bluuebunny/crossref_metadata_2025 huggingface dataset:
Biocatalytic Reduction of Formaldehyde to Methanol: Effect of pH on Enzyme Immobilization and Reactive Membrane Performance
Authors: Rahman Norhayati Abdul , Marpani Fauziah , Othman Nur Hidayati , Alias Nur Hashimah , Jai Junaidah , Nik Him Nik Raikhan
Published: May 2021

Thermodynamic stabled CO2 molecules can be biocatalytically reduced to methanol via three cascade dehydrogenases (formate, formaldehyde and alcohol) with the aid of cofactor as the electron donor. In this study, Alcohol dehydrogenase (EC 1.1.1.1), the third step of the cascade enzymatic reaction which catalyzed formaldehyde (CHOH) to methanol (CH3OH) will be immobilized in an ultrafiltration membrane. The enzyme will be immobilized in the support layer of a poly(ether)sulfone (PES) membrane via a technique called fouling induced enzyme immobilization. The objective of this study is to evaluate the effect of varying pH (acid (pH 5), neutral (pH 7) and alkaline (pH 9)) of the feed solution during immobilization process of ADH in the membrane in terms of permeate flux, observed rejection, enzyme loading and fouling mechanism. The experiment was conducted in a pressure driven, dead-end stirred filtration cell. Reaction conversion and biocatalytic productivity will be also evaluated. The results showed that permeate flux for acid solution were the lowest during immobilization. High concentration polarization and fouling resistance cause lower observed rejection for pH 7 and 9. Enzyme loading for pH 5 give 73.8% loading rate which is the highest compared to 62.4% at pH 7 and 70.1% at pH 9. Meanwhile, the conversion rate during the reaction shows that reaction on fouled membrane showed more than 90% conversion for pH 5 and 7. The fouling model predicted that irreversible fouling occurs during enzyme immobilization at pH 7 with standard blocking mechanism while reversible fouling occurs at pH 5 and 9 with intermediate and complete blocking, respectively. Copyright © 2021 by Authors, Published by BCREC Group. This is an open access article under the CC BY-SA License (https://creativecommons.org/licenses/by-sa/4.0).
Evidence for the presence of high‐Mr microtubule‐associated proteins and their Ca2+‐dependent proteolysis in synaptosomal cytosol
Authors: Burgoyne Robert D. , Cumming Richard
Published: July 2002

Calcium‐dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M
r 340 000 and 300 000. These latter polypeptides comigrated with high‐M
r microtubule‐associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca2+‐dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M
r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca2+.
Properties of brain spectrin (fodrin)
Authors: Burns N.R. , Ohanian V. , Gratzer W.B.
Published: July 2002

Fodrin, a protein from bovine brain, immunologically related to spectrin, is shown, unlike some other proteins of generally similar appearance in the electron microscope, to resemble spectrin closely in its most distinctive structural characteristic, the very high α‐helix content. Like spectrin, it is also insoluble below pH 5. One of the subunits only is phosphorylated by the cAMP‐independent red cell membrane kinase, that phosphorylates the smaller subunit of spectrin. Fodrin also forms a ternary complex with F‐actin and the third constituent of the red cell membranes cytoskeleton, protein 4.1. In the presence of 4.1 the interaction between fodrin and F‐actin is enhanced. It is surmised that fodrin plays an analogous functional role in neuronal cells to that of spectrin in the red cell.
Definitive 15N NMR evidence that water serves as a source of ‘O’ during nitrite oxidation by Nitrobacter agilis
Authors: Kumar Sharad , Nicholas D.J.D. , Williams E.H.
Published: July 2002

Using 18O isotope shifts in 15N NMR it has been shown that during oxidation of nitrite to nitrate by Nitrobacter agilis, the third ‘O’ in nitrate originates from water.
Binding specificities of the lectins from Helix pomatia, soybean and peanut against different glycosphingolipids in liposome membranes
Authors: Månsson Jan-Eric , Olofsson Sigvard
Published: July 2002

The binding specificities of the lectins from Helix pomatia, soybean and peanut against glycosphingolipids containing β‐linked terminal D‐galactose and N‐acetyl‐D‐galactosamine and the role of sialic acid as a modulator of the binding specificity were investigated. The test system used consisted of liposomes containing the glycosphingolipids and lectins coupled to gel columns. Of the investigated glycosphingolipids only ganglioside GM2 bound to soybean agglutinin while Helix pomatia agglutinin was found to bind only GA2. Peanut agglutinin showed good affinity both for ganglioside GM1 and its asialoderivative GA1.
A membrane‐bound protein kinase from mouse liver stimulated by iron
Authors: Loeb Jacques E. , Creuzet Claudine , Komano Odile , Boissel Jean-Paul
Published: July 2002

At μM levels, iron stimulates strongly the in vitro phosphorylation of a membrane protein from mouse liver. This phosphorylation also occurs in the absence of other added divalent cations but to a lower degree. In SDS gels the phosphorylated protein has app. M
r 250 000 in the absence of mercaptoethanol and M
r 130 000 in its presence. It is phosphorylated on threonine residues.
The limiting rate of the ATP‐mediated dissociation of actin from rabbit skeletal muscle myosin subfragment 1
Authors: Millar N.C. , Geeves M.A.
Published: July 2002

The ATP‐induced dissociation of actoS1 has been studied at temperatures between −10°C and +30°C in a stopped‐flow apparatus using ethylene glycol as antifreeze. At temperatures at and below 0°C the observed rate of the dissociation of actin shows a hyperbolic dependence on ATP concentration. This is interpreted in terms of a rapid binding of ATP followed by an isomerisation of the ternary complex which results in actin dissociation. Ethylene glycol weakens ATP binding but the rate of the isomerisation is unaffected. The second order rate constant for the dissociation shows a break in the Arrhenius plot.
Calmodulin binding and protein phosphorylation in adrenal medulla coated vesicles
Authors: Geisow Michael J. , Burgoyne Robert D.
Published: July 2002

Coated vesicles from bovine adrenal medulla contained clathrin and major detergent‐insoluble polypeptides of 120‐100, 51 and 49 kDa. Intact coated vesicles and vesicles lacking clathrin light chains were bound by immobilized calmodulin in the presence of Ca2+. Clathrin in the form of 700 Å cages was not bound. The calmodulin binding components in intact coated vesicles are therefore contributed by the enclosed vesicle or by the 120‐100, 50 or 49 kda polypeptides. The 51 kDa component incorporated 32Pi from labelled ATP by an endogenous kinase activity; no other coat or vesicle membrane protein was phosphorylated in vitro, either by intrinsic or exogenous kinases.
Reconstitution of photosynthetic charge accumulation and oxygen evolution in CaCl2‐treated PS II particles
Authors: Imaoka Akiko , Yanagi Masayuki , Akabori Kozo , Toyoshima Yoshinori
Published: July 2002

The reconstitution of high O2 evolution in CaCl2‐treated PS II particles was achieved by the simultaneous addition of the 17‐, 23‐ and 34‐kDa proteins and total thylakoid lipids in the presence of 25% glycerol and 15 mM sodium cholate. The activity of the reconstituted membranes recovered to 85% of that of the non‐depleted original PS II particles at the optimal condition. By means of this reconstitution method, evidence for the cooperation of the three proteins in the recovery of O2 evolution in the CaCl2‐treated PS II particles was found by changing the concentration of NaCI in the assay medium, and the relationship between the amount of manganese retained in the water‐splitting complex and the O2 evolving activity was examined by using the partially solubilized PS II particles with n‐octyl‐β‐D‐glucoside.
Neurokinin B is hydrolysed by synaptic membranes and by endopeptidase‐24.11 (‘enkephalinase’) but not by angiotensin converting enzyme
Authors: Hooper Nigel M. , Turner Anthony J.
Published: July 2002

The major site of hydrolysis was the Gly8‐Leu9 bond. Angiotensin converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1) from pig kidney hydrolysed substance P releasing the C‐tenninal tripeptide Gly‐Leu‐MetNH2 but failed to hydrolyse neurokinin B. Pig brain striatal synaptic membranes hydrolysed neurokinin B producing a similar pattern of products as did endopeptidase‐24.11. Substantial inhibition of this activity was achieved with the selective inhibitor phosphoramidon. A combination of phosphoramidon and bestatin abolished the hydrolysis of neurokinin B by synaptic membranes. Thus, a bestatin‐sensitive aminopeptidase may play a role in the synaptic metabolism of neurokinin B in addition to endopeptidase‐24.11. This aminopeptidase appears to be distinct from aminopeptidase N (EC 3.4.11.2).
Effects of manno‐1‐deoxynojirimycin and 2,5‐dihydroxymethyl‐3,4‐dihydroxypyrrolidine on N‐linked oligosaccharide processing in intestinal epithelial cells
Authors: Romero Pedro A. , Friedlander Paula , Fellows Linda , Evans Stephen V. , Herscovies Annette
Published: July 2002

The effects of manno‐1‐deoxynojirimycin (ManDJN) and 2,5‐dihydroxymethyl‐3,4‐dihydroxypyrrolidine (DMDP) were compared in IEC‐6 intestinal epithelial cells in culture. ManDJN caused complete inhibition of N‐linked complex oligosaccharide synthesis whereas a maximum of 80% inhibition was obtained with DMDP. HPLC showed similar endo H‐sensitive oligosaccharides for control and treated cells. ManDJN caused a large increase in the levels of labeled Man7–9 GlcNAc and a decrease in Man5GlcNAc. DMDP produced similar changes except that the increase in Man7–9GlcNAc was less pronounced and some increase in glucosylated oligosaccharides was observed. Since the major oligosaccharides found in DMDP‐treated cells were non‐glucosylated, its primary effect on complex oligosaccharide synthesis is not due to inhibition of glucosidases, in contrast to what has been reported for influenza virus‐infected MDCK cells [(1984) J. Biol. Chem. 259, 12409‐12413].
Fractionation of chromatin fragments on columns of Biogel A50‐m at different salt concentrations
Authors: Huang Hui-Chuan , Cole R.David
Published: July 2002

Nuclease fragmented chromatin was chromatographed on Biogel at various NaCl concentrations. The yield of eluted chromatin, and its H1/core histone ratio was minimal at 0.18 M NaCl where the ratio of H1 subtypes H1c/H1 ab was maximal. Therefore, the eluted material was aggregation‐resistant chromatin while aggregatable chromatin remained on the columns. Previous results were interpreted as Hl depletion of chromatin by ion‐exchange properties of Biogel, but the primary phenomenon is now seen as a separation of classes of chromatin that differ in sensitivity to salt‐induced aggregation. At very low salt concentrations, Biogel chromatography can be used without concern for H1 depletion.
Picosecond fluorescence of reaction centres from Rhodospirillum rubrum
Authors: Freiberg A.M. , Godik V.I. , Kharchenko S.G. , Timpmann K.E. , Borisov A.Yu. , Rebane K.K.
Published: July 2002

The fluorescence kinetics of isolated Rhodospirillum rubrum reaction centres was studied. In the picosecond time range the decay was well approximated by two exponential components. The short‐lived component was observed only when P870 was reduced and had a maximal amplitude at about 910 nm. Its lifetime was equal to 7 ± 2 ps both at room temperature and at 77 K. It is inferred that this fluorescence component is emitted by excited photochemically active P870 before the formation of an intermediate radical‐pair state. The second, long‐lived component with a lifetime of about 100–200 ps was observed in a wider spectral range and had maxima at about 850 and 900 nm both under reducing and oxidizing conditions. The bulk of this luninescence seemed to be emitted either by photochemically inactive P870 and/or by traces of antenna bacteriochlorophyll.
The prosthetic groups of succinate dehydrogenase: 30 years from discovery to identification
Authors: Singer Thomas P. , Johnson Michael K.
Published: July 2002

Recent studies using magnetic circular dichroism at cryogenic temperatures, electron paramagnetic resonance (EPR) and linear electric field effect‐EPR (LEFE) of succinate dehydrogenase in membranes and in soluble, homogeneous preparations demonstrated the presence of 3 different Fe‐S clusters in the mammalian enzyme, as well as in a similar bacterial enzyme, fumarate reductase from Escherichia coli. There is one each of the 2Fe, 3Fe, and 4Fe clusters. Thus, succinate dehydrogenase is the first enzyme which has been shown to contain all 3 of these Fe‐S clusters. The enzyme also contains 1 mol 8α‐[N(3)‐histidyl]‐FAD. It has taken the combined expertise of many laboratories and 15 years of effort to identify the flavin component, and nearly 3 decades to identify the Fe‐S clusters. The data from physical methods appear to be internally consistent, in harmony with the results of chemical analysis, and provide a rational explanation for earlier results by the cluster extrusion method. There remains, however, a number of interesting and substantive questions for future investigations. This review traces the tortuous path, the many pitfalls and false leads, which have led us from the discovery of nonheme iron and ‘bound’ flavin in the enzyme to elucidation of their structures.
Close evolutionary relationship between the chromosomally encoded β lactamase gene of Klebsiellapneumoniae and the TEM β‐lactamase gene mediated by R plasmids
Authors: Arakawa Yoshichika , Ohta Michio , Kido Nobuo , Fujii Yasuaki , Komatsu Takayuki , Kato Nobuo
Published: July 2002

Sixty‐three percent homology of nucleotide sequence and 67% homology of deduced amino acid sequence were found between the chromosomally encoded β‐lactamase gene of Klebsiella
pneumoniae and the TEM β‐lactamase of transposon Tn3. Moreover, 22 out of 24 amino acid residues are identical around the predicted active site. It is therefore suggested that these two kinds β‐lactamases share a common evolutionary origin. The 0.5 kb DNA fragment of the cloned gene hybridized specifically with the chromosomal DNA of all the K. pneumoniae strains tested which had been isolated in Japan, USA and Europe.
In vitro interaction of the photoactive anticancer porphyrin derivative photofrin II with low density lipoprotein, and its delivery to cultured human fibroblasts
Authors: Candide C. , Morliére P. , Maziére J.C. , Goldstein S. , Santus R. , Dubertret L. , Reyftmann J.P. , Polonovski J.
Published: July 2002

Low density lipoprotein (LDL) doped with the anticancer mixture of hematoporphyrin derivatives Photofrin II (P2) competes with native LDL for binding to fibroblast receptors, despite a slight increase in the negative net charge related to the presence of acidic residues of porphyrins. P2 delivery to fibroblasts can be achieved by LDL, HDL3 or albumin doped with P2 (LDL‐P2, HDL‐P2 or A‐P2, respectively). P2 delivery to cells assessed by fluorescence measurement, is much more efficient, at low protein concentrations (10–20 ) by LDL‐P2 than by HDL‐P2 or A‐P2. Moreover, P2 delivery to cells by LDL‐P2 as a function of protein concentration is a saturable process, whereas P2 delivery by HDL‐P2 or A‐P2 is a linear process. Finally, reduction of the LDL‐receptor number by preincubation of fibroblasts in medium supplemented with lipoproteins results in a decrease of P2 delivery by LDL‐P2. These results suggest a special role of the LDL‐receptor pathway in P2 delivery to cells and could be of interest in cancer phototherapy by porphyrins.
A class I (Schiff base) fructose‐1,6‐bisphosphate aldolase of halophilic archaebacterial origin
Authors: Dhar Nenoo M. , Altekar Wijaya
Published: July 2002

The presence of a salt‐dependent class I type (schiff base‐forming and no metal requirement) fructose bisphosphate aldolase activity is shown for the first time in a halophilic archaebacterium.
Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts
Authors: Sowers Arthur E. , Lieber Michael R.
Published: July 2002

Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field‐induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent‐tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius of at least 8.4 nm the electropores resealed within 200 ms to a radius of about 0.5 nm and stayed at that radius thereafter. Video sequences clearly show that pores are induced preferentially in the cathodal hemisphere. Pores induced in the hemisphere facing the positive electrode were either (i) never greater than 0.5 nm in radius, (ii) much smaller in number if they were greater than 0.5 nm in radius, or (iii) shorter lived. Calculations indicated that an upper limit of 700 electropores were induced per membrane.
Human chorionic gonadotropin activates the inositol 1,4,5‐trisphosphate‐Ca2+ intracellular signalling system in bovine luteal cells
Authors: Davis John S. , West Leigh A. , Weakland Laura L. , Farese Robert V.
Published: July 2002

Human chorionic gonadotropin, hCG, a hormone which increases intracellular cAMP, provoked rapid (30 s) and sustained (up to 30 min) increases in the levels of inositol mono‐, bis‐ and trisphosphates (IP, IP2 and IP3, respectively) in bovine luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to hCG. Concentration‐dependent increases in inositol phosphates, cAMP and progesterone accumulation were observed in hCG‐treated luteal cells. hCG also induced rapid and concentration‐dependent increases in cytosolic free Ca2+ as measured by quin 2 fluorescence. These findings demonstrate that hCG stimulates the phospholipase C‐IP3 and diacylglycerol ‘second messenger’ system in the bovine corpus luteum.
Type 1 M protein of Streptococcus pyogenes
Authors: Morávek Ladislav , Kühnemund Otto , Havlíček Jiří , Kopecký Petr , Pavlík Manfred
Published: July 2002

Limited proteolysis of the surface of type 1 Streptococcus pyogenes by pepsin gives rise to fragment Pep M1 of M

r
20270 as the main product which covers the N‐terminal part of the M protein. The amino acid sequence was determined of the N‐terminal region of the M protein representing the most exposed part of the molecule on the surface fibrils of streptococcal cells, which seems to be very important for the differentiation of the individual serological types. The sequence differs from the homologous N‐terminal sequences of types 5, 6 and 24, and shows a homology with sequences repeating in the chain of type 24. Fragment Pep M1 binds to fibrinogen; the absence of its 30 N‐terminal amino acid residues, however, abolishes this interaction which is believed to play a role in the virulence of S. pyogenes.
A 1H‐NMR study of the solution conformation of secretin resonance assignment and secondary structure
Authors: Gronenborn Angela M. , Bovermann Gunter , Clore G.Marius
Published: July 2002

The solution conformation of the 27 residue polypeptide hormone secretin has been investigated by 1H‐NMR spectroscopy under conditions where it adopts a fully ordered structure as judged by circular dichroism spectroscopy, namely in an aqueous solution of 40% (v/v) trifluoroethanol. Using a combination of two‐dimensional NMR techniques the 1H‐NMR spectrum of secretin is completely assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. It is shown that under these conditions secretin adopts a conformation consisting of an N‐terminal irregular strand (residues 1–6) followed by two helices (residues 7–13 and 17–25) connected by a ‘half‐turn’ (residues 14–16); the last two residues (26 and 27) are again irregular. This conformation is shown to be very similar to that of glucagon in perdeuterated dodecylphosphocholine micelles and to that of the active 1–29 fragment of growth hormone releasing factor in 30% (v/v) trifluoroethanol.
Production of superoxide anions by a CNS macrophage, the microglia
Authors: Colton Carol A. , Gilbert Daniel L.
Published: July 2002

Microglia have been implicated in both physiological and pathological processes of the brain. Their possible roles have been compared to those of macrophages and granulocytes. Here we demonstrate the specific ability of microglia to secrete the superoxide radical ion in response to a complement activated agent, opsonized zymosan, and to phorbol myristate acetate. As in other organs, this endogenously produced reactive oxygen intermediate could have both beneficial and deleterious effects.
Evidence for structural distinction between PS IIα and PS IIβ reaction centers
Authors: Hsu Ban-Dar , Lin Wen-Jye , Lee Jee-Yau , Long Pan Rong
Published: July 2002

The technique of (γ‐ray) radiation inactivation has been used to estimate the functional sizes of the PS II centers (PS IIα and PS IIβ) responsible for the biphasicity of the fluorescence induction curve from DCMU‐inhibited spinach chloroplasts. It is found that the size of PS IIα (266 ± 5 kDa) is almost twice as large as that of PS IIβ (135 ± 5 kDa). The result clearly indicates that the two types of PS II are structurally distinct and furthermore, the difference exists in the reaction center per se. Implications on the organization of PS II reaction centers are discussed.
Recognition of inter‐transmembrane regions of acetylcholine receptor α subunit by antibodies, T cells and neurotoxins implications for membrane‐subunit organization
Authors: Atassi M.Zouhair , Manshouri Taghi , Yokoi Tsuyoshi
Published: July 2002

Three regions of the α chain of Torpedo californica acetylcholine receptor (AChR), corresponding to residues α262–276, α388–408 and α427–437 were synthesized, purified and characterized. The first two peptides have been proposed to occupy inter‐transmembrane regions while the third represented the C‐terminal segment, proposed by various models to be either extracellular or intracellular. Peptide α388–408 stimulated a good response in the AChR‐primed T cells of H‐2s haplotype mice, a low response in the H‐2q haplotype and no response in the H‐2b haplotype. Peptide α427–437 stimulated AChR‐primed T cells of the H‐2s haplotype, but caused no response in the q and b haplotypes. Peptide α262–276 evoked no in vitro stimulation in any of the s, q or b haplotypes. In antibody binding studies, peptide α388–408 bound antibodies raised against free AChR or against membrane‐bound AChR. The other two peptides showed little or no binding activity. Further, peptide α388–408 bound specifically both 125‐I‐labelled bungarotoxin and cobratoxin, while the other two peptides had no binding activity. These results were consistent with only one of the models for subunit organization with‐in the membrane.
Quantitation and identification of carbon‐13 isotopomers by homonuclear polarization transfer NMR experiments
Authors: Boudot Denis , Canet Daniel , Brondeau Jean , Martin Francis
Published: July 2002

The simplest pulse sequence (π/2)x—τ— (π/2)y (Acquisition) for nuclear spin polarization transfer is used in a selective fashion: transfer only flows from a nucleus A to any nucleus X J‐coupled to A, provided that the carrier frequency coincides with the A resonance and that τ is slightly varied around a properly chosen value, relevant free induction decays being co‐added. According to the value selected for τ (1/2J or 1/4J, J: one bond carbon‐carbon coupling constant), it proves possible to sort out and compare subspectra pertaining to isotopomers involving one, two, three, and four consecutive 13C. The method has been applied to an extract of mycelium grown on a medium containing 13C‐labeled acetate, with the aim of delineating metabolic pathways from proportions of the detected isotopomers.
Identification of a novel protein phosphatase catalytic subunit by cDNA cloning
Authors: da Cruz e Silva Odete B. , da Cruz e Silva Edgar F. , Cohen Patricia T.W.
Published: July 2002

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.
2‐Mercaptoethylamine, a competitive inhibitor of spermidine synthase in mammalian cells
Authors: Hibasami Hiroshige , Kawase Masahiro , Tsukada Tetsuya , Maekawa Satoru , Sakurai Masaki , Nakashima Kunio
Published: July 2002

Spermidine synthase from rat ventral prostate was inhibited by 2‐mercaptoethylamine (MEA). Inhibition of spermidine synthase by MEA was competitive with respect to one of the substrates putrescine, but not competitive with respect to the other substrate decarboxylated S‐adenosylmethionine. MEA markedly depressed spermidine and spermine contents in human erythroid leukemia K562 cells, suggesting that these changes resulted from the inhibitory effect of MEA on spermidine synthase in situ.
A new strategy for primary structure determination of proteins: Application to bovine β‐casein
Authors: Carles Christophe , Huet Jean-Claude , Ribadeau-Dumas Bruno
Published: July 2002

A new approach has been developed for sequencing proteins. A radioactive label is attached specifically to the C‐terminus of the protein. The labelled molecule is subjected to varying proteolysis conditions. From the electrophoretic patterns (SDS‐PAGE) of the hydrolysates, appropriate cleavage conditions are selected, giving labelled peptides of different lengths which are purified. The labelled peptides are sequenced in order of increasing size (from 1 to n), peptide (i) being sequenced until the N‐terminal sequence of peptide (i‐1) is encountered. This approach allows the determination of a complete protein sequence with a minimal number of Edman cycles. The method was successfully applied to bovine β‐casein (209 residues) which was completely resequenced with only 239 Edman cycles.
Sequencing of peracetylated oligosaccharides by rotating‐frame nuclear Overhauser enhancement spectroscopy
Authors: Dabrowski Janusz , Ejchart Andrzej , Bruntz Renate , Egge Heinz
Published: July 2002

A combination of two‐dimensional total correlation spectroscopy (TOCSY) and rotating‐frame nuclear Overhauser enhancement spectroscopy (ROESY) is suggested as the optimum strategy for determining the primary structure of peracetylated oligosaccharides.
Evidence for reversible multiple redox transformations of [3Fe‐4S] clusters
Authors: Armstrong Fraser A. , Butt Julea N. , George Simon J. , Hatchikian E.Claude , Thomson Andrew J.
Published: July 2002

Analysis of the diffusionless cyclic voltammetry of ferredoxin III from Desulfovibrio africanus, co‐adsorbed with neomycin as an electroactive film on pyrolytic graphite ‘edge’ electrodes, shows that the reduced [3Fe‐4S]0 cluster undergoes further fast, chemically reversible, two‐electron reduction, at a potential E
0' = ca. −720 mV (pH 7.15, 0°C). The pH dependence of E
0' (pH 6.25–7.80) indicates net transfer of two H+. Observation of similar voltammetric waves in other proteins specifically containing [3Fe‐4S] centres suggests that extensive redox activity and stabilisation of what is formally an all‐Fe(II) species, may be a common, perhaps characteristic, feature of this cluster type.
Metallothionein genes from the flowering plant Mimulus guttatus
Authors: de Miranda J.R. , Thomas M.A. , Thurman D.A. , Tomsett A.B.
Published: July 2002

In response to excess metal, higher plants produce metal‐binding peptides ([γEC]nG) whose biosynthesis is believed to be mediated by enzymes involved in glutathione (γECG) metabolism. In contrast, animals synthesize metallothioneins, gene‐encoded low molecular weight cysteine‐rich metal‐binding proteins. In an investigation of copper‐regulated genes in the copper‐tolerant flowering plant Mimulus guttatus, we have isolated a series of cDNA clones identifying two genes which encode a protein with class I metallothionein domains. This represents the first description of a metallothionein gene in a flowering plant.
Enhancement of FGF‐like polypeptides in the retinae of newborn mice exposed to hyperoxia
Authors: Nyberg Fred , Hahnenberger Rudolph , Jakobson Åke M. , Terenius Lars
Published: July 2002

Retinae from neonatal mice exposed to prolonged hyperoxia (100% oxygen), show marked vasoproliferation. Extracts of such retinae were chromatographed on a heparin‐Sepharose column and the absorbed material subjected to HPLC fractionation. Two components, approx. 10 and 18 kDa, respectively, were found to have angiogenic activity, which was higher than in corresponding extracts from animals exposed to air. Both fractions and an additional 5 kDa component reacted with an antibody to basic fibroblast growth factor (bFGF) and showed higher levels in hyperoxia. The data suggest that hyperoxia activates angiogenic factors belonging to the heparin binding family.
Absence of tryptophan fluorescence quenching by metal cations in delipidated bacteriorhodopsin
Authors: Jang Du-Jeon , van den Berg R. , El-Sayed M.A.
Published: July 2002

The addition of metal cations to deionized bacteriorhodopsin (bR) quenches the steady‐state tryptophan (Trp) fluorescence intensity and reduces the decay times of some of its picosecond components. Similar quenching processes of the Trp emission are not observed in deionized 75% delipidated bR (dLbR). The results are discussed in terms of conformational changes taking place in the protein upon delipidation.
Cycle‐related LHRH responsiveness of superfused pituitary cells in a Phenol red free medium
Authors: O'Conner James L. , Kellom Theresa A.
Published: July 2002

Anterior pituitary cell cultures are frequently used in studying the control of gonadotropin secretion. Historically, many (if not most) of these studies have been performed in the presence of Phenol red as a pH indicator. Phenol red preparations, because of their potential estrogenic activity, may have influenced the results of previous studies defining the relative luteinizing hormone releasing hormone responsiveness of rat anterior pituitary cells derived from various stages of the estrous cycle. We therefore felt it of interest to investigate this possibility by repeating our previous cycle‐related superfusion studies [(1988) Life Sci. 42, 61‐72] in the absence of these Phenol red preparations. Comparisons of data obtained in the presence or absence of Phenol red revealed cells derived from late proestrous (19.00) and cultured in the absence of Phenol red continued to evidence the highest LH responsiveness. However, diestrous 1 08.00 cells cultured in the absence of Phenol red were lower in responsiveness than previously observed in the presence of the substance and the responsiveness of proestrous 08.00 and 15.00 in the presence was lower in comparison to the same stages in the absence of Phenol red. The results suggest that Phenol red preparations are capable of modulating LHRH responsiveness in superfusion and that the effect is more pronounced at certain cycle stages than at others.
Submicromolar Ag+ increases passive Na+ permeability and inhibits the respiration‐supported formation of Na+ gradient in Bacillus FTU vesicles
Authors: Semeykina Anna L. , Skulachev Vladimr P.
Published: July 2002

The effect of Ag+ on Na+ pumping by Na+‐motive NADH‐quinone reductase and terminal oxidase has been studied in Bacillus FTU inside‐out vesicles. Very low concentrations of Ag+ (C

= 1 × 10−8M or 2 × 10−12 g ion · mg protein−1) are shown to inhibit the uphill Na+ uptake coupled to the oxidation of NADH by fumarate or of ascorbate + TMPD by oxygen but exert no effect on the H+ uptake by the H+‐motive respiratory chain. Low Ag+ also induces a specific increase in the Na+ permeability of the vesicles. HQNO, added before and not after Ag+, prevents the Ag+‐induced permeability increase, with effective HQNO concentrations being similar to those inhibiting the uphill Na+‐uptake coupled to the NADH‐fumarate oxidoreduction. Reduction of terminal oxidase by ascorbate + TMPD in the presence of cyanide sensitizes the Na+ permeability to Ag+. It is suggested that low [Ag+], known as a specific inhibitor of electron transport by the Na+‐motive NADH‐quinone reductase, uncouples the electron and Na+ transports so that the Ag+‐modified NADH‐quinone reductase operates as an Na+ channel rather than an Na+ pump. This effect is discussed in connection with the antibacterial action of Ag+.
Purification, cDNA‐cloning and expression of human diacylglycerol kinase
Authors: Schaap Dick , de Widt John , van der Wal Jose , Vandekerckhove Joel , van Damme Jose , Gussow Detlef , Ploegh Hidde L. , van Blitterswijk Whim J. , van der Bend Rob L.
Published: July 2002

Diacylglycerol (DG) kinase attenuates the level of the second messenger DG in signal transduction, and therefore possibly modulates protein kinase C (PKC). DG kinase was purified to homogeneity from human white blood cells, showing an M
1 of 86 kDa as determined by SDS‐PAGE and gel filtration. Two amino acid sequences of tryptic peptides from DG kinase were determined and degenerate oligonucleotides were prepared and used in the polymerase chain reaction. An amplified DNA fragment was subsequently used to clone the full‐length human DG kinase cDNA. This sequence is the human homolog of porcine DG kinase cDNA sequence reported recently [1]. The sequence contains a double EF‐ hand structure typical for Ca2+ binding proteins. DG kinase further contains a double cysteine repeat that is present in all PKC isoforms, where it constitutes the phorbol ester (and most likely diacylglycerol) binding site. Therefore we speculate that the double cysteine repeat in DG kinase is involved in DG binding. DG kinase is transcribed as a single mRNA of 3.2 kb, that is highly expressed in T‐lymphocytes. The human DG kinase cDNA when transfected in mammalian cells (COS‐7) results in a 6–7‐fold increase of DG kinase activity.
Streptavidin‐induced lysis of homologous biotinylated erythrocytes Evidence against the key role of the avidin charge in complement activation via the alternative pathway
Authors: Muzykantov Vladimir R. , Smirnov Michael D. , Samokhin Gennady P.
Published: July 2002

It is shown that non‐covalent attachment of streptavidin, as well as of avidin, to biotinylated human erythrocytes induces homologous hemolysis by complement. Rabbit antiserum against human C3 is found to inhibit the lysis specifically as compared with non‐immune rabbit serum. Efficiency of lysis inhibition is greater for avidin‐ and streptavidin‐induced lysis of biotinylated human erythrocytes than for antibody‐sentitized sheep erythrocytes. In contrast to positively charged avidin (pI II), streptavidin is a neutral protein. Hence, hemolysis of streptavidin‐carrying erythrocytes is inconsistent with the suggestion on the crucial role of avidin charge in lysis. Membrane alterations (cross‐linking and clusterization of biotinylated components) induced by avidin (streptavidin) seem to be a more plausible explanation for the lysis.
Identification of the protein and cDNA of the cardiac Na+ /H+ exchanger
Authors: Fliegel Larry , Sardet Claude , Pouyssegur Jacques , Barr Amy
Published: July 2002

We examined the myocardial form of the Na+ /H+ exchanger. A partial length cDNA clone was isolated from a rabbit cardiac library and it encoded for a Na+ /H+ exchange protein. In comparison with the human Na+ /H+ exchanger, the sequence of the 5′ end of the cDNA was highly conserved, much more than the 3/t' region, while the deduced amino acid sequence was also highly conserved. To further characterize the myocardial Na+ /H+ exchange protein, we examined Western blots of isolated sarcolemma with antibody produced against a fusion protein of the Na+ /H+ exchanger. The antibodies reacted with a sarcolemma protein of 50 kDa and with a protein of 70 kDa. The results show that the rabbit myocardium does possess a Na+ /H+ exchanger protein homologous to the known human Na+ /H+ exchanger.
Folding around the C‐terminus of human carbonic anhydrase II Kinetic characterization by use of a chemically reactive SH‐group introduced by protein engineering
Authors: Freskgård Per-Ola , Carlsson Uno , Mårtensson Lars-Göran , Jonsson Bengt-Harald
Published: July 2002

We are characterizing the process of refolding of the enzyme human carbonic anhydrase II from the denatured state in guanidine hydrochloride. To describe the folding in defined parts of the protein we use protein engineering to introduce cysteine residues as unique chemically reactive probes. The accessibility of the cysteine SH‐group to the alkylating reagent iodoacetate, at different stages during refolding, is used to give a kinetic description of the folding process. The structuration of the C‐terminal part of the polypeptide chain, which is involved in a unique ‘knot’ topology, was investigated. Our results show that the structure around the C‐terminal, composed of the outermost β‐strands in a dominating β‐structure that extends through the entire protein, is formed relatively late during refolding. In contrast, it was found that β‐strands located in the interior of the protein were structured very rapidly. The final native structure is formed in a process that is slower than those observed for formation of β‐structure.
Cis‐trans isomerization is rate‐determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase
Authors: Fransson Cecilia , Freskgård Per-Ola , Herbertsson Helena , Johansson Åsa , Jonasson Per , Mårtensson Lars-Göran , Svensson Magdalena , Jonsson Bengt-Harald , Carlsson Uno
Published: July 2002

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t½=9 min). Kinetic data from ‘double‐jump’ measurements indicate that proline isomerization might be rate determining, in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics; the presence of isomerase during reactivation lowers the half‐time or the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis‐peptidyl‐Pro bonds at Pro30 and Pro202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) wore constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPlase the P202N mutant behaved exactly like the unmutaled enzyme, Thus, cis‐trans isomerization of the Pro202
cis‐peptidyl bond is not rate determining in the reactivation process, The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.
Solubilization steps of dark‐adapted purple membrane by Triton X‐100 A spectroscopic study
Authors: Meyer Olivier , Ollivon Michel , Paternostre Marie-Thérèse
Published: July 2002

Ultraviolet‐visible spectroscopy has been used to follow the solubilization of the dark‐adapted purple membrane of Halobacterium halobium by Triton X‐100. Turbidity of purple membrane fragments and absorbance of bacteriorhodopsin variations during continuous addition of detergent give solubilization profiles exhibiting several break points corresponding to different equilibrium stages of the solubilization process. The present method allows the determination of the detergent to protein + lipid ratio in mixed aggregates at the corresponding break points. It was concluded that, when performed systematically, this technique is a very convenient and powerful tool for the quantitative study of biomembrane‐to‐micelle transition.
Expression of human collagen type IV genes is regulated by transcriptional and post‐transcriptional mechanisms
Authors: Schmidt Cornelia , Pollner Reinhold , Pöschl Ernst , Kühn Klaus
Published: July 2002

The molecules of the basement membrane specific collagen type IV are heterotrimers consisting of two α1(IV) and one α2(IV) polypeptide chains. Comparison of the ratios of transcription by nuclear run‐on analysis and mRNAs by RNase protection assay indicates the involvement of transcriptional as well as post‐transcriptional events in the control of overall collagen type IV expression. The relative ratios of transcription of the respective genes COL4A1 and COL4A2 remained near 2:1 in most cells, whereas the ratio of mRNA steady‐state levels α1(IV)/ α2(IV) varied from 0.3:1 to 1:1 and did not parallel the subunit structure of the protein. Nevertheless, secreted protein shows a 2:1 ratio of the subunit polypeptides. This indicates that post‐translational processes during chain selection, aggregation and secretion finally determine the amount of secreted protein.
Localization of myosin IIB at the leading edge of growth cones from rat dorsal root ganglionic cells
Authors: Cheng Toni P.O. , Murakami Noriko , Elzinga Marshall
Published: July 2002

Primary cultures of rat dorsal root ganglionic (DRG) cells were stained With isoform‐specific antibodies against non‐muscle myosin II. Antibodies against the brain type myosin (MIIB) stained the peripheries of growth cones and non‐neuronal cells. Double staining of the cells with the anti‐myosin antibodies and rhodamine‐phalloidin or anti‐actin antibodies indicated that MIIB co‐exists, with F‐actin, at the leading edge. Antibodies against platelet myosin stained neither leading edges nor neurites, but stained the cell bodies of neurons and the stress fibers of non‐neuronal cells. These results suggest that MIIB functions in the motility of the leading edge of DRG cells.
Iron released from an erythrocyte lysate by oxidative stress is diffusible and in redox active form
Authors: Ferrali Marco , Signorini Cinzia , Ciccoli Lucia , Comporti Mario
Published: July 2002

The incubation of a ghost‐free erythrocyte lysate with the oxidizing agent phenylhydrazine resulted in both methemoglobin formation and release of iron in a desferrioxamine (DFO)‐chelatable form. The released iron was diffusible, as shown by a dialysis carried out simultaneously with the incubation. When the dialysate was added to erythrocyte ghosts or to microsomes from liver or brain, lipid peroxidation developed in the membranes, indicating that the diffusible iron was in a redox active form. The addition of ATP to the lysate markedly increased both iron diffusion and lipid peroxidation in the membranes subsequently added to the dialysate. The possible implication of these data in some well known pathologies is discussed.
Stable changes in expression or activation of G protein αi or αq subunits affect the expression of both β1 and β2 subunits
Authors: Hermouet Sylvie , Murakami Takeshi , Spiegel Allen M.
Published: July 2002

G proteins consist of three subunits: α,β and γ. Four β subunits have been cloned: β1 and β4 (36 kDa), and β2 and β3 (35 kDa). We studied endogenous β subunits in mouse NIH 3T3 fibroblasts stably expressing high levels of G protein α subunits after transfection with cDNAs encoding αi1, αi2, αi3 and αq. Immunoblots showed that NIH 3T3 cells express β36 and β35 subunits; in these cells,β35 subunits are four times more abundant than β36 subunits. We could detect β1 and β2 mRNA, but neither β3 norβ4 mRNA. We found that a stable increase in expression of wild‐type αi1, αi2, αi3or αq subunits is always accompanied by an increase in β1 and β2 mRNA and protein levels. There was no evidence of selectivity for an increase in β1 rather than β2 subunits depending on the type of α subunit overexpressed. However, constitutive activation or inactivation of α subunits induced specific changes in β subunits. Expression of constitutively inactivated αi2 subunits was accompanied by an increase in mRNA and protein levels of both β subunits. In contrast, cells expressing constitutively activated αi2 subunits did not show any change in the amount of β proteins expressed in membranes, despite a significant increase in β1 and β2 mRNA. We conclude that stable changes in the levels of expression or degree of activation of Gα subunits affect the level of expression, and possibly the turn‐over, of β subunits, without selectivity among β1 and β2 subunits.
Functional expression of a MAP kinase kinase in COS cells and recognition by an anti‐STE7/byrl antibody
Authors: Haystead Clare M.M. , Wu Jie , Gregory Peter , Sturgill Thomas W. , Haystead Timothy A.J.
Published: July 2002

Mitogen‐activated protein (MAP) kinases 042mapk and p44mapk are activated by dual tyrosine and threonine phosphorylation in vivo. Both MAPKs are phosphorylated and activated in vitro by an activator recently identified as a protein‐tyrosine/threonine kinase. We have isolated a putative cDNA for a MAP kinase kinase (MAPKK) and determined its structure [Proc. Natl. Acad. Sci. USA, in press]. The protein encoded by this cDNA shares sequence homology with two yeast protein kinases byrl and STE7. We now report that stimulation with serum of COS cells expressing this protein amplifies MAPK activator activity markedly. The increased activity co‐migrates during chromatography with the expressed 45 kDa protein, recognized by an anti‐STE7/byrl antibody, and is abrogated by treatment with phosphatase 2A. Thus, this cDNA encodes a functional MAPKK. The anti‐STE7/byrl antibody also recognized a 46 kDa COS cell protein that was resolved from the expressed MAPKK by anion‐exchange chromatography. This immunoreactive protein co‐eluted with endogenous MAPKK activity, suggesting identification of the immunoreactive band as monkey MAPKK.
Inclusion body formation by interleukin‐1β depends on the thermal sensitivity of a folding intermediate
Authors: Wetzel Ronald , Chrunyk Boris A
Published: July 2002

We show that sequence and growth temperature effects on IB formation in the small, monomeric β‐barrel protein interleukin‐1β (IL‐1β) can be quantitatively reproduced in an in vitro system in which IL‐1β is refolded from denaturant at different temperatures. The results suggest that temperature and mutational effects on IB formation may be based on intrinsic properties of the protein sequence rather than interactions with chaperones or other cellular factors. We also report striking correlations of IB formation with mutation‐dependent changes in residue hydrophobicity. The nature of these trends differs considerably with residue position, however, suggesting that they are mediated by particular local environments created by an ordered structure.
Molecular characterization of a class IV human alcohol dehydrogenase gene (ADH7)
Authors: Yokoyama Shozo , Matsuo Yoshinori , Ramsbotham Rebecca , Yokoyama Ruth
Published: July 2002

Class IV alcohol dehydrogenase (ADH) is a form preferentially expressed in stomach. We report here the isolation and sequence determination of a novel human ADH gene (ADH7). Phylogenetic analysis strongly suggests that ADH7 is a functional class IV ADH gene.