and Embryonic Chicken Muscles
Authors: SUMIKAWA Katumi , BARNARD Eric A. , DOLLY J. Oliver
Published: March 2005

The native (9‐S) form of the acetylcholine receptor, purified from chick embryonic or adult innervated or denervated chicken muscles, was shown in each case to contain three subunits of Mr about 40000 (predominant), 50000 and 54000. The 40000‐Mr subunit (termed α) appears to exist in two forms; the minor variant form of it, with Mr about 41000, may have a different carbohydrate content. Bromo[3H]acetylcholine, an affinity alkylating reagent for this receptor, labels the 40000‐Mr subunit and the accompanying 41000‐Mr variant. The apparent ratio of the subunits varies with the several methods of detection used (protein staining, radio‐iodination or tritiation) but the α subunit always predominates.

The α subunit was separated in urea gel isoelectric focussing; its isoelectric point was identical there for the three receptors from innervated, denervated and embryonic muscles. Their respective peptide maps for the α subunit were identical, and likewise for the 54000‐Mr subunit. All of the evidence obtained shows that the subunit structures of the junctional, extra‐junctional and embryonic chick muscle receptors are identical.

Although the α subunit in the receptor from Torpedo electric organ, cat muscle and chicken muscles is distinguishable in all three cases in dodecylsulphate gel electrophoresis, it is affinity‐labelled in each case. The peptide map of this subunit after limited proteolysis is also very similar in all three cases. It is concluded that the α subunits from these three diverse sources are closely related structurally.
Structural Studies on Microvillus Aminopeptidase from Pig Small Intestine
Authors: SVENSSON Birte , SJÖSTRÖM Hans , NORÉN Ove
Published: March 2005

Two amphiphilic forms of intestinal microvillus aminopeptidase were obtained by purification of the enzyme in the absence and in the presence of aprotinin respectively. By subsequent treatment with trypsin in vitro a hydrophilic form was obtained. These three enzyme preparations all contained three subunits, A, B and C (molecular weights 168000, 118000 and 54000 respectively). The individual subunits were purified by gel filtration on Ultrogel AcA 22 in the presence of sodium dodecyl sulphate. Subunits A and B from aminopeptidase prepared in the presence of aprotinin contained both N‐terminal lysine and alanine, whereas N‐terminal alanine and glycine were found for both of these subunits from the other amphiphilic form. Conversion of amphiphilic aminopeptidase to the hydrophilic form in vitro led only to a change in the N terminus of the B subunit to a valine. N‐terminal serine was found in subunit C from all three forms of the aminopeptidase. The amino acid composition and peptide maps of the isolated subunits from one of the amphiphilic forms are reported. It is suggested that subunit B and C arise by cleavage of subunit A, whereas it seemed unlikely that C originates from B.

Brush border membrane vesicles were cross‐linked by dimethyl‐3,3′‐dithiobispropionimidate, and the aminopeptidase subsequently isolated. Analysis of molecular weights and subunit composition of its constituent polypeptide complexes by two‐dimensional polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulphate showed that AB, BBC, BCC, BB, BC and CC complexes had been formed. The subunit structures ABC and BBCC were thus proposed in agreement with the maximum molecular weight of the membrane‐bound enzyme previously determined as 330000.
Expression of the Gene Coding for the Small Subunit of Ribulosebisphosphate Carboxylase during Differentiation of Tobacco Plant Protoplasts
Authors: VERNET Thierry , FLECK Jacqueline , DURR Andrée , FRITSCH Christiane , PINCK Monique , HIRTH Léon
Published: March 2005

A hybridization probe was used to study the regulation of expression of the gene coding for the small subunit of ribulose 1,5‐bisphosphate carboxylase, during functional differentiation of protoplasts. A library of cDNA from poly(A)‐containing RNA extracted from specially treated tobacco leaves was constructed in the plasmid pBR322, by blunt‐end ligation. This library was screened by colony hybridization with 32P‐labelled cDNA prepared from mRNA coding for the precursor of the small subunit. A positive colony was identified containing recombinant plasmids with a nucleotide sequence homologous to this mRNA. These plasmids, bound to diazobenzyloxymethylated cellulose paper, were then used as a hybridization probe. The results showed unambiguously that the small subunit was not transcribed in protoplasts but was transcribed in undifferentiated white and chlorophyll‐containing green callus cultures derived from protoplasts. The discrepancy between these results and those obtained with classical techniques is discussed.
Characterization of the Ca2+ Binding Sites of Calmodulin from Bovine Testis Using 43Ca and 113Cd NMR
Authors: ANDERSSON Thomas , DRAKENBERG Torbjörn , FORSÉN Sture , THULIN Eva
Published: March 2005

The exchange rates of Ca2+ ions to the two classes of sites on calmodulin have been determined from the temperature dependence of the 43Ca NMR line width. The exchange rates were found to differ by a factor of about 40 at room temperature. The apparent pK value for one of these classes was estimated from the pH dependence of the 43Ca line width.

The PK values of the two‐high‐affinity sites were found to differ about 0.1–1.

Zn2+ ions were shown to bind to calmodulin and affect the exchange rates of Ca2+ and Cd2+ for all four sites.
Evolutionary Divergence of Chloroplast and Cytosolic Glyceraldehyde‐3‐phosphate Dehydrogenases from Angiosperms
Authors: CERFF Rüdiger
Published: March 2005

Extracts from 13 different angiosperm species (spinach, mustard, pea, bean, tomato, cucumber, pumpkin, maize, sorghum, rye, wheat, oats, barley) were submitted to electrophoresis under nondenaturing conditions and stained for enzyme activities of cytosolic and chloroplast glyceraldehyde‐3‐phosphate dehydrogenases by a modified tetrazolium test of high sensitivity. Zymograms of the cytosolic enzyme revealed a single band of similar electrophoretic mobility for all but one species, the tomato, which displayed an ordered set of five different bands. In contrast, zymograms of the chloroplast dehydrogenase are highly different, containing between two and five distinct bands of variable electrophoretic mobilities according to the plant species examined. This variability of the native chloroplast enzyme is paralleled by a remarkable interspecific heterogeneity of the enzyme with respect to subunit size and number, as shown by dodecylsulfate electrophoresis of the purified chloroplast enzyme from 11 different angiosperm species.

The present data suggest that cytosolic and chloroplast glyceraldehyde‐3‐phosphate dehydrogenases belong to two separate protein families of different evolutionary rate. While the cytosolic enzyme is probably an extremely conservative protein like the corresponding enzymes from animals, yeast and bacteria, the chloroplast enzyme seems to change rather rapidly during evolution.
An Affinity Label for α2‐Adrenergic Receptors in Rat Brain
Authors: ATLAS Daphne , PLOTEK Yael , MISKIN Ian
Published: March 2005

Clonidine, a potent and highly selective α2‐adrenergic agonist of the central nervous system, was modified. Insertion of the strong alkylating isothiocyanate group (NCS) group, at its aromatic residue, makes clonidine a potential affinity label of the α2‐adrenergic receptors. In displacement of [3H]clonidine and p‐[3H]aminoclonidine from rat brain membrane preparations, clonidine‐NCS demonstrates high affinity for the α2‐adrenergic receptors (Kd= 50 mM). The covalent labelling of the central α2‐receptors requires higher concentrations of the irreversible ligand (1–70 μM), thus indicating possible non‐productive interactions at the environment of the receptor site. Only partial protection of the receptors is observed with a reversible α2‐agonist. The new clonidine analog appears to be a general ligand for the α2‐adrenergic receptors and might serve as a potential affinity probe for these receptors.
Lectin‐Induced Changes among Polyadenylated and Non‐polyadenylated mRNA in Lymphocytes
Authors: KECSKEMETHY Nicolàs , SCHÄFER Klaus P.
Published: March 2005

The differentiation of T‐cell‐enriched concanavalin‐A‐stimulated bovine lymphocytes was studied in vitro. mRNA was isolated from resting and stimulated cells. The amount of polyadenylated RNA increases from 3.6 × 10−9 to 12 × 10−9μg/cell during 40 h concanavalin A stimulation. The estimated maximum length of the 3′‐poly(A) tract in this RNA is reduced from 240 to 220 residues in stimulated cells. During translation in the rabbit reticulocyte lysate in vitro mRNA from stimulated cells consistently incorporates about 1.6–3 times more radioactivity/μg RNA into proteins than mRNA from resting cells. Three translation products have been identified on two‐dimensional gels as actin, tubulin and calmodulin. Large quantitative shifts are seen between proteins translated from mRNAs isolated from resting cells and stimulated cells respectively. Actin and calmodulin are already major products from resting cell mRNA. Actin, however, increases about fivefold after stimulation while calmodulin does not change. Tubulin appears in substantial amounts only among stimulated cell mRNA products. Tubulin and calmodulin, on the other hand, remain mainly in the polyadenylated RNA fraction after stimulation while 50% of the actin together with a group of about six other major products is found among proteins translated from non‐polyadenylated RNA. We conclude that in lectin‐stimulated lymphocytes, besides a general increase in the amount of mRNA, alterations in post‐transcriptional processing reactions are active in determining the fate of individual mRNAs.
Oxidase and Oxygenase Function of the Microsomal Cytochrome P450 Monooxygenase System
Authors: KUTHAN Hartmut , ULLRICH Volker
Published: March 2005

The rates of the NADPH‐dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital‐pretreated rats. Correcting a quenching of O2− radicals by microsomes, a stoichiometry of O2− to H2O2 close to 2:1 was obtained. This, and the fact that pseudo‐substrates of microsomal cytochrome P450 like perfluoro‐n‐hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase‐inhibited assay, rules out a two‐electron reduced oxygen species as the source of H2C2.

The rates of O2− as well as H2O2 generation in the presence of 7‐ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%.

It was concluded that most of the O2− originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton‐assisted decomposition of oxy‐cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 μM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.
Solubilization and Partial Characterization of a Multienzyme Complex of DNA Synthesis from Human Lymphoblastoid Cells
Authors: WICKREMASINGHE R. Gitendra , YAXLEY John C. , HOFFBRAND A. Victor
Published: March 2005

Gently lysed human lymphoblastoid cells (HPB‐ALL) catalysed incorporation of the distal DNA precursors [3H]thymidine, [3H]dTMP, and [3H]dUMP into DNA. Measurement of the [3H]dTTP, produced during the incorporation reactions, provided kinetic evidence for the channelling of the distal precursors into DNA by a multienzyme complex consisting of precursor‐synthesizing enzymes and of DNA polymerase.

In the presence of the DNA polymerase inhibitor, 1‐β‐d‐arabinofuranosylcytosine triphosphate, incorporation of [3H]dTMP into DNA was abolished. However, there was no accumulation of [3H]dTTP, suggesting that the flow of substrates through the complex was regulated by the activity of DNA polymerase.

Multienzyme complexes were detected by chromatography of a lysate on a column of Sepharose 6B. These complexes retained the ability to channel [3H]thymidine and [3H]dTMP into DNA.
Model for the Interaction of Membrane‐Bound Substrates and Enzymes
Authors: SCHEEL Günther , ACEVEDO Enrique , CONZELMANN Ernst , NEHRKORN Heidi , SANDHOFF Konrad
Published: March 2005

Microsomes and synaptosomes isolated from calf brain contain a sialidase which cleaves ganglioside substrates. The hydrolysis of [3H]ganglioside GD1a by the membrane‐bound enzyme has been studied under various conditions.

The reaction rate decreased with increasing ionic strength in the incubation mixture, and was progressively enhanced by increasing concentrations of the primary alcohols n‐pentanol to n‐octanol. This stimulation correlates quantitatively with an increase in membrane ‘fluidity’ caused by these alcohols as measured by fluorescence depolarization employing l,6‐diphenyl‐1,3,5‐hexatriene as probe. The dependence of the reaction rate on the amount of enzyme in the incubation mixture was linear only with water‐soluble substrates but not with the lipophilic ganglioside substrate. Evidence is presented that lipophilic substrate and enzyme interact mainly within the plane of the membrane presumably by lateral diffusion. Taking this into consideration Michaelis‐Menten theory was modified accordingly.

As predicted, apparent Km values increased linearly with the amount of membrane‐bound enzyme added and decreased with the concentration of n‐hexanol in the incubation mixture. In the presence of varying n‐hexanol concentrations the apparent Km‐value decreased with increasing membrane ‘fluidity’, as measured by fluorescence depolarization of l,6‐diphenyl‐1,3,5‐hexatriene. On the other hand, as expected, V values were not affected by membrane ‘fluidity’ and increased linearly with the amount of membrane protein.
Dissociation and Assembly of Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii
Authors: VOORDOUW Gerrit , de HAARD Hans , TIMMERMANS Johannus A. M. , VEEGER Cees , ZABEL Pim
Published: March 2005

Thermal inactivation studies of pyridine nucleotide transhydrogenase (55°C, pH 7.5, ionic strength 0.1 M) show a biphasic loss of enzyme activity with time. Approximately half of the activity is lost in both the rapid and the slow phase of the inactivation reaction.

With electron microscopy it is found that the quaternary structure of transhydrogenase does not change during thermal inactivation. Using Naja naja venom phosphodiesterase to monitor the release of FAD it is shown that dissociation of FAD and activity loss are proportional at every degree of inactivation. The biphasic loss of enzyme activity thus indicates non‐equivalent positions for the subunits (Mr 54000, 1 FAD per subunit) in the transhydrogenase polymer.

The process of thermal inactivation is greatly accelerated when the enzyme‐bound flavin is reduced by addition of NADH. The apoenzyme, obtained by gel filtration of transhydrogenase in the presence of NADH at 37°C, can be partly (10–15%) reactivated by addition of excess FAD. The dissociation of FAD under non‐reducing conditions at 55°C is irreversible.

In a guanidine hydrochloride denaturing solvent (cGuHCl > 2 M, 25°C, pH 7.5) transhydrogenase dissociates into subunits. These are highly unfolded as judged by hydrodynamic criteria. Retransfer of the enzyme to conditions stabilizing the native conformation (cGuHcl < 0.3 M) leads to reactivation provided that FAD is also present in the reactivation mixture. The yield of the reactivation is low, 3–8% of the theoretical maximum, and the process is slow. Delaying the time of addition of FAD to the reactivation mixture causes a decrease in reactivation yield. The results indicate that FAD is inserted in an intermediate stage in the assembly path leading to active transhydrogenase.

Polyacrylamide gel electrophoresis in combination with the Western blotting technique shows the presence of a covalent dimer in purified transhydrogenase preparations, which is not dissociated by treatment with dodecylsulphate and 2‐mercaptoethanol. Since a dimer or other form of high relative molecular mass is not detected in cell extracts it follows that such forms do not play a role in the assembly of the complex quaternary structure of the enzyme in vivo.
Characterization of an ATPase on the Inside of Rat‐Liver Nuclear Envelopes by Affinity Labeling
Authors: KONDOR‐KOCH Claudia , RIEDEL Norbert , VALENTIN Reiner , FASOLD Hugo , FISCHER Helmut
Published: March 2005

Nuclear envelope membranes from rat liver cells contain ATPases, one of which can be inhibited and irreversibly labeled by (S‐dinitrophenyl)‐6‐mercaptopurine riboside triphosphate. Inhibition and covalent substitution of the ATPase are achieved only after disruption of the nuclei, the ATP analogue is inactive on the ATPase activity of whole nuclei or on vesicles of the membrane prepared after a modified heparin method of Bornens and Courvalin. Electron micrographs and scanning micrographs helped to establish the characterization of closed vesicles and intact nuclei. With the aid of (α‐32P)‐labeled, and of the (β,γ‐32P)‐labeled analogue, it was possible to demonstrate the incorporation of the nucleotide into a few protein regions of the nuclear membrane disc electrophoresis pattern.
An RNA‐Dependent Nucleoside Triphosphate Hydrolase from Krebs‐II Ascites Tumor Cells
Authors: CHUMAKOV Konstantin M. , ROZANOV Mikhail N. , AGOL Vadim I.
Published: March 2005

A novel enzymatic activity, RNA‐dependent, NTPase, was isolated from Krebs‐II ascites tumor cells. This activity is associated with ribosomes and can be detached from them by washing in KCl solutions of a higher than 0.3 M concentration. The enzyme hydrolyzes all the four nucleoside triphosphates to the corresponding nucleoside diphosphates and orthophosphate. The rate of NTP hydrolysis increases about 10‐fold in the presence of natural RNAs and synthetic polyribonucleotides [except poly(G)]. Natural DNAs, both double and single‐stranded, are poor cofactors, although pol(dA) and poly(dT) stimulate, to a certain extent, the rate of ATP hydrolysis. Possible involvement of RNA‐dependent NTPase in protein biosynthesis is discussed.
Dynamic Properties of Microtubules at Steady State in the Presence of Taxol
Authors: CAPLOW Michael , ZEEBERG Barry
Published: March 2005

The dynamic properties of steady‐state microtubules in the presence of the antitumor drug taxol and GTP, but in the absence of microtubule‐associated proteins have been studied. The molecular rate constants for the loss or gain of subunits at steady state was found to be dramatically decreased as compared with that for microtubules formed in the presence of GTP and microtubule‐associated proteins but in the absence of taxol [Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891–98991. In light of this change it was surprising to find that the degrees of directionality for subunit flux into the microtubule at steady state are nearly identical within 1.5% of each other) in the two systems. One mechanism to account for this would be for taxol to cause a nearly identical decrease in the rate constants for subunit dissociation at both ends of the microtubule, with no effect on the rate constants for subunit addition.

Similar results have previously been found in studies with an endogenous effector of the microtubule steady state, a protein kinase [Jameson, J. L. and Caplow, M. (1981) Proc. Natl Acad. Sci. USA, 78, 3413–34171. In this case it was found that phosphorylation of microtubule‐associated proteins altered the molecular rate constants for tubulin subunit addition and dissociation, but had no effect on the degree of directionality for subunit flux. It will be of interest to determine whether other exogenous or endogenous effectors also act in a manner such as to leave the directionality unaltered.