bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-strand breaks or alkali-sensitive sites in the DNA of human myeloma plasma cells and chronic lymphocytic leukemia lymphocytes
Authors: Brox L. , Ng A. , Pollock E. , Khaliq A. , Belch A.
Published: December 2009

Alkaline-elution studies showed significant levels of either DNA single-strand breaks or alkali-sensitive sites in the plasma cells of six out of six myeloma patients and in the lymphocytes of two out of four patients with chronic lymphocytic leukemia as compared with normal human lymphocytes. The increased rate of DNA elution was variable from sample to sample with a range that would correspond to that observed with 100–1000 rad (1 rad = 10 mGy) of X-ray irradiation. This alteration in DNA structure was observed in both new and advanced patients, did not appear to be related to prior therapy, and did not affect the in vitro viability of these cells. Repetitive alkaline-elution profiles obtained with tumor cells from three patients were similar on subsequent samples obtained 1 month apart. Altered DNA elution was not evident in peripheral blood lymphocytes from myeloma patients with altered plasma cell DNA elution. These observations are interesting in light of the recent hypothesis that breaking and rejoining of DNA, regulated by poly(ADP-ribosyl)ation, may be a general mechanism of altering gene expression during differentiation.
Contrasting effect of phosphorylcholine-binding protein from rat and rabbit on heparin–lipoprotein interaction: a role of sialic acid
Authors: Saxena Uday , Nagpurkar Arun , Mookerjea Sailen
Published: December 2009

The effect of rat phosphorylcholine-binding protein (PCBP) and rabbit C-reactive protein (CRP) on the serum lipoprotein–heparin–Ca2+ precipitation reaction has been examined. Rabbit CRP, a nonglycosylated acute-phase protein, has been isolated from the serum after turpentine-induced inflammation following an isolation procedure similar to that used for rat PCBP. The mobilities of rabbit CRP and rat PCBP on polyacrylamide gel electrophoresis and immunoelectrophoresis have been found to be different. Rabbit CRP, even though a P-choline binding protein, was found to have no inhibitory effect on the serum lipoprotein–heparin–Ca2+ precipitation reaction. Desialylated rat PCBP obtained by enzymatic desialylation of rat PCBP no longer inhibited the precipitation reaction, even though it retained its P-choline binding property and immunochemical identity with rat PCBP. The results suggest that the binding of rat PCBP and rabbit CRP to the P-choline moiety is not a sufficient requirement, and it now appears that the sialic acid moiety must also be present on these proteins to observe the inhibition of lipoprotein precipitation. This requirement is fulfilled by rat PCBP, but not by rabbit CRP which is nonglycosylated.
The ribosomal RNA genes of Locusta migratoria: copy number and evidence for underreplication in a polyploid tissue
Authors: Oishi M. , Locke J. , Wyatt G. R.
Published: December 2009

From libraries of Locusta migratoria genomic DNA in bacteriophage λ, clones have been isolated that hybridize with one or both of the 18S and 28S components of locust rRNA. Six clones studied show common patterns of restriction sites in the coding sequences and some include an intron in the 28S region. Subcloned sequences from within the 18S and 28S coding regions were used as probes in dot hybridization assays of the rRN A gene copy number in DNA prepared from several locust tissues and stages. The 18S and 28S probes gave similar results, showing about 4000 copies/haploid genome in testis, embryo, and fat body from larvae and immature adults of both sexes. This is by far the greatest rRNA gene copy number yet reported for any insect. DNA from reproductively mature adult female fat body showed a consistent reduction in the copy number by about one-third. Mature adult male fat body DNA showed no significant reduction in the copy number. It therefore appears that the final round of DNA replication in the adult female fat body, which produces 8-ploid and 16-ploid cells active in vitellogenin synthesis, involves underreplication of rDNA.
Steroid sulfotransferases and steroid sulfate sulfatases: characteristics and biological roles
Authors: Hobkirk R.
Published: December 2009

This review discusses the biological roles of steroid sulfotransferase enzymes (ST's) and steroid sulfate sulfohydrolases (sulfatases) mainly in mammalian tissues. In addition, some consideration is given to certain characteristics of these enzymes and, where possible, to their biological control. A considerable number of ST's of varying specificities, substrate affinities, and kinetics appear to exist. Several of these possess the properties of regulatory enzymes. ST's which act upon estrogen in reproductive tissues, such as uterus, are of particularly high affinity, appear to be under some biological control, and may exert important effects upon estrogen action. Although biosynthetic pathways involving steroid sulfate intermediates have been described, their importance is difficult to determine. The presence of an esterified sulfate group on a steroid molecule may markedly affect the action of enzymes, such as hydroxylases, upon the steroid structure in both a qualitative and quantitative sense. The number of different steroid sulfatases is not well understood. A sterol sulfatase present in the female reproductive tract appears capable of destabilizing the sperm head membrane by hydrolyzing sterol sulfates necessary for its integrity, and hence enabling the fusion of sperm and ovum. Other sulfatases may utilize blood-borne steroid sulfates for the ultimate production of estrogen which, in fetal membranes, could play a role in parturition and, in breast tumours, could function as a growth promoting agent. Brain sulfatases could possibly produce steroid hormones for purposes of tissue differentiation and (or) feedback control mechanisms, but this is not firmly established.
Purification and properties of an exocellular β-glucosidase of Candida molischiana (Zikes) Meyer and Yarrow capable of hydrolyzing soluble cellodextrins
Authors: Gondé Pierre , Ratomahenina Robert , Arnaud Alain , Galzy Pierre
Published: December 2009

The exocellular enzyme β-glucosidase of Candida molischiana was studied. This strain is able to ferment soluble cellodextrins. The enzyme was partially purified by ion-exchange chromatography and gel filtration. The molecular weight of this enzyme was 120 000; its optimum pH was between 4 and 4.5 and its optimum temperature was 60 °C. This enzyme was active against different soluble glucosides and was inhibited by p-chloromercuribenzoate, gluconolactone, and glucose. A "glucosyltransferase" activity appeared in the presence of ethanol. The biosynthesis of the enzyme was constitutive but repressed by glucose.
Glycoproteins on the surface of smooth muscle cells involved in their interaction with type V collagen
Authors: Leushner James R. A. , Haust M. Daria
Published: December 2009

Type V collagen is a major component of the pericellular coat of smooth cells (SMC). The purpose of the present study was to assess biochemically the nature of an in vitro interaction between bovine aortic SMC and type V collagen from the same source. This interaction was originally shown to be mediated by a cell-surface glycoconjugate. Data obtained in the present study suggests that the binding system consists of integral membrane glycoproteins which act alone or in combination with a surface glycolipid in type V attachment. The nature of this system was indicated by the finding of 80 000 and 50 000 components in the plasma membrane fractions which were specifically retained by type V collagen – Sepharose columns and incorporated both methionine and mannose label. Moreover, inhibition of protein synthesis lowered SMC attachment by 25%. The mannose label associated with these components was probably in the form of a simple oligosaccharide at the attachment site since it bound to concanavalinA (ConA) and was sensitive to endoglycosidase H. Iodinated ConA labelling indicated elevated levels of these components were associated with SMC – type V collagen interaction. The attachment region on the type V molecule was localized within the cyanogen bromide peptide 6 of the α2 (V) chain.
The subcellular distribution of free fatty acids released during post-decapitative ischemia in rat cerebral cortex
Authors: Baker R. Roy , Loh Zou Dao
Published: December 2009

After periods of 5 and 30 min following decapitation, rat cerebral cortices were removed and subcellular fractions were prepared. Fractions P1A (large myelin), P1B (nuclei), P1C (cells and debris), P2A (small myelin), P2B (synaptosomes), P2C (mitochondria), and P3 (microsomes) were isolated. Free fatty acid levels of 1.0 and 1.4 μmol/g tissue were found in the homogenates at the early and late times of ischemia. In the 30-min samples, P1A, P1C, and P2A had relatively high specific contents of total free fatty acids in comparison with other subfractions. At this time P2C was relatively enriched in arachidonate, P1A and P2A were enriched in palmitate, and P2B and P3 were enriched in stearate in comparison with the homogenate. P2C had the highest ratio of polyunsaturates/saturates in its free fatty acid pool. Comparing the 5- and 30-min samples, a large increase in the quantity of free fatty acids was found in fractions P1A and P2A, so that at the later time P1A + P2A contained 60 mol% of the free fatty acid in the total subfractions derived from cerebral cortex. In comparison with the homogenate, the lack of accumulation of free fatty acids in certain membranes known to possess phospholipase activities (e.g., phospholipase A2 in P2C) and the buildup of free fatty acids in P1A and P2A led to the hypothesis that free fatty acids may be migrating outwards from intracellular sites of production and accumulating in the multilamellar structure of myelin.
Influence of maternal mineral deficiency on the hepatic metallothionein and zinc in newborn rats
Authors: Gallant Karen R. , Cherian M. George
Published: December 2009

The effects of maternal Zn, Cu, or Fe deficiencies during late gestation on hepatic levels of metals and metallothionein (MT) and the binding of Zn and Cu to protein fractions were investigated in newborn rats. Timed pregnant rats were fed one of the following diets: Zn deficient (Zn-D), Cu-D, Fe-D, or control from day 12 of gestation until birth. The specific nutritional deficiency status of the dams was confirmed by low plasma levels of the deficient metal. Livers from pups were analyzed for MT, metal content, and metal–protein binding. Maternal Zn-D resulted in a greater than 50% reduction of hepatic MT levels in pups, whereas Cu-D and Fe-D had no significant effects. Pups in each deficient group showed a significant decrease in the hepatic levels of the respective metals. Fractionation of hepatic cytosols from the pups by Sephadex G-75 gel filtration showed that in both Fe-D and Cu-D pups the respective metals were depleted from the high molecular weight protein fraction, whereas in Zn-D pups the Zn was depleted mainly from the MT fraction (Ve/V0 ~ 2). Incorporation of [35S]cysteine into MT fractions was significantly lower in Zn-D pups as compared with control pups. These results indicate that there is a specific effect of the maternal Zn-D on the hepatic storage of Zn as MT in newborn rats.
A sensitive fluorographic method for the detection of nopaline and octopine synthase activities in Brassica crown-gall tissues
Authors: Holbrook Larry A. , Haffner Margaret , Miki Brian L. A.
Published: December 2009

L-[guanido-14C]Arginine has been used as a precursor for nopaline and octopine syntheses in the in vitro assay for opine synthase activities. Fluorographic detection of [14C]nopaline and [14C]octopine was optimized following their separation from other labelled compounds by paper electrophoresis. Compared with detection by phenanthrenequinone, fluorography was more specific for the guanidine compounds and severalfold more sensitive. Alternate precursors such as α-[14C]ketoglutarate or [14C]pyruvate were poor substitutes for [14C]arginine because they generated compounds that comigrated with opines. Four nonopine radioactive spots recurred in the in vitro assays. These have not been identified but have been distinguished from both octopine and nopaline. This technique has provided clear evidence for transformation of Brassica species by Ti plasmids of Agrobacterium tumefaciens and will be valuable in the analysis of extremely small tissue samples.
Flow kinetics of immobilized β-glucosidase
Authors: Hsuanyu Yuchiong , Laidler Keith J.
Published: December 2009

The enzyme β-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 °C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 °C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.
Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides
Authors: Schachter Harry
Published: December 2009

Detailed studies on the enzyme machinery responsible for the biosynthesis of protein-bound oligosaccharides of the Asn-GlcNAc and Ser(Thr)-GalNAc linkage types have allowed the formulation of some general rules which explain, at least in part, the branching patterns and microheterogeneity of these structures. These rules are discussed under the following headings: (i) competition of two or more enzymes for a common substrate; (ii) controls at the level of enzyme substrate specificity (e.g., critical sugar residues which turn enzyme activity on or off, branch specificity, and the role of the polypeptide in the glycoprotein substrate); (iii) substrate availability.
Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties
Authors: Squires E. James , Haard N. F. , Feltham L. A. W.
Published: December 2009

The zymogens of three gastric proteases of the Greenland cod (Gadus ogac) were isolated by exclusion chromatography and chromatofocusing. The cod zymogens were activated more rapidly at lower temperatures than porcine pepsinogen and, after activation, were further purified by exclusion chromatography. The cod proteases had more alkaline pH optima and were active over a wider range of pH than porcine pepsin. The specific activity of porcine pepsin on protein substrates was greater than that of the individual cod proteases. However, the cod proteases had cumulative activity on protein substrates that was greater than the sum of their individual activities. Cod protease I was active on pepsin-specific substrates, and cod proteases 2 and 3 were active as gastricsin-specific substrates. All three cod proteases had greater milk-clotting activity and hydrolysed hemoglobin to a greater extent than porcine pepsin. The Vmax and Km,app of the cod proteases were dependent upon the substrate, and Vmax/Km,app values of the cod proteases were generally lower than porcine pepsin. It is suggested that the cod proteases together exhibit broad substrate specificity and maintain activity over a wide range of conditions to enhance protein digestion in the cod stomach.
Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor
Authors: Walker Peter
Published: December 2009

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+-specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4, 0.4 μg/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 ± 0.9%, mean ± SEM) than in that of control mice (7.3 ± 0.5%, P < 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (Mrs) of 12 000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with α-chymotrypsin. Autoradiograms, following high-voltage electrophoresis and ascending chromatography on thin-layer cellulose, showed a marked similarity of the peptide maps of purified EGF, the IMMP of a SMG extract, and the translation product IMMPs. Furthermore, the peptide maps of the Mr 12 000, 9000, and 6000 IMMPs were highly concordant, suggesting that the Mr 12 000 and 9000 species are structurally related to the Mr 6000 EGF species. These observations support the hypothesis that thyroid hormones increase SMG EGF concentrations by increasing the number of poly(A)+ RNA species coding for EGF.
Osteonectin is a minor component of mineralized connective tissues in rat
Authors: Zung Paul , Domenicucci Carmelo , Wasi Safia , Kuwata Fumiyuki , Sodek Jaro
Published: December 2009

Osteonectin is a major glycoprotein of porcine and bovine bones and teeth that is found associated with hydroxylapatite crystal surfaces. From the ability of osteonectin to bind calcium ions, it has been proposed as a possible nucleator of hydroxylapatite crystal formation. Analysis of hydroxylapatite-bound proteins of rat bone and dentine, however, has revealed that osteonectin represents only 2.5 ± 1.5% of the hydroxylapatite-bound protein in long bones, 0.9 ± 0.5% in calvariae, and < 0.1% in incisor dentine of animals of different ages. Further, in vivo pulse–chase studies carried out in young adult rats have shown osteonectin to be synthesized at low levels in these tissues. Similarly, low levels of osteonectin were synthesized by rat calvarial cells in vitro. In contrast, fibroblastic cells from periodontal ligament and gingiva synthesized significantly greater amounts of osteonectin. These studies indicate that the low quantities of osteonectin in rat mineralized tissues are a consequence of low rates of formation rather than being due to rapid turnover. The virtual absence of osteonectin in incisor dentine correlates with the lack of peritubular dentine in rat, whereas the low osteonectin content of rat bones may reflect differences in their structure and biophysical properties compared with bones of larger mammals.
Production and characterization of monoclonal antibodies specific for the functional domains of poly(ADP-ribose) polymerase
Authors: Lamarre Daniel , Talbot Brian , Leduc Yvan , Muller Sylviane , Poirier Guy
Published: December 2009

Monoclonal antibodies were developed against poly(ADP-ribose) polymerase and analyzed for their reactivity against the NAD+- and DNA-binding fragments. Two fusions were performed to obtain hybridomas and the resulting anti-poly(ADP-ribose) polymerase antibodies were further screened by characterization of their immunoglobulin light chains. Five different hybridomas were isolated which produced different immunoglobulin light chains, all of which were specific for poly(ADP-ribose) polymerase. The specificities of these antibodies were determined by immunoblotting against the purified poly(ADP-ribose) polymerase, its autodegradation fragments, and the fragments prepared by limited proteolysis with chymotrypsin and papain. These fragments have been suggested to contain the NAD+-binding site, the DNA-binding site, and the automodification site, respectively. All the monoclonal antibodies reacted with the 116 kdalton (kDa) band corresponding to the purified enzyme. Four antibodies reacted exclusively with antigenic site(s) on the 46-kDa fragment which contains the DNA-binding site. A fifth antibody reacted exclusively with a clearly different antigenic site on the 74- and 54-kDa fragments which possess the NAD+ (substrate) binding site. The immunoreactivity with the major autodegradation products (69- and 46-kDa fragments) of the purified enzyme confirms this difference between the two groups of antibodies. The 22-kDa fragment corresponding to the automodification site does not show any immunoreactivity with the antibodies.
Effects of fatty acids on phosphatidylcholine biosynthesis in isolated hamster heart
Authors: Mock Thomas , Slater Tracy L. , Arthur Gilbert , Chan Alvin C. , Choy Patrick C.
Published: December 2009

The effects of stearic, oleic, and arachidonic acids on phosphatidylcholine biosynthesis in the hamster heart were investigated. When hamster hearts were perfused with labelled choline in the presence of fatty acids, biosynthesis of phosphatidylcholine was stimulated only by stearic acid. Stearic acid was found to accumulate in unesterified (free) form in the hamster heart after perfusion. The stimulation by stearic acid was mediated in vivo by an enhancement of CTP:phosphocholine cytidylyltransferase activity in the microsomal fraction of the hamster heart and the enzyme activity in the cytosolic fraction was not affected. In contrast with the observations in rat hepatocytes, cytidylyltransferase from the hamster heart was not stimulated directly by stearic acid. The selective activation of the microsomal enzyme when the heart was perfused with stearic acid suggests that activation of the enzyme was mediated via the modification of the membrane by stearic acid.
Synthesis of stress-induced protein in isolated and perfused rat hearts
Authors: Currie R. William
Published: December 2009

Isolated and perfused rat hearts were examined by two-dimensional gel electrophoresis and liquid scintillation counting for alterations in protein synthesis following incubation with L-[3H]leucine at 0.5–2.5, 2.5–4.5, or 4.5–6.5 h of perfusion. When 35-mL volumes of three different buffers were recycled for a 2-h period from 0.5 to 2.5 h, by fluorography little effect was seen on the normal patterns of protein synthesis and there was a moderate synthesis of a stress-induced protein (heat-shock protein) with a molecular mass of 71 × 103 daltons (SP71). However, hearts perfused with Krebs-improved Ringer 1 bicarbonate had the highest incorporation of L-[3H]leucine. When buffers were recycled for 30-min periods from 0.5 to 2.5 h, SP71 was synthesized in hearts perfused with Krebs–Henseleit original Ringer bicarbonate. Hearts perfused in a similar fashion with Krebs-improved Ringer 1 bicarbonate had the lowest incorporation of label into SP71 and in fact SP71 was undetectable on fluorograms. Overall protein synthesis was decreased and the ratio of SP71 to the total synthesis was increased at 4.5–6.5 h of perfusion when 35-mL volumes of Krebs-improved Ringer 1 bicarbonate was recycled for 2-h periods. A similar result was observed at 2.5–4.5 h of perfusion when this buffer was recycled for either the duration of the experiment or 30-min periods.
Partial purification and characterization of phosphoenolpyruvate carboxylase from the cyanobacterium Anabaena variabilis
Authors: Lem Nora W. , Penrose Donna M. , Glick Bernard R.
Published: December 2009

The partial purification and characterization of phosphoenolpyruvate (PEP) carboxylase from the cyanobacterium Anabaena variabilis is reported. Spheroplasts made from photoautotrophically grown cells were lysed to produce a crude cell extract which was fractionated by (i) (NH4)2SO4 precipitation and (ii) gel filtration through Sephacryl S-300. The peak of enzyme activity that was eluted from the column corresponded to a molecular mass of approximately 365 000 daltons; the molecular mass of the sub-units was found to be approximately 100 500 daltons as assessed by polyacrylamide gel electrophoresis. The pH optimum for PEP carboxylase activity was pH 7.0. The response of this A. variabilis enzyme to effectors distinguished this enzyme from that found in C3 plants and in bacteria. Malate inhibited enzyme activity but was a much more effective inhibitor at pH 7.0 (50% inhibition at 1.6 mM malate) than at pH 8.0 (15% inhibition at 10 mM malate). Glycine stimulated PEP carboxylase activity at both pH 7.0 and 8.0, while glucose 6-phosphate had no significant effect. The Km for HCO3− was calculated to be 14.5 ± 9.2 μM. These results suggest that PEP carboxylase from A. variabilis is kinetically similar to maize PEP carboxylase and may participate in inorganic carbon uptake for photosynthesis.
Reducing conditions induce a total degradation of the major zymogen granule membrane protein in both its membranous and its soluble form. Immunochemical quantitation of the two forms
Authors: Paquette Jean , Leblond François A. , Beattie Marlyne , LeBel Denis
Published: December 2009

The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 × 103 daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-α-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels. It is concluded that as much of the 92-kDa protein is soluble in the content as it is anchored in the membrane. The protease responsible for its degradation upon addition of DTT seems to be closely associated with the protein and could be involved in its posttranslational solubilization leading to its secretion.
Cerebrosides and psychosine disrupt mitochondrial functions
Authors: Strasberg Paula
Published: December 2009

Glucocerebroside and galactocerebroside increased the respiratory rate of liver and brain mitochondria by 33–400% and produced an average 30% decrease in oxidative phosphorylation. Psychosine stimulated mitochondrial respiration 66–700%. At concentrations over 100 μg/mg mitochondrial protein, oxidative phosphorylation was completely inhibited. Atractyloside did not prevent the rspiratory stimulation. Ca2+ transport was blocked and addition of ATP could not overcome this inhibition. The possible deleterious effect of glycosphingolipids on the conformation of the mitochondrial membrane and cellular bioenergetics is discussed in relation to the toxicity of accumulating glycosphingolipids in Gaucher and Krabbe diseases.
Photoinactivation of acetylcholinesterase by erythrosin B and related compounds
Authors: Tomlinson George , Cummings Maxwell D. , Hryshko Larry
Published: December 2009

Acetylcholinesterase was rapidly inactivated when exposed to light in the presence of xanthene dyes. Photosensitizing efficiency paralleled the dye triplet state quantum yields, increasing in the order fluorescein < eosin B < eosin Y < erythrosin B < rose bengal. The observed first-order rate constants of photoinactivation increased hyperbolically with dye concentration. Evidence for the formation of a dye–enzyme complex prior to inactivation was obtained from spectrophotometric and protein fluorescence quenching methods. The latter technique allowed estimates of the dye–enzyme dissociation constants for rose bengal (20 μM) and erythrosin B (30 μM). After photoinactivation, a portion of the dye became covalently bound to the enzyme. The photoinactivation reaction occurs in both aerobic (air saturated) and anaerobic (argon saturated) solution, with the rates of photoinactivation being about three to five times greater under the latter conditions. The aerobic reaction exhibits a large deuterium isotope enhancement effect and is largely (but not completely) quenched by 10−2 M azide. The anaerobic reaction is unaffected by azide and exhibits only a small deuterium isotope effect. These results indicate that the photoinactivation reaction proceeds mainly by a type II (singlet oxygen mediated) pathway under aerobic conditions and by a type I (radical) pathway under anaerobic conditions. The enzyme was protected from inactivation by edrophonium, a competitive inhibitor, but not by d-tubocurarine, a peripheral-site ligand, indicating that destruction of a crucial residue at or near the catalytic site is an important component of the inactivation process. Extensive destruction of tryptophan undoubtedly occurs, at least under aerobic conditions. The destruction of smaller amounts of other photosensitive residues cannot be ruled out. The pH dependence of the photoinactivation reaction appears to eliminate histidine as a primary target under both aerobic and anaerobic conditions; an additional group which appears to titrate in the region of pH 7–9 is observed under anaerobic but not aerobic conditions.
Properties of the alfalfa–Rhizobium meliloti symbiotic nitrogenase enzyme system in vivo and in vitro
Authors: Miller R. W. , Al-Jobore A. , Berndt W. B.
Published: December 2009

A comparison of hydrogen production and acetylene reduction by excised alfalfa nodules, nitrogenase proteins isolated from Rhizobium meliloti bacteroids, and nitrogenase proteins purified from Klebsiella pneumoniae revealed several differences in the catalytic properties of the in vivo and in vitro systems. In all cases, assays for nitrogenase activity were carried out at near-physiological enzyme concentrations. Intact nodules maintained high nitrogenase activity to much lower reaction temperatures than the isolated enzymes. The Q10 value for acetylene reduction was 1.4 for nodules, but exceeded 5 for isolated R. meliloti nitrogenase in the temperature range 12–28 °C. The reduction of substrates requiring more than two reducing equivalents by the isolated enzyme system was severely restricted by the effects of reaction temperature on electron flux, as shown by very low values for electron allocation to dinitrogen determined indirectly at 12 °C. Temperature-induced lags in the initial reaction velocity of the reconstituted Klebsiella nitrogenase system were not observed with the isolated, but unseparated, R. meliloti nitrogenase proteins when acetylene was the substrate. The respiration-supported nodule system was not saturated with oxygen under air at reaction temperatures between 12 and 25 °C. Measurements of the adenine nucleotide levels of whole nodules indicated that the observed effects of reaction temperature on nitrogenase in vitro may be partially compensated in vivo by increases in adenylate energy charge and marked decreases in the ratio of ADP/ATP which occur as the nodule temperature is lowered. From the results reported here, it is clear that the R. meliloti – alfalfa nitrogenase system is limited in activity by oxygen supply. However, electron flux is sufficient within the physiological temperature range to maintain the Mo–Fe protein in the catalytically active state.
Spontaneous cholesterol movement between lipid vesicles and monkey small intestinal brush border membrane
Authors: Sadana Tarun , Sanyal Sankar N. , Majumdar Siddartha , Dhall Kamla , Chakravarti Rabindra Nath
Published: December 2009

[14C]Cholesterol movement between egg phosphatidylcholine–cholesterol lipid vesicles and vesicles prepared from monkey small intestinal brush border membrane (BBMV) was studied in physiological buffer at 37 °C. The rate of cholesterol transfer from sonicated unilamellar vesicles (ULV) to BBMV follows apparently first-order kinetics. Intermembrane cholesterol movement was strikingly similar in both the directions. However, from BBMV to ULV, the transfer rate was three times faster than that of ULV to brush border membrane (BBM). Similarity in the rate constant was observed when cholesterol transfer was studied using either large multilamellar lipid vesicles or ULV as the donor and BBMV as the acceptor membrane. Rate constant was also the same when the acceptor membrane used was either intact BBMV or ULV prepared from BBM lipids. The rate of transfer of label was not affected even when the acceptor vesicle concentration was increased over fivefold, indicating the first-order nature of the reaction. Transfer of cholesterol from ULV to BBMV was accelerated by the presence of acetone, dimethyl sulfoxide (DMSO), deoxycholate, and papain. Partially purified nonspecific lipid-exchange protein increased the rate of cholesterol transfer by about threefold. Reduction in BBM cholesterol and phospholipid content was noted by DMSO, acetone, and deoxycholate, while papain caused a small depletion of membrane protein. Cholesterol transfer is temperature dependent with an activation energy of 31 kJ∙mol−1, which is almost identical in the presence or absence of nonspecific lipid-exchange protein. The molecular mechanism of intermembrane cholesterol movement is discussed in view of the kinetic data obtained.
Inhibition of microtubule assembly by poly(L-glutamic acid) and the site of its action
Authors: Fujii Toshihiro , Suzuki Tatsuo , Fujii Michiyo , Hachimori Akira , Kondo Yoshiyuki , Ohki Kosuke
Published: December 2009

Poly(L-glutamic acid) (PGA) suppresses the polymerization of porcine brain microtubule proteins and induces the depolymerization in vitro in a concentration-dependent manner. The extent of inhibition increases with increasing molecular weight of the PGA tested. A 50% inhibition of the protein polymerization was observed at a PGA (molecular weight = 60 000) to microtubule protein ratio of 0.04 (w/w), and complete inhibition was obtained at a ratio of 0.07. Such an inhibition on the polymerization by PGA is greatly decreased when Mg2+ is present at a higher concentration. The addition of PGA raises the critical concentration of microtubule proteins necessary for assembly. During incubation with PGA, microtubule proteins retain the ability to assemble, i.e., substoichiometric amounts of taxol considerably relieve the inhibition of assembly by PGA. PGA interacts with microtubule-associated proteins (MAPs) preferentially, because the amount of MAPs binding to PGA-Sepharose 4B is much larger than that of tubulin. Tau proteins were observed only in adsorbed fractions, while MAP-2 was present in both unbound and adsorbed fractions.
Purification and characterization of catalase HPII from Escherichia coli K12
Authors: Loewen Peter C. , Switala Jacek
Published: December 2009

Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts. The purified HPII was found to have equal amounts of two subunits with molecular weights of 90 000 and 92 000. Only a single 92 000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit. The apparent native molecular weight was determined to be 532 000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase. HPII was very stable, remaining maximally active over the pH range 4–11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea. The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility. On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit.
Effects of triorganotin-mediated anion–hydroxide exchange upon reconstituted cytochrome c oxidase proteoliposomes
Authors: Singh A. P. , Nicholls P.
Published: December 2009

Proteoliposomes containing cytochrome c oxidase and an internally trapped fluorescent pH probe (pyranine) were used to monitor respiration-dependent internal alkalinization and membrane potential formation. A maximum steady-state pH gradient of about 0.4 pH unit (vesicle interior alkaline) was obtained during active respiration in presence of reducing substrates and cytochrome c. This pH gradient was abolished by the triorganotin compounds tripropyl-, tributyl-, and triphenyl-tin chloride. At the same time, the membrane potential, measured by carbocyanine dye uptake, was slightly increased in value. Valinomycin, which abolishes the membrane potential, restores the value of ΔpH at low trialkyltin concentrations. The organotin compounds acted as electroneutral ionophores which exchanged intravesicular OH− ions with external SCN−, I−, and Cl− ions, but not [Formula: see text] or [Formula: see text] ions. Abolition of ΔpH is accompanied by an increase in respiration rate, but full resiratory stimulation only occurs when both Δψ and ΔpH are abolished by addition of both triorganotin and valinomycin. The triorganotin–valinomycin combination leads to active KC1 accumulation by the respiring proteoliposome, and it is necessary to postulate an electrically neutral KC1 efflux process to explain the continued steady respiration of the proteoliposomes in the presence of this ionophore combination.
The primary structure of the ribosomal A-protein (L12) from the moderate halophile NRCC 41227
Authors: Falkenberg Paul , Yaguchi Makoto , Roy Camille , Zuker Michael , Matheson Alastair T.
Published: December 2009

The complete amino acid sequence of the ribosomal A-protein (equivalent to L7/L12 in Escherichia coli) from a moderate halophile, NRCC 41227, has been determined using an automatic Beckman sequencer and by the manual Edman cleavage of peptides obtained from selective proteolytic cleavage of the ribosomal A-protein. The protein contains 122 amino acids and has a composition of Asp5, Asn2, Thr6, Ser6, Glu21, Gln2, Pro2, Gly12, Ala21, Val14, Met4, Ile4, Leu9, Phe2, Lys11, and Arg1, and a molecular weight of 12 537. It has a net negative charge of −14 and is, therefore, slightly more acidic than other eubacterial ribosomal A-proteins, The phylogenetic tree, obtained by computer analysis of the amino acid sequence of this and other eubacterial A-proteins, indicate these proteins form five subgroups within the eubacterial kingdom. The moderate halophile NRCC 41227 is part of a group of Gram-negative bacteria that include E. coli and another moderate halophile Vibrio costicola. The sequence data provides further evidence that the moderate and extreme halophiles have evolved by separate pathways.
Persulfide sulfur is a growth factor for cells defective in sulfur metabolism
Authors: Toohey John I.
Published: December 2009

Several systems which generate persulfide sulfur promote in vitro proliferation of L1 210 murine lymphoma cells. The systems include cysteine disulfides and pyridoxal, cystamine and diamine oxidase, β-mercaptoalcohol disulfides and an alcohol dehydrogenase, and sulfide-treated proteins and a thiol. Persulfide sulfur is very unstable at pH near 7 and an essential feature of the growth-supporting systems is the ability to generate persulfide sulfur at a very low rate for long periods of time. Methyl disulfides (R—S—S—CH3) also support growth of L1210 cells and are more stable than persulfides (R—S—S—H). The requirement for these sulfur groups by L1210 cells may be related to the fact that these cells are defective in at least two enzymes of sulfur metabolism, cystathionase and 5′-methylthioadenosine phosphorylase. These findings provide the first evidence that persulfide sulfur may have a physiological role.
A steryl glycoside fraction from Momordica charantia seeds with an inhibitory action on lipid metabolism in vitro
Authors: Ng T. B. , Wong C. M. , Li W. W. , Yeung H. W.
Published: December 2009

A saponin fraction was isolated from Momordica charantia seeds by delipidation, saline extraction, ammonium sulfate precipitation, and extraction of the resulting supernatant with n-butanol. Thin-layer chromatography, in the upper phase of the n-butanol – ethyl acetate – water (4:1:5, by volume) system on plastic sheets coated with silica gel 60 F254, revealed the presence of a single spot after spraying with 10% sulfuric acid. The lack of contamination of the saponin preparation with proteins was judged by the absence of protein bands in sodium dodecyl sulfate – polyacrylamide gel electrophoresis, agarose electrophoresis, and agarose diffusion, and by the absence of an absorption maximum around 278 nm. The saponin acted as a noncompetitive inhibitor of corticotropin, glucagon, and epinephrine in lipolysis in isolated rat adipocytes, and it also antagonized dibutyryl cAMP induced lipolysis. The antilipolytic activity was resistant to heat, trypsin, chymotrypsin, pronase, and glutathione, in keeping with the chemical nature of saponin. Incorporation of [3-3H]glucose into lipid was inhibited. Adipocyte viability and ATP content were not affected by the saponin, suggesting that its inhibitory effects on lipolysis and lipogenesis were not due to an adverse effect on cell viability.
Effect of different neutral phospholipids on apolipoprotein binding by artificial lipid particles in vivo
Authors: Tong Ming-for , Kuksis Arnis
Published: December 2009

Soybean triacylglycerol particles, stabilized with egg yolk sphingomyelin (SPH), phosphatidylcholine (PC), phosphatidylethanolamine (PE), or PC–PE mixtures, with diameters ranging from 170 to 550 nm were prepared by sonication and isolated by ultracentrifugation. Binding of apoproteins to the lipid particles was studied in vivo using the strategy of Connelly and Kuksis. The recoveries of the injected particles, which had decreased in size and undergone minimal changes in lipid composition, ranged from 70% and 57% for SPH- and PC-stabilized particles to 14% for particles stabilized with egg yolk PC–PE mixtures. The apoprotein (apo) composition of the recovered particles showed qualitative and quantitative differences, which were affected by the number of washes during isolation. After four washes, the SPH and PC particles contained apoE, apoC-II, and apoC-III as major components and apoA-IV as minor components. In addition, all particles, except those stabilized with egg yolk PC, contained large amounts of albumin. In contrast to egg yolk PC, the dipalmitoyl PC particles bound albumin as a major component. The recovered PC-PE and PE particles were characterized by a relative decrease of apoC and greatly increased binding of albumin. The higher rate of clearance of the PE-containing particles was attributed to a relative absence of apoC-III, which is known to delay hepatic uptake of lipid particles containing it, and to a more rapid hydrolysis of PE by lipoprotein lipases. Since PE occurs as a minor and variable component of chylomicrons and plasma lipoproteins, the present observations are of physiological interest.
The purification and characterization of a β-glucosidase from Alcaligenes faecalis
Authors: Day Anthony G. , Withers Stephen G.
Published: December 2009

The β-glucosidase from Alcaligenes faecalis has been purified to homogeneity (880-fold purification, 11% yield) using a combination of classical techniques and medium pressure ion-exchange chromatography. It is a dimeric enzyme of monomer molecular weight 50 000 and has no specific requirement for divalent metal ions. It has a high specificity for β-glucosides and hydrolyses a wide variety of different chemical types with retention of configuration at the anomeric centre. It has no exo-β-1,4-glucanase activity. It is reversibly inhibited by a variety of sugars which have been shown previously to be very active against glucosidases, suggesting a normal mechanism of action. Measured Km values for cellobiose and p-nitrophenyl β-D-glucopyranoside are quite low (0.70 and 0.08 mM, respectively), making this a good choice for cocloning into a cellulase system optimized for glucose production.
Domain unfolding and the stability of thermolysin in guanidine hydrochloride
Authors: Corbett Ronald J. T. , Ahmad Faizan , Roche Rodney S.
Published: December 2009

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)–S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.
Colocalization of sulfogalactosylacylalkylglycerol (SGG) and its binding protein during spermatogenesis and sperm maturation. Topology of SGG defines a new testicular germ cell membrane domain
Authors: Lingwood C. A.
Published: December 2009

By use of double-labelling indirect immunofluorescence, we have shown that the major mammalian testicular glycolipid sulfogalactosylacylalkylglycerol (SGG) and a membrane protein, previously shown to bind specifically to SGG in vitro, are colocalized on the surface of rat testicular germ cells during spermatogenesis. SGG is restricted to convoluted membrane domains within these cells. Thus, the binding affinity in vitro is reflected in the cell surface topology. The topological relationship between these two antigens was also studied during epididymal sperm maturation. Whereas these antigens were colocalized in caput spermatozoa (on the middle and principal piece of the tail and on the concave surface of the head), the distribution of the binding protein was altered for cauda sperm in that the convex surface of the sperm head was now strongly labelled. These studies illustrate the dynamic nature of protein–glycolipid interactions during germ cell differentiation.