bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the unmasking of membrane-bound alkaline phosphatase during the differentiation of Dictyostelium discoideum
Authors: Das D. V. Mohan , Weeks Gerald
Published: December 2009

Evidence has been presented recently to suggest that the 5′-nucleotidase and alkaline phosphatase activities of culmination phase cells of Dictyostelium discoideum are due to a single protein. However, we find that the membrane bound 5′-nucleotidase activity is only marginally activated by either 50 °C treatment or by dialysis, conditions that markedly activate alkaline phosphatase activity. In contrast, the 5′-nucleotidase activity of Triton X-100 extracts is activated by dialysis to the same extent as alkaline phosphatase activity. The available evidence suggests that, although a single protein is responsible for both alkaline phosphatase and 5′-nucleotidase activities, the characteristics of binding of the two substrates p-nitrophenyl phosphate and AMP to this enzyme are somewhat different. The alkaline phosphatase activity of dialyzed vegetative cell membranes is inhibited by the addition of concentrated dialyzate and this inhibition is reversed by further dialysis. The culmination phase enzyme is also inhibited by concentrated dialyzate from vegetative cells, suggesting that the removal of inhibitor from the enzyme can completely account for the developmental regulation. Dialyzates from culmination phase membranes do not inhibit enzyme activity, indicating the absence of inhibitor in these preparations. Alkaline phosphatase and 5′-nucleotidase activities of a partially purified enzyme preparation are equally inhibited by the addition of the concentrated dialyzate.
Purification and properties of rice germ lectin
Authors: Shen Zhao-Wen , Sun Ce , Zhu Zheng , Tang Xi-Hua , Shen Rui-Juan
Published: December 2009

An electrophoretically homogenous lectin was obtained from rice germ by extraction with acidified saline, precipitation with ammonium sulfate, and affinity adsorption on chitin. The molecular weight of the lectin was ascertained by gel filtration on Sephadex G-75 to be 23 000. Sodium dodecyl sulfate – polyacrylamide electrophoresis with 1% mercaptoethanol gave three bands, with molecular weights of 11 300, 13 700 and 19 000, indicating disulfide cleavage to form subunits. The intact lectin was found to have (i) cell agglutinating activity on erythrocytes and lymphocytes, sperm cells, sweet potato mesophyll cells, and callus cells of rice, all specifically inhibited by N-acetylglucosamine and (ii) mitogenic activity when tested on human peripheral lymphocytes and mouse spleen lymphocytes. The amino acid composition, N-terminal residues glycine and lysine, and isoelectric point (pH 6.5–6.8) of the lectin were determined.
Phosphate distribution and transport in mycoplasma
Authors: Lajeunesse Daniel , Grimellec Christian Le
Published: December 2009

In Mycoplasma capricolum 75% of the total cell phosphate is found in the nucleic acids and polyphosphate fraction. The cold acid soluble fraction consisted primarily of orthophosphate (40 mM) in resting organisms. In metabolizing cells, phosphate esters, probably organophosphates, represented the largest part of that fraction (40 mM) whose content in orthophosphate is lowered to 10–15 mM. Phosphate is accumulated against its concentration gradient after its entry as orthophosphate. It is taken up by the cell by two different, energy-stimulated transport systems. The response of both systems to metabolizable substrates resembles what is described for other microorganisms. The high affinity system had a Km of 12.8 μM and the low affinity system had a Km of 1.1 mM when cells were grown in a phosphate-poor medium. Addition of phosphate (30 mM) to that medium modified both system kinetic properties. Inhibition of phosphate influx by arsenate is limited to the energy-stimulated portion of the influx in both systems.
An in vitro model to study lipolysis of rat hepatic very low density lipoprotein using cardiac lipoprotein lipase
Authors: Brissette Louise , Noël Simon-Pierre
Published: December 2009

The purpose of this work was to design an in vitro model for studying the lipolysis of very low density lipoprotein (VLDL) under conditions that approximate those likely to exist in the rat circulation. We first measured the total lipase activity available in the circulation of normal fasted rats. Knowing the VLDL-triacylglycerol concentration and the circulating time of VLDL in rat serum, we calculated that incubating 80 mU of lipoprotein lipase activity per milligram of VLDL triacylglycerols at 37 °C, for 50 min, approximated the in vivo conditions of lipolysis in normal fasted rats. Under these conditions, different concentrations of albumin were tested. The degree of lipolysis gradually increased from 25 ± 10% without albumin to 75 ± 5% (mean ± SD, n = 3) with 4% albumin. No further increase occurred above 4% albumin. The rate of triacylglycerol hydrolysis was faster with VLDL having a high triacylglycerol/protein ratio. However after 50–60 min of incubation, the extent of hydrolysis tended to become similar for all VLDL (75–90%). This suggests that the rate of VLDL hydrolysis differs from one particle to another (depending on the size), but the extent of hydrolysis ends up being approximately the same. Furthermore, addition of high density lipoprotein to the incubation medium did not affect the rate nor the extent of triacylglycerol hydrolysis. We conclude that a large proportion of VLDL triacylglycerols can be hydrolyzed in the normal fasted rat if there is no limitation in the capacity of fatty acid removal from the lipolysis site. The physiological significance of these results obtained in vitro is discussed.
Inhibition of DMSO-induced differentiation by hyperthermia in a murine erythroleukemia cell system
Authors: Raaphorst G. P. , Azzam E. I. , Borsa J. , Einspenner M. , Vadasz J. A.
Published: December 2009

Friend erythroleukemia cells were induced by dimethyl sulfoxide (DMSO) into erythroid differentiation, as characterized by the production of hemoglobin. Induction increased with DMSO concentrations up to 1.5% v/v, at which point about 90% of the cell population produced hemoglobin as measured by a benzidine-staining technique. Heat treatment at 39.0–40.5 °C during a 7-day-incubation period, for differentiation in the presence of DMSO, resulted in the inhibition of hemoglobin induction. Also, acute heat treatments at 41.5–46.0 °C before or after the addition of DMSO resulted in the inhibition of DMSO induction. This effect was greatest when DMSO was present during heating. The results support the conclusion that hyperthermia inhibits the differentiation process which is induced by DMSO treatment.
Cross-reactivity of a monoclonal anti-[Met5]enkephalin antibody with cholecystokinin peptides
Authors: Deschodt-Lanckman Monique , Pontarotti Pierre
Published: December 2009

The hypothesis of a conformational similarity between the unsulfated form of cholecystokinin C-terminal heptapeptide and [Met5]enkephalin was proposed by Schiller and colleagues. To test this possibility, we investigated the competition of cholecystokinin C-terminal peptides for the binding to a monoclonal anti-[Met5]enkephalin antibody. We observed that the antibody immunoprecipitated cholecystokinin peptides having at least the six C-terminal amino acids. Unlike the opiate receptors which had no affinity for the sulfated heptapeptide, the monoclonal antibody recognized the sulfated form of the C-terminal octapeptide of cholecystokinin.
Effect of phase transitions on the interaction of peptides and proteins with phospholipids
Authors: Epand Richard M. , Surewicz Witold K.
Published: December 2009

This review is not a comprehensive study of the broad area of lipid–protein interactions, but rather concentrates on the question of the effect of phase transitions on the interaction of peptides and proteins with phospholipids. Before considering the relative ability of peptides and proteins to incorporate into phospholipid bilayers in the gel or the liquid crystalline state, we briefly review the variety of effects of proteins on lipid phase transitions. It is essential to be cognizant of these effects when discussing how-phase transitions affect protein incorporation. The effect of proteins on lipid order and motion above and below the phase transition is discussed and the current state of knowledge on this topic is briefly reviewed. In discussing the thermodynamics of lipid–protein association and the effect of phase transitions one must deal with systems at equilibrium, a state often difficult to achieve with high molecular weight aggregates. It is demonstrated that there are a variety of effects of phase transitions on protein incorporation. Some proteins incorporate more readily into liquid crystalline state lipid; others incorporate only in a narrow temperature region around the phase transition temperature, while still others interact with lipid over a broad range of temperatures while exhibiting some preference for interacting with gel state lipid. The molecular basis for preferential interaction with gel state lipid is suggested to be the ability of proteins to self-associate, probably at defect sites, below the phase transition temperature and thereby increase protein–protein interactions while maintaining protein–lipid and lipid–lipid interactions. In liquid crystalline state lipid, the protein would be more dispersed, the system would become less stable as a result of decreased protein–protein interactions, and the dispersed protein would also lessen lipid–lipid interactions. The ability of several peptides and proteins to intercalate more readily into gel rather than liquid crystalline state lipid is a more common phenomenon than is generally appreciated.
The functional architecture of band 3, the anion transport protein of the red cell membrane
Authors: Rothstein Aser
Published: December 2009

Band 3 is an abundant, intrinsic, transmembrane peptide of about 95 000 daltons that mediates anion exchanges across the red blood cell membrane. It is present in the membrane as a dimer with each monomer arranged so that it crosses the bilayer at least five times. Based on proteolytic dissection and the use of covalent probes to "mark" specific sites, it is proposed that the crossing strands form an assembly of α-helices, with hydrophobic residues exposed so they are closely associated with the fatty acid side chains of the phospholipids and with hydrophilic residues internalized to form an aqueous core through which transport occurs. Anions cannot freely diffuse through the transport pathway, but are constrained by a "gating" mechanism that requires an electroneutral one-for-one exchange. The process involves the binding of the anions to specific transport sites followed by a local conformational change, such that the sites (with the bound anion) alternate between states that are topologically in and out, with kinetics consistent with a "ping-pong" mechanism. Large organic anions can bind to the transport sites resulting in competitive inhibition. Those such as 4,4′-diisothiocyano-2,2′-stilbene sulfonic acid that can react covalently have been used to mark the transport site in the primary structure of band 3. Sequence data will ultimately allow a much more detailed assessment of the functional architecture of band 3.
CDP-diglyceride hydrolase from pig liver mitochondria
Authors: Nicholson D. W. , McMurray W. C.
Published: December 2009

A CDP-diglyceride hydrolase activity, measured by the release of [3H]CMP from labeled CDP-diglyceride, has been identified in pig liver mitochondria. A modified preparatory method for the synthesis of [3H]CDP-diglyceride of high specific activity and purity is also reported. Activity of the hydrolase is enriched 2.5-fold in mitochondrial membranes (over whole mitochondria) and can be solubilized by nonionic detergents such as Triton X-100 with further enrichment of activity (i.e., 7.9-fold). The CDP-diglyceride hydrolase has a Km of 12.8 μM for CDP-diglyceride and a broad pH range with optimum activity at approximately pH 6.2. Of the CDP-diglycerides tested, the hydrolytic rate is highest for dioleoyl CDP-diglyceride. Activity is inhibited by all divalent cations in whole mitochondria, except in the presence of phosphatidylglycerol in which CMP release is stimulated by Co2+ and Mn2+. The increase in CMP release in the presence of Co2+ or Mn2+ can be accounted for entirely by diphosphatidylglycerol synthase activity which requires either cation. This effect is not seen in Triton X-100 solubilized mitochondrial membranes which contain no diphosphatidylglycerol synthase. All preparations are inhibited by mixed phospholipids (Asolectin) and by Trixon X-100 which abolishes activity completely at concentrations greater than 0.5% (w/v). CDP-diglyceride hydrolase is also inhibited by AMP (46%) and by cytidine nucleotides (CTP > CDP > cytidine) except CMP. A role for this activity in the regulation of biosynthesis of mitochondrial polyglycerophosphatides is proposed.
Hexose transport in plasma membrane vesicles of rat myoblast L6
Authors: Cheung Matthias O. , Lo Theodore C. Y.
Published: December 2009

To determine the molecular mechanism of hexose transport in rat myoblasts, transport studies were carried out with purified plasma membrane vesicles. Rat myoblasts were homogenized and fractionated by differential and sucrose gradient centrifugation. Six different fractions were obtained. Studies with marker enzymes revealed that two fractions (A and B) were composed of only plasma membrane. These two fractions differed considerably in their physical properties. Fraction A was composed of large multilaminated vesicles, with an intravesicular volume of 50 μL/mg protein, whereas fraction B was composed of membrane fragments and much smaller vesicles, with an intravesicular volume of 7 μL/mg protein. Based on the response of the ouabain-sensitive Na+,K+-ATPase activity to sodium dodecyl sulfate and ionophore treatments, it seemed likely that fraction A was composed of a significant amount of sealed right-side-out vesicles, whereas fraction B was composed of mainly membrane sheets or leaky vesicles. The initial rate of hexose influx into the membrane vesicles was determined by the flow dialysis technique. The optimal conditions for 2-deoxyglucose (2-DG) uptake into the plasma membrane vesicles were either 50 mM phosphate buffer or 10 mM 2-(N-2-hydroxyethylpiperazin-N′-yl)ethanesulfonic acid buffer at pH 7.0. In the presence of 500 μM 2-DG, the initial rates of 2-DG influx were 295 and 49 nmol/min per milligram protein for fractions A and B, respectively. In other words, after 1 min of incubation, the intravesicular concentration of 2-DG was around 6 mM, about 10 times the extravesicular concentration. D-Glucose was taken up to a similar extent (333 nmol/min per milligram protein), whereas L-glucose only equilibrated across the plasma membrane. Analysis of the fate of 2-DG revealed that the substrate was not phosphorylated upon incubation with the vesicles. Transport activity can be abolished either by disruption of the membrane vesicles or by reduction of the electrical potential across the membrane.
The hydrolysis of phosphatidylcholine by phospholipase A2 in hamster heart
Authors: Tam Stanley W. , Man Ricky Y. K. , Choy Patrick C.
Published: December 2009

The hydrolysis of acyl esters in phosphatidylcholine and phosphatidylethanolamine by phospholipase A in hamster heart subcellular fractions was investigated. Phosphatidylcholine was found to be a much poorer substrate than phosphatidylethanolamine for the cardiac phospholipase A. The rate of hydrolysis of phosphatidylcholine by microsomal phospholipase A was 10-fold less than with phosphatidylethanolamine as substrate. When 1-[1-14C]palmitoyl-2-acyl phosphatidyl-[Me-3H]choline was used as substrate, both phospholipase A1 and A2 activities were detected in all subcellular fractions, but the highest specific activities for both enzymes were located in the microsomal fraction. However, phospholipase A2 activity in all hamster heart particulate fractions was three to six times higher than phospholipase A1 activity. The hydrolysis of phosphatidylcholine by microsomal phospholipase A2 displayed an alkaline pH optimum and an absolute requirement for Ca2+ or Mg2+. The enzyme also depicted high specificity towards polyunsaturated acyl groups at the C-2 position of phosphatidylcholine.
Release of ferrous iron from ferritin by liver microsomes: a possible role in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin
Authors: Rowley Bruce , Sweeney George D.
Published: December 2009

Nonheme iron is synergistic with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in producing hepatotoxicity in mice. Fe2+ rather than Fe3+ is the probable toxin and we speculated that TCDD, an inducer of microsomal electron transport, might favour reduction of iron. We have defined a system which will release Fe2+ from ferritin (Fe3+) under anaerobic conditions and in the presence of added flavin mononucleotide (FMN). The rate of reduction of ferritin iron was proportional (a) to microsomal protein from 0.5 to >3 mg/mL, (b) to the activity of NADPH–cytochrome c reductase over 0.1 U/mL, (c) to ferritin at concentrations exceeding iron concentrations >200 μmol/L, and (d) to the concentration of FMN when it was less than 125 μmol/L. The system was approximately twice as active with NADPH as with NADH as electron donor. The linear phase of iron release did not commence immediately, but followed a delay (±0.5 min) after adding FMN to an anaerobic mixture containing microsomes, ferritin, an NADPH-generating system, and an oxygen-scavenging system. When microsomes from untreated, Phenobarbitaltreated (3 days), or TCDD-treated (1 or 3 weeks) rats were compared, iron release correlated most closely with the cytochrome P-450 concentration. However, when the microsomal proteins were solubilized and the NADPH–cytochrome c reductase and cytochrome P-450 activities were separated, reduction of ferritin iron was shown to be a function only of the reductase fraction, except that the delay in initiating release of Fe2+ was increased with purified reductase and decreased when a monooxygenase system was reconstituted with cytochrome (phenobarbital or TCDD induced) and lipid. These studies have defined a potentially important hepatic microsomal system able to release Fe2+ from ferritin iron, but have failed to indicate any feature unique to the dioxin-induced monooxygenase system.
A comparison of four sulfhydryl cathepsins (B, C, H, and L) from porcine spleen
Authors: Lynn K. R. , Labow Rosalind S.
Published: December 2009

Four sulfhydryl cathepsins, B, C (dipeptidyl aminopeptidase I), H, and L were isolated from porcine spleen. They are all glycoproteins of similar amino acid compositions, which are comparable with those of cathepsins B and H from other sources and so with papain. All four cathepsins exist in multiple charged forms: B, C, H, and L have isoelectric points in the range 4.3–5.4, 5.3 and 5.9, 5.2–5.7, and 7–8.7, respectively. The molecular weights of cathepsins B and H were 24 000 and 26 000. Anomalous behaviour of cathepsin L on both conventional gel filtration and high pressure liquid chromatography precluded a precise assessment of its weight which is between 22 000 and 28 000. The isolated mercurial derivative of cathepsin C has a molecular weight of 56 000 (an active dimer formed on reduction). Cathepsins B and H also aggregate.
Somatomedin-like activity from cultured embryo-derived cells: partial characterization and stimulation of production by epidermal growth factor (urogastrone)
Authors: Atkison Paul R. , Bala R. Marvin , Hollenberg Morley D.
Published: December 2009

We have measured the appearance in cell culture media of a somatomedin-like activity (SLA) that cross-reacts in a basic-somatomedin radioreceptor assay and radioimmunoassay and that is produced by cultures of normal fetal-derived human (WI-38 fibroblasts) and mouse (embryonic palate) tissues and by cultures of a Simian virus 40 transformed WI-38 fibroblast cell line (WI-38 VA13/2RA subline). In addition to human growth hormone (hGH), epidermal growth factor (urogastrone) (EGF-URO), human placental lactogen (hPL), and porcine insulin (INS) were able to stimulate the production of SLA in serum-free cultures of the normal WI-38 fibroblasts; human prolactin (hPRL) caused only a marginal stimulation of SLA production in these cultures. In order of the magnitude of the stimulation of SLA production, the effects of the several hormones tested were [Formula: see text]. The half-maximal stimulatory effect of EGF-URO in the WI-38 cultures was observed at a concentration of about 1 nM. In the mouse palate organ cultures, EGF-URO also stimulated SLA production; the effect of EGF-URO was more pronounced in palate organ cultures derived from day 13 embryos compared with tissue obtained from embryos at days 14 and 15. The SLA produced by the human VA13/2RA subline was characterized further by gel filtration under acid conditions, by Procion dye column chromatography, and by high pressure liquid chromatography (HPLC). In terms of their immunoreactivity, adsorption to the dye column, and their apparent molecular weights (about 45 000), the SLA's produced by the VA13/2RA cells and the WI-38 cell cultures did not appear to differ. However, in terms of molecular weight and HPLC behaviour, the VA13/2RA-derived SLA was distinct from human basic somatomedin. We conclude that fetal-derived cells are capable of producing SLA in a manner that can be regulated both by a variety of polypeptide hormones and by the stage of embryonic development. Further, we conclude that the SLA produced by the fetal-derived human VA13/2RA cell line is indistinguishable from the SLA produced by WI-38 cells and that this SLA differs from the immunologically cross-reacting somatomedin that has been previously isolated from human plasma Cohn fraction.
Chemical modification and hybrid enzyme formation as probes of the active site and subunit interactions in Escherichia coli succinyl-CoA synthetase
Authors: O'Connor-McCourt Maureen D. , Bridger William A.
Published: December 2009

Succinyl-CoA synthetase from Escherichia coli has an α2β2 subunit structure and is known to display half-of-the-sites reactivity with respect to its phosphorylation by ATP. The studies reported herein are a component of our attempts to rationalize the heterologous tetrameric structure in terms of catalytic function. The isolated refolded β subunit interacts specifically with an affinity column of agarose–hexane–CoA, consistent with the idea that the CoA-binding subsite of the active center is located on the β subunit. The enzyme is inactivated by phenylglyoxal according to biphasic kinetics; saturating levels of the substrates CoA and ATP, alone or in combination, give only partial protection against such inactivation. Treatment of the enzyme with the sulfhydryl reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in rapid inactivation, accompanied by the reaction of three to four -SH groups per molecule; prolonged incubation with NBD-Cl eventually results in reaction of 16 of the 24 sulfhydryl groups of the tetramer. Hybrid enzyme preparations have been constructed by refolding mixtures containing β and various ratios of native and NBD-Cl-modified a subunits. The loss of activity associated with the incorporation of chemically modified α is not that predicted by a simple model based on binomial distribution, but is complex and consistent with the kind of intersubunit communication that may be expected for catalytic cooperativity between the two active sites of the enzyme molecule.
Cerulenin inhibition of lipid synthesis and its reversal by exogenous fatty acids in Mycobacterium smegmatis ATCC 607
Authors: Mahajan Sandeep , Khuller G. K.
Published: December 2009

Cerulenin inhibited the lipid synthesis of Mycobacterium smegmatis ATCC 607 over the range of 0.5–1.8 μg/mL with complete inhibition at 1.8 μg/mL, as monitored by [14C]glycerol incorporation into lipids. Exogenous fatty acids failed to restore the lipid synthesis at 1.8 μg/mL; however, the addition of palmitic acid to the growth medium partially restored the lipid synthesis when cerulenin concentration was decreased to 1.6 μg/mL. Fatty acid analysis of cerulenin plus palmitic acid supplemented cultures revealed that exogenously supplied fatty acid was incorporated into cellular phospholipids. Further investigations with 1.6 μg/mL of cerulenin and [14C]acetate and [32P]orthophosphoric acid showed that cerulenin inhibited the synthesis of saturated plus unsaturated fatty acids and phospholipids. Pulse–chase studies with [14C]acetate revealed decreased synthesis and degradation of each of the phospholipid components.
Thymus metallothionein: regulation of zinc-thionein in the aging mouse
Authors: Olafson R. W.
Published: December 2009

The presence of a zinc-inducible metallothionein in mouse thymus tissue has been shown, together with an age-related modulation of zinc, metallothionein, and thymus mass. Metallothionein (MT) and zinc concentrations were found to be maximum prior to completion of thymic growth and to be at a minimum when development of the thymus had ceased. Subsequent transient outgrowths of the thymus during involution showed similar associated fluctuations of MT and zinc levels. A marked difference in the latter parameters was found on comparison with New Zealand Black mice known for development of early onset autoimmunities. These findings provide a new avenue for both investigation of the regulation of zinc in thymus tissue, as well as the possible elucidation of the molecular basis for certain age-related immune dysfunctions.
Rapid isolation of a triacylglycerol synthetase complex from rat intestinal mucosa
Authors: Manganaro F. , Kuksis A.
Published: December 2009

A triacylglycerol synthetase complex made up of acyl-CoA synthetase, acyl-CoA:monoacylglycerol acyltransferase, and acyl-CoA:diacylglycerol acyltransferase has been solubilized by sodium taurocholate and isolated by chromatography on phenyl-Sepharose. For this purpose microsomes of the villus cells of rat intestinal mucosa were dissolved in 2% sodium taurocholate prepared in 1 M (NH4)2SO4 and 25 mM Tris–HCl (pH 8.5) (buffer A). After dialysis against buffer A, the sample was loaded on a phenyl-Sepharose column and the enzyme complex was eluted with 25 mM Tris–HCl (pH 8.5) (buffer B). The enzymatically active fractions were pooled and rechromatographed on a Bio-Gel A-0.5 m column equilibrated with buffer B and 150 mM NaCl. The recovered triacylglycerol synthetase complex accounted for over 75% of the original enzyme activity and represented a 145-fold purification from villus cells and a 10-fold purification from microsomes. It exhibited maximal activity at pH 8.0–9.0. On the basis of Bio-Gel A-0.5 m chromatography the three enzymic activities appeared as a single fraction in the molecular weight range of 350 000–375 000. The complex migrated as a single peak on high performance liquid chromatography on an ion-exchange column using a NaCl gradient. The ratio of the activities of the three enzymes remained constant during the purification. The purified enzyme complex lost about 50% of its diacylglycerol acyltransferase activity on storage for 2 weeks at −20 °C in presence of phenylmethylsulfonyl fluoride.
Deacylation of endogenously deuterated rat liver microsomal phospholipids by endogenous phospholipases
Authors: Pind S. , Kuksis A. , Myher J. J. , Marai L.
Published: December 2009

The relative deacylation of microsomal phospholipid molecular species was reexamined. Microsomal membranes were prepared from the livers of rats injected, over a period of 20 h, with perdeuterated ethanol. The phosphatidylcholine and phosphatidylethanolamine were isolated by thin-layer chromatography of the total lipid extracts and the distribution of deuterium among the molecular species of the diacylglycerol moieties was determined by reversed-phase high pressure liquid chromatography in combination with chemical ionization mass spectrometry. Deuterium was found to be incorporated into newly formed glycerol and newly synthesized palmitic and stearic acids (4–22% replacement) which were distributed throughout the molecular species in proportion to their relative rates of turnover (10–45% replacement). Within each unsaturation class, the palmitoyl species were labelled more extensively than the corresponding stearoyl species. Following in vitro incubation of the membranes with 10 mM Ca2+ at pH 8.5, there occurred a rapid degradation of the phosphatidylethanolamines (to 60% of control values after 90 min), while the phosphatidylcholines remained essentially unaffected. The various molecular species of the phosphatidylethanolamines were degraded linearly and in proportion to their masses. The deuterium content of the phosphatidylethanolamine and phosphatidylcholine remained constant throughout the incubation. It was concluded that under the present experimental conditions all molecular species of phosphatidylethanolamine, both old and newly synthesized, are equally accessible to the endogenous phospholipase.
The cross-linking of human Met-hemoglobin with [14C]dimethyl adipimidate
Authors: Ferris Robert J. , Smith D. B.
Published: December 2009

Met-hemoglobin, cross-linked with [14C]dimethyladipimidate (cross-linking span 9 Å; 1 Å = 0.1 nm), yields four distinct molecular weight products (monomer, dimer, trimer, and tetramer) as observed on sodium dodecyl sulfate – polyacrylamide gels. The dimer species was purified to homogeneity by gel filtration. It was proteolytically degraded using a combination of Staphylococcus aureus V8 protease, pepsin, trypsin, and chymotrypsin. The resultant peptides were fractionated using gel filtration and ion-exchange chromatography methods. Two cross-links were unambiguously identified: (i) α1Lys7−α1Lys11 and (ii) β1Lys82−β2Lys82. The identified cross-links correlated well with the known structure of hemoglobin. However, attempts to isolate and identify a greater number of cross-linked peptides were unsuccessful owing to the complexity of the peptide mixtures. The complexity was a direct result of the chemical modification of the hemoglobin molecule. Therefore, attempts to employ chemical cross-linking as a means of examining sites of protomer contact within large oligomeric proteins should be approached with caution.
Transfer reactions catalyzed by β-D-xylosidase from Penicillium wortmanni
Authors: Deleyn François , Claeyssens Marc , Bruyne Clement K. De , Bruyn André De
Published: December 2009

The β-D-xylosidase from Penicillium wortmanni catalyses (i) the hydrolysis of β-D-xylopyranosides and α-L-arabinopyranosides, (ii) the transfer of the corresponding glycosyl residue to alcohols, and (iii) the dismutation of aryl β-D-xylopyranoside substrates. These reactions all occur with retention of configuration and can be rationalized by a symmetrical reaction scheme. The key intermediate is an enzyme–glycosyl complex with a lifetime that is sufficient for the diffusion away of the leaving (aglycon) group and the binding of an acceptor group before water reacts with the intermediate. The exact nature of this complex is unknown, but it must contain an aglycon site which binds preferentially alcohols and sugar molecules having the D-xylose structure.
A statistical comparison of parameter estimation for the Michaelis–Menten kinetics of human placental hexosaminidase
Authors: Tommasini R. , Endrenyi L. , Taylor P. A. , Mahuran D. J. , Lowden J. A.
Published: December 2009

To enable the most effective method of kinetic discrimination between a group of isozymes such as those of human placental hexosaminidases (HEX), three methods estimating the parameters of the Michaelis–Menten equation were evaluated. Computer-simulated experiments were performed under various conditions. They indicated that, in the presence of constant absolute or relative errors, the method of unweighted nonlinear least squares yielded slightly more precise and accurate parameters than the method of the direct linear plot. Parameters calculated from the Lineweaver–Burk plot were very imprecise and inaccurate. The direct linear plot was comparatively resistant to outlier observations; however, only when outliers were substantial did the method become superior to nonlinear least squares. The calculation of a confidence limit is necessary for the evaluation of any resulting differences in the kinetic parameters for a set of isozymes. This can easily be calculated from either the Lineweaver–Burk plot or the nonlinear least-squares method. However, those calculated from the Lineweaver–Burk plot are biased, especially at higher levels of experimental errors. Therefore, the nonlinear least-squares method is the one most suited for the discrimination of a group of enzymes based on their kinetic parameters.
Mapping of proteolytic and cyanogen bromide peptides from subunits of intestinal maltase–glucoamylase: evidence for significant homology
Authors: Lee L. , Forstner G.
Published: December 2009

Rat intestinal maltase–glucoamylase was purified in the presence of detergent and proteolytic inhibitors, and the 130 000 and 145 000 subunits were separated and isolated by preparative sodium dodecyl sulfate – polyacrylamide gel electrophoresis and electrophoretic elution. Amino acid analyses were very similar, with a small excess of apolar amino acid residues in the 145 000 subunit. Peptide mapping with Staphylococcus aureus V8 protease revealed eight similar peptide products for each, with apparent elongation of the five larger peptides in the 145 000 subunit by a relative mass (Mr) of 2000–5000. α-Chymotrypsin maps showed at least eight identical cleavage products plus one large, shared product which was larger by a Mr of 5000 in the 145 000 subunit. Cyanogen bromide cleavage of the 145 000 subunit produced a single peptide of Mr 75 000. A peptide of Mr 66 000, also indicative of a central cleavage, was generated from the 130 000 subunit, but a second cleavage into 43 000 and 23 000 segments was also evident. Several sets of antibodies formed against both antigens consistently gave reactions of identity without spurring on immunodiffusion. These results indicate extreme homology between the central segment of the 145 000 subunit and the 130 000 subunit. The cyanogen bromide cleavage results suggest, however, that the two central sequences are not absolutely identical and therefore that one subunit may not be a posttranslational derivative of the other.
Cone-shaped lipids increase the susceptibility of phospholipids in bilayers to the action of phospholipases
Authors: Buckley James Thomas
Published: December 2009

Addition of cardiolipin or diacylglycerol to dispersions of phosphatidylcholine greatly increased hydrolysis by snake venom or pancreatic phospholipase A2, as well as by a microbial phospholipase. Monogalactosyl diglyceride which, like cardiolipin and diacylglycerol, will form nonbilayer hexagonal II structures also caused an increase in the breakdown of phosphatidylcholine. Addition of digalactosyl diglyceride, a bilayer lipid from the same source, had a much smaller effect on the three phospholipases, indicating that stimulation by the nonbilayer lipids was not due to their fatty acid compositions. Stimulation of the microbial phospholipase by cardiolipin did not require the presence of calcium, leading to the conclusion that the formation of nonbilayer structures was not necessary. The results suggest that cone-shaped lipids increase the accessibility of lipids in bilayers to phospholipases by decreasing the packing of the polar head groups.
Phosphatidylcholine and 4-methylumbelliferyl phosphorylcholine hydrolysis by purified placental sphingomyelinase
Authors: Freeman Stuart J. , Shankaran Prema , Wolfe Leonhard S. , Callahan John W.
Published: December 2009

We present evidence which indicates that highly purified placental acid sphingomyelinase hydrolyses [14C]phosphatidylcholine ([14C]PC) and the synthetic phosphodiester 4-methylumbelliferyl phosphorylcholine (4-MUPC). Hydrolysis was achieved by phospholipase C phosphodiesterase action. Of the several detergents tested, sodium taurocholate alone was necessary for PC hydrolysis, while 4-MUPC was hydrolysed independent of any detergent requirement. The pH optima for the reactions were 4.6–4.8 for PC hydrolysis and 4.8–5.0 for 4-MUPC hydrolysis. As with sphingomyelin hydrolysis, degradation of both PC and 4-MUPC was inhibited by 5′-, 3′-, and 2′-AMP, 5′-AMP being the most effective of the three. Furthermore, the phosphodiesterase activity against PC and 4-MUPC copurified with sphingomyelinase from human placenta and cross-reacted with a specific anti-sphingomyelinase monoclonal antibody, strongly indicating identity of the phosphodiesterases. This explains phospholipase C deficiency in sphingomyelinase-deficient Niemann-Pick disease cells.
The Ribosome: A biochemist's mechano set
Authors: Nazar Ross N.
Published: December 2009

A ribosome, the cellular site for protein synthesis, is a very complex organelle composed of a myriad of macromolecular substructures. As models for this complex structure, we have been examining the structures and interactions of eukaryotic 5S and 5.8S rRNAs using adaptations of rapid RNA gel sequencing techniques. Estimates for their higher order structures have been proposed or evaluated, sites of interaction with other ribosomal components have been delineated, and the topography of these RNAs within the intact ribosome or 60S subunit have been examined. The results indicate that a universal structure for the ribosomal RNAs may only be present within the ribosome, that these molecules are probably present, at least in part, within the ribosomal interface, and that the bases for interactions with other ribosomal components are strongly dependent on their higher order structure. The experimental approaches which underlie these studies are considered in this review and the significance of the results with respect to the function and evolution of the ribosome are briefly discussed.
Secretion and export of amylolytic activities in Schwanniomyces alluvius
Authors: Lusena C. V. , Champagne C. C. , Calleja G. B.
Published: December 2009

We define secretion as the passage from the cytoplasm, across the cell membrane, to the periplasmic space. By contrast, export is the passage across the cell wall into the medium. Operationally we distinguish the two processes by comparing the molecular species in the medium, in whole cells, and in spheroplasts. Two techniques make the task possible: complete spheroplast preparation and detection of activities in bands obtained by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The capability of Schwanniomyces alluvius to export α-amylase during stationary phase gradually increased with continual successive transfers from a slant culture to a liquid medium containing starch until a maximum was reached. Only cells which had developed full capability to export α-amylase were used in these studies. About 1 h after the end of the log phase of growth, α-amylase and glucoamylase start to be exported above constitutive levels and a concentration 10 times the constitutive level is reached 3 h later. Electrophoretic results show that at least three active molecular species of α-amylase appear in the cytoplasm at the end of log phase and that the smaller component (52 000 daltons) is secreted into the periplasm 0.5 h later and starts to be exported 1 h after that. The sequence of events suggests that the larger species are precursors of the 52 000 dalton molecules. Amylolytic activities in the cytoplasm and periplasm in late log phase are not detectable.
Molecular biology of interleukin 2
Authors: Paetkau Verner
Published: December 2009

Study of the lymphokine interleukin 2 (IL2) began with several diverse qualitative observations in the middle 1970s. Rationalization of early work led to a focused program of research involving a number of laboratories. This recently culminated in the molecular description of both murine and human IL2. The glycoprotein, containing about 16 000 daltons of protein, is secreted in response to activators of T helper lymphocytes. A number of complex immune responses is stimulated by pure, recombinant IL2. The induction of IL2 and other lymphokines is apparently specific, in that only a small subset of proteins shows altered expression in the response. Moreover, the immunosuppressive agent cyclosporin A specifically inhibits the induction of mRNA for IL2 and certain other lymphokines, without affecting the expression of the majority of other proteins. Understanding of IL2 and its biological context has recently been enhanced by an intense activity on the part of biotechnology companies.
Characteristics of myoblasts isolated from golden Syrian and dystrophic (strain CHF-146) hamsters
Authors: Ng Stephen K. , Lewis Keith E.
Published: December 2009

A method for dissociating and culturing myoblast cells from normal (golden Syrian) and myopathic (CHF-146) hind leg muscles of 8- to 10-day-old hamsters has been described. We have compared the fusion ability of the myoblasts from normal tissue with that of myopathic myoblasts and, further, we have studied this process with respect to gelatin-coated and uncoated plastic culture dishes. Our data show that myoblasts from normal tissue grows well on both surfaces, although fusion is enhanced when these cells are grown on a gelatin-coated surface. Myoblasts from dystrophic tissue, however, appear to have an impaired ability to adhere to an uncoated surface, although fusion is enhanced when these myoblasts are grown on coated plates. The fusion percentage of the dystrophic cells is consistently lower than that of myoblasts from normal tissue. At low concentrations of 5-azacytidine tested, there seems to be no enhancement of fusion capability in either the normal or dystrophic lines.
The presence of a heat-stable cytosolic factor in rat submandibular gland which activates Na+,K+-ATPase
Authors: Pon D. J. , Stewart D. J. , Sen A. K.
Published: December 2009

A low molecular weight substance is present in the soluble fraction of the rat submandibular gland which activates Na+,K+-ATPase. Varying amounts of cytosolic protein from rat submandibular gland were added to a heavy microsomal preparation of the same tissue. A dose-dependent activation of the Na+,K+-ATPase was observed, with a peak activation of approximately 82% occurring when 9.0 μg/mL of cytosolic protein was included in the assay. The activating factor is heat stable and soluble following heat treatment. The factor elutes at a molecular mass of 600 daltons as determined by molecular sieve column chromatography. Ultraviolet–visible scanning of the eluted material results in an absorbance at 210 nm, which is characteristic of a low molecular weight peptide.
The in vitro formation of sulfates and glucuronides of estrogens by adult and fetal ovine tissues
Authors: Hobkirk R. , Cardy Catherine A.
Published: December 2009

Incubation of nanomolar concentrations of [3H]estrone with ovine liver slices from adult and fetal animals demonstrated, in particular, the production of estrogen sulfates together with smaller amounts of glucuronides, even although microsomal estrogen glucuronyltransferase (GT) and sulfatase activities were high, especially in adult tissue. [3H]Estriol was conjugated almost exclusively as sulfate under the same experimental conditions. Slices of maternal and fetal kidney medulla were also strikingly active in promoting estrogen sulfate production as were slices of fetal kidney cortex. Adult kidney cortex conjugated estrogen only in the glucuronide form. These data indicate the possibility that maternal and fetal liver and kidney might contribute to the high circulating level of estrone sulfate in the pregnant sheep. Through the use of [3H]estrone and [3H]estrone sulfate as substrates, it was possible to demonstrate that adult slices of kidney medulla possessed relatively low sulfatase, considerable sulfotransferase (ST), and virtually no GT activity, whereas cortex had high sulfatase, little or no ST, and low, though demonstrable, GT activity. The ST activity of kidney high-speed supernatants was stimulated by the presence of sulfhydryl groups, whereas that in liver was not. Enzymic reduction of estrone and (or) estrone sulfate by liver and kidney slices indicated that, in the former, 17α-reduction prevailed and, in the latter with the exception of the maternal medulla, 17β-reduction was the main pathway, particularly in the fetus.
Hydrodynamic and optical properties of the wheat germ Em protein
Authors: McCubbin William D. , Kay Cyril M. , Lane Byron G.
Published: December 2009

The size and shape of the Em protein from wheat embryos have been examined by gel filtration, densitometry, ultracentrifugation, and viscosity. Circular dichroism and fluorescence measurements have also been made. Em has an intrinsic sedimentation coefficient, [Formula: see text], of 1.11S and a Stokes radius, RS, of 28.2 Å (1 Å = 0.1 nm) as determined by high performance liquid chromatography gel filtration on a TSK 2000SW column. The partial specific volume [Formula: see text] from density measurements is 0.683 mL/g, a much lower than typical value. The molecular weight from sedimentation equilibrium is 11 200, with no indication for protein aggregation. The intrinsic viscosity [η] of Em is 6.02 mL/g. Circular dichroism shows the molecule to be about 70% random coil. The fluorescence emission spectrum is typical for a tyrosine-containing protein. The hydrodynamic data indicates a poor fit to either a prolate or oblate ellipsoid model; excess hydration or flexibility of the polypeptide chain caused by the rather unusual amino acid composition may be a possible cause. The implications that the low value of [Formula: see text], the high value of RS, and the random-coil configuration of the Em protein may have on its ability to bind water and to contribute to the maintenance of a minimal level of hydration compatible with the sustained viability of the "dry" organism are subjects of an extended discussion. Briefly, it is suggested that Em may provide a matrix of bound water which opposes denaturation of proteins in the "desiccated" cytoplasm of dry plant embryos.
Translatability of rat kidney mRNA after mercury administration
Authors: Samji Shahebina , Kuliszewski Maciej J. , Girgis George R. , Nicholls D. McEwen
Published: December 2009

Young male rats received an intraperitoneal injection of 0.5 mg HgCl2/kg body weight and 16 h later the kidneys were removed and homogenized to prepare the polysomal fraction from which the poly(A)+ RNA was obtained. The activity of this fraction was assessed by translating the poly(A)+ RNA in a mRNA-dependent rabbit reticulocyte lysate and the activity was markedly elevated relative to preparations from control rat kidneys. The incorporation of labelled leucine and cysteine, but not phenylalanine, into a low molecular weight protein (approximately 10 000 as judged by denaturing polyacrylamide gel electrophoresis) accounted for the increased mRNA activity. The mobility during electrophoresis of the denatured labelled product and carboxymethylated product, as well as their acidic isoelectric points, provided evidence that it is metallothionein mRNA which exhibits increased translatability in preparations derived from mercury-treated rats.