bluuebunny/crossref_metadata_2025 huggingface dataset:
Bile acid synthesis in isolated rat hepatocytes
Authors: Yousef I. M. , Ho J. , Jeejeebhoy K. N.
Published: December 2009

Normal adult rat hepatocytes were incubated for 48 h and the concentration of total and individual bile acids in homogenized samples of the culture was measured at intervals during the incubation, using radiogas chromatography and isotope derivative assay. The net increase in bile acids over the value observed at the start of the culture was taken as synthesis. The results showed that bile acid synthesis was linear up to 24 h of incubation, at a rate of 20 nmol/g hepatocytes per hour, and that 85% of the newly synthesized bile acid was cholic acid. The bile acid synthesized was mainly conjugated with taurine. These results suggest that isolated hepatocytes cultured in the way described could be a useful in vitro model for the study of bile acid synthesis.
Properties of poly(ADP-ribose) synthetase from rat pancreas and poly(ADP-ribosylation) of basic nuclear proteins
Authors: Poirier G. G. , Savard P. , Rajotte D. , Morisset J. , Lord A.
Published: December 2009

The isolated nuclei of rat pancreas contain an enzyme system that will incorporate 3H-labeled NAD into an acid-insoluble product, which is shown to be poly(ADP-ribose). The enzyme has an optimum pH of 7.8 and the optimum temperature is between 20 and 30 °C. Optimum Mg2+ concentration is 8 mM and dithiothreitol also stimulates the enzyme at a concentration of 8 mM. Under standard conditions, the Km value for the reaction is 0.25 mM and an inhibition by the substrate is observed at high substrate concentrations. It has also been found that only one basic nuclear protein, that is, histone H1, is modified by the synthetase. An average chain length of 5.0 is found in the nuclei and of 4.5 on histone H1. Radioautographic studies show that poly(ADP-ribose) is closely associated with chromatin.
Determination of the rates of synthesis and degradation of adenosine 3′,5′-cyclic monophosphate in Escherichia coli CRP− and CRP+ strains
Authors: Fraser Ann D. E. , Yamazaki Hiroshi
Published: December 2009

We have developed a method for estimating the rates of synthesis and degradation of adenosine 3′,5′-cyclic monophosphate (cAMP) in Escherichia coli during balanced growth. Applying this method, we have found that an E. coli CRP− mutant 5333 (deficient for cAMP receptor protein) synthesizes cAMP about 25 times faster than does its CRP+ parent 1100. This accounts for the abnormally high intracellular and extracellular cAMP accumulation in 5333.
Sphingomyelinases in human tissues. IV. Purification of sphingomyelinase from human placenta and effect of Triton X-100
Authors: Callahan John W. , Shankaran Prema , Khalil Mary , Gerrie Jacqualine
Published: December 2009

Sphingomyelinase was purified about 1700-fold from human placenta. The major steps in the procedure included chromatography on Concanavalin A - Sepharose, Sepharose 6B, and carboxymethyl-Sepharose (CM-Sepharose). The final preparation was stable for at least 3 months when stored at 4 °C.The enzyme was found to be heterogeneous on CM-Sepharose and isoelectric focusing. Triton X-100 which was present in most buffers used during the purification appears to be partially responsible for the heterogeneity. When Triton X-100 is removed by treatment with Bio Beads, heterogeneity was reduced. However, removal of the detergent also leads to loss of enzyme activity which could not be restored by readdition of Triton X-100. The data suggest that sphingomyelinase has a high hydrophobic character and that both its stability and electrofocusing behaviour are influenced by interaction with the nonionic detergent.
Microheterogeneity and sialic acid in human plasma angiotensinogens in various physiological states
Authors: Faiers A. A. , Loh A. Y. , Osmond D. H.
Published: December 2009

Pooled or individual plasmas from normal men, women, pregnant women (third trimester), anephric women, and rat liver perfusates were used as sources of angiotensinogen. The plasmas were fractionated and desalted by Sephadex gel filtration, then subjected to isoelectric focusing in a pH 4 to 6 gradient on 40 × 4 cm slabs of polyacrylamide gel. The gels were cut transversely into 0.5-cm-wide strips, the pH measured, and their angiotensinogen concentrations determined by incubation with excess human renin and radioimmunoassay of the product, angiotensin I. This revealed several peaks of angiotensinogen concentration indicative of microheterogeneity in all cases. Contrary to other claims, the isoelectric pH profiles of angiotensinogens in the various physiological states were substantially alike. Major peaks were found at pH 4.75 to 4.85 and 4.9 to 5.0 and minor peaks at pH 4.5 to 4.7 and 5.0 to 5.2; this resolution was greater than that achieved with rat liver angiotensinogens. Incubation of human angiotensinogens with neuraminidase for 3 or 16 h raised their isoelectric pH by about 0.5 U, probably due to removal of sialic acid. Since microheterogeneity persisted after desialylation, it is probably determined by structural characteristics other than sialic acid composition.
Properties of a unique catalase isolated from Aspergillus niger
Authors: Gruft H. , Ruck R. , Traynor J.
Published: December 2009

The properties of an unusual catalase isolated from Aspergillus niger are reported. The enzyme, which has a molecular weight of 323 000, consists of four subunits and is highly resistant to sodium azide.
N-Terminal amino acid sequence of rat tonin: homology with serine proteases
Authors: Seidah N. G. , Routhier R. , Caron M. , Chrétien M. , Demassieux S. , Boucher R. , Genest J.
Published: December 2009

In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residue s of the single polypeptide chain composed of 272 amino acids. The se results showed an extensive homology with the sequence of many serine proteases of the trypsin–chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family.
Potato spindle tuber viroid: circular dichroism spectrum and physical chemical studies of its interaction with ethidium bromide
Authors: Singh R. P. , Williams R. E.
Published: December 2009

The presence and extent of base-paired secondary structure in the protein-free potato spindle tuber viroid RNA has been investigated by spectroscopic techniques. Dye-binding studies with the intercalating dye, ethidium bromide, indicate the presence of base-paired regions in the molecule. Variation of the induced chirality in the dye with the dye to RNA phosphate ratio indicate the presence of two base-paired regions in the molecule.
Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid–protein and lipid–lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10–12 °C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19–21 °C approximately and at 20–30 °C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.
3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in beef adrenal cortex
Authors: Preiss B. , Tan L. , Lehoux J.-G.
Published: December 2009

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity (mevalonate:NADP+ oxidoreductase (CoA-acylating) EC 1.1.1.34) was demonstrated in beef adrenal cortex. Most of the HMG-CoA reductase activity is in the microsomal fraction while a small percentage of the activity is associated with the mitochondria, Mitochondria purified on a linear sucrose gradient are enriched in HMG-CoA reductase and cytochrome c oxidase activities. The reductase present in microsomal preparations from the whole adrenal cortex has an apparent Km of 5.6 × 10−5 M for (R,S)-HMG-CoA. Reductase activities found in the microsomal fractions from the zona glomerulosa, the zona fasciculata, and the zona reticularis were 1.32, 7.37, and 9.74 nmol mevalonate formed per milligram protein in 30 min respectively.
Heterogeneity of human plasma low density lipoprotein
Authors: Rubenstein Bernard
Published: December 2009

Low density lipoprotein (LDL) was fractionated into subspecies by the use of DEAE-agarose column chromatography and the peptide compositions of the LDL subspecies which eluted at different NaCl concentrations were determined. LDL which elutes at low NaCl concentration has relatively less non-B apoprotein than does LDL which elutes at high salt concentration. The LDL subspecies which elute at high NaCl concentration contain more apo A-1 than do those which elute at the lower NaCl molarity. These results indicate that LDL consists of subfractions which differ in their peptide compositions.
Etude du métabolisme du 3-propyl-éther de l'estradiol 17β comparativement à l'estradiol 17β dans le foie de rat perfusé
Authors: Tresca Jean-Pierre , Ponsard Geneviève , Degrelle Hervé , Jayle Max-Fernand
Published: December 2009

The metabolism of 6,7-3H-tabelled 3-propyl ether estradiol (PE2) and of [6,7-3H]estradiol was studied by rat liver perfusion under different experimental conditions. In all cases, 90% of the radioactivity was retained in the liver, indicating an active uptake by the liver. The hepatic radioactivity was slowly released at a constant rate in the efferent perfusate. The proportion of radioactive metabolites in the perfusate was approximately the same as in the liver.3-Propyl ether estrone (PE1), a dehydrogenation product of PE2, and 3-propyl ether estriol (PE3), a 16α-hydroxylated derivative of PE2, were identified. Propylated metabolites more polar than PE2 were found. A low amount of propylated metabolites was conjugated with the exception of PE1. From the appearance of phenolic steroids including estrone, estradiol, and estriol, it was concluded that cleavage of the 3-propyl ether group had occurred.Compared with perfusion under oxygen, the overall metabolism was significantly reduced when the perfusion was carried out under nitrogen which demonstrates that oxygen plays a part in all the enzymatic systems involved. When animals were stimulated by phenobarbital, their entire metabolism was activated. These results suggest a metabolism mainly located in the hepatic microsomes.Our results show that the propylated hormone is metabolized like the free hormone. However, the transformations of PE2 are slower when compared with estradiol: thus, the 3-propyl ether group provided some hormone protection against hepatic degradation.
Inorganic salt denaturants stabilize ribonuclease against denaturation by urea
Authors: Ahmad Faizan , Bigelow Charles C.
Published: December 2009

The isothermal denaturation of ribonuclease A by mixed denaturant systems was investigated at 25 °C. It was observed that low concentrations of lithium chloride stabilize the protein against denaturation by urea, even though the salt itself is a denaturant. This study also provides, for the first time, the most convincing evidence that the lithium chloride denatured ribonuclease A contains some of the native secondary and tertiary structure.
Bis-intercalative binding to DNA of novel bis(10-methyl)acridinium chlorides and its dependence on chain length of linker
Authors: Lown J. William , Gunn Brian C. , Chang Rwei-Ying , Majumdar Krishna C. , Lee Jeremy S.
Published: December 2009

The synthesis of a series of novel bis(10-methyl)acridinium compounds (both unsubstituted and the 6-chloro-2-methoxy substituted) linked by methylene bridges of lengths from (CH2)4 to (CH2)12 and in one case by spermine is described. Their ability to bind to duplex DNA was compared by their relative inhibition of E. coli DNA polymerase catalyzed DNA synthesis. It was determined that they function as DNA template inhibitors and do not affect the DNA polymerase directly. Their ability to function as bis-intercalators was assessed by a novel and convenient topoisomerase fluorescent assay. It was concluded that whereas the (CH2)4-linked compounds act only as monofunctional intercalators because of steric constraints the (CH2)6-, (CH2)8-, and (CH2)10-linked substituted bisacridinium compounds, as well as the (CH2)10- and (CH2)12-unsubstituted analogues, function as bis-intercalators with DNA.
An immunological comparison of rye and calf histones
Authors: Roland Birgitte , Pallotta Dominick
Published: December 2009

The rye and calf histones were fractionated by differential solubility and exclusion chromatography. Antibodies were produced against histones H1, H2A, H2B, and H3 of both species and the extent of cross-reaction was measured by microcomplement fixation. The results allowed the identification of rye histones H2A and H2B. The near structural identity of the H3 histones was confirmed by the small immunological distance which indicated an amino acid difference of less than 2% between rye and calf. The interspecific differences found for both H2A and H2B were greater, the amino acid difference estimated from the immunological distance being between 10 and 20%. H1 gave no immunological cross-reaction, indicating a large (greater than 40%) difference in the structure of this protein in the two species. The extent of variation that a histone fraction shows between plant and animal species is probably related to its role in chromatin organization.
Enzymatic sulfation of steroids. V. Partial purification and some properties of sulfotransferase III, the major glucocorticoid sulfotransferase of liver cytosols from male rats
Authors: Singer Sanford S. , Gebhart James , Hess Edward
Published: December 2009

This manuscript describes purification of sulfotransferase III (STIII), the major hepatic glucocorticoid sulfotransferase of male rats, 77.8 ± 16 fold from cytosol. This represents a probable 250–345 fold enrichment, compared with homogenates. Purified STIII has a molecular weight of 61 500 ± 2500 from Sephadex G-100 chromatography. It is markedly activated by 5 mM divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts; inhibited strongly by 5 mM divalent Zn and Cd; and unaffected by 8 mM ADP, ATP, and AMP. Comparison of the ability of purified STIII to sulfate equimolar Cortisol, estradiol-17β, testosterone, and dehydroepiandrosterone suggests that the enzyme may sulfate glucocorticoids preferentially. However, its Cortisol sulfotransferase activity is inhibited by a variety of steroids. Of these, dehydroepiandrosterone, dexamethasone, and progesterone were tested extensively. They were found to be competitive inhibitors. STIII has a sharp pH optimum at pH 6.0 ± 0.1. However, it is routinely assayed at pH 6.8, as explained in the text. It exhibits a sequential mechanism and Km values of 6.82 ± 1.2 and 6.28 ± 0.64 μM for Cortisol and 3′-phosphoadenosine-5′-phosphosulfate, respectively. It also possesses essential sulfhydryl groups, as shown by p-hydroxymercuribenzoate inhibition studies.
DNA sequence organization in the common Pacific starfish Pisaster ochraceous
Authors: Smith Michael J. , Boal Robin
Published: December 2009

The sequence arrangement of the genomic DNA from the common sea star Pisaster ochraceous has been examined. Reassociation kinetics at DNA fragment lengths of 300 base pairs (bp) indicate the presence of at least three repetitive components in this DNA. The majority of these repetitive sequences are reiterated over the range from 10 to 100's. Approximately one-third of the nucleotides are found in repetitive sequences. Analysis of the reassociation kinetics of 3000-bp DNA fragments demonstrates the interspersion of repetitive and unique DNA sequences. The hyperchromicity of 3000-bp fragments reassociated to low Cot values (the product of moles of nucleotide per litre and time in seconds), and the size distribution of S1 nuclease resistant DNA duplex in these reassociation products, indicate a short-period interspersion pattern in the starfish genome. Repetitive segments (400 ± 100 bp) are interspersed with longer unique DNA sequences. At a fragment length of 3000 bp the major fraction of the single-copy DNA is found in such an arrangement. In addition to short repetitive segments a substantial portion of the repetitive DNA nucleotides are found in segments excluded by Sepharose CL-2B (≥ 2000 bp). As much as one-quarter of the repetitive sequence nucleotides can be assigned to long segments.
Nonexistence-of N-acetylseryl-tRN A in rat liver
Authors: Green R. M. , Elce J. S.
Published: December 2009

Reports of the existence of N-acetylseryl-tRNA in rat liver were reinvestigated. Methods were developed to permit recovery of N-acetylserine in a yield of 30–40% from N-acetylseryl-tRNA added to liver homogenates and cell-free incubations. [14C]Serine and [3H]acetate were injected into rats pretreated with iron and into rats after partial hepatectomy, and aminoacyl-tRNA was isolated from their livers. The amount of radioactivity associated with N-acetylserine in the amino acids released by hydrolysis from the aminoacyl-tRNA was negligible. No formation of N-acetylseryl-tRNA could be observed in incubations of acetyl CoA and seryl-tRNA or tRNA with enzyme fractions from liver of rats pretreated with iron. It is concluded that previous reports of the existence of N-acetylseryl-tRNA in rat liver are erroneous.
Bile acids of a 3200-year-old Egyptian mummy
Authors: Kuksis A. , Child P. , Myher J. J. , Marai L. , Yousef I. M. , Lewin P. K.
Published: December 2009

The bile acids of the gall bladder and hepatic tissue of a 3200-year-old Egyptian mummy were isolated by thin-layer chromatography and identified by combined gas–liquid chromatography and mass spectrometry. Except for complete deconjugation and extensive dehydration, the bile acids were found to correspond in their qualitative and quantitative composition to the gall bladder bile acids of modern man. The secondary bile acids constituted about 50% of the total and were identified as the normal bacterial oxidoreduction products of the primary bile acids and their dehydration products. In addition a series of unsaturated bile acids were identified, which corresponded to the dehydration products of cholic and chenodeoxycholic acids. It is suggested that both bile acid deconjugation and the limited oxidoreduction were probably brought about by the Clostridium organisms identified in the tissue. On the basis of the bile acid composition it is concluded that the ancient man metabolized cholesterol along the same pathways as modern man.
Evidence for an in vivo conformational change in ribulose bisphosphate carboxylase–oxygenase from Puma rye during cold adaptation
Authors: Huner N. P. A. , Macdowall F. D. H.
Published: December 2009

Ribulose bisphosphate carboxylase–oxygenase (RUBPCase) from leaves of cold-hardened and unhardened Puma rye was purified by gel filtration and ion exchange chromatography. Chemical properties that might be associated with a previously demonstrated difference in molecular charge of purified RUBPCase from cold-hardened and unhardened Puma rye were investigated. Amino acid analyses indicated no significant differences in amino acid composition or average hydrophobicity per residue. Enzymes from hardened and unhardened rye were reversibly coid inactivated at 0 °C. However, the former was more stable at this temperature than the latter. Titration with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) indicated the presence of fast and slow titrating sulfhydryl groups in both enzyme preparations but there were 50% fewer SH groups titrated in the enzyme from hardened rye in 30 min than in the enzyme from unhardened rye. Activation of both enzymes by HCO3− enhanced the reactivity of sulfhydryl groups to titration with DTNB. According to the kinetics of the slow titrating SH groups, the enzyme from hardened rye was less susceptible to denaturation by sodium dodecyl sulfate than was the same enzyme from unhardened rye. It is concluded that the tertiary structure of RUBPCase from Puma rye is affected during low-temperature adaptation.
Characterization of proteins associated with nuclear ribonucleoprotein particles by two-dimensional polyacrylamide gel electrophoresis
Authors: Suria D. , Liew C. C.
Published: December 2009

Rat liver nuclear ribonucleoprotein particles were prepared by two different methods and defined as 40S ribonucleoprotein (40S RNP) and heterogeneous nuclear ribonucleoprotein (HnRNP) particles. The RNP particles were either solubilized in 8 M urea – 6 mM 2-mercaptoethanol – 20 mM glycine – 20 mM Tris–HCl (pH 8.4) or subjected to removal of RNA by phenol extraction prior to solubilizing the proteins in the urea buffer. The proteins associated with 40S RNP and HnRNP were heterogeneous and very similar in their electrophoretic patterns when analyzed by two-dimensional PAGE, except a protein with molecular weight of 62 000 and an isoelectric point (pI) of 6.2 was present only in HnRNP particles. At least 12 major and 22 minor components could be identified in both preparations. The major proteins were found at pI values varying from 6.0 to 8.5 and with molecular weights from 32 000 to 42 000, and a group of proteins with molecular weight approximately 65 000 were more prominent in HnRNP than in 40S RNP. The other components were found mainly at pI ranges from 5.0 to 6.5 with molecular weights from 43 000 to 65 000. The phenol method extracted essentially all proteins associated with either 40S RNP and HnRNP, but was less effective in extracting a group of proteins with pI values from 5.0 to 5.5 and more efficient for proteins with pI values from 7.5 to 8.5. When chromatin proteins isolated by phenol extraction were compared with HnRNP particle proteins isolated by the same method, the electrophoretic mobilities of the HnRNP particle proteins were found to be identical with a fraction of nonhistone chromatin proteins. The 40S RNP particles were further purified by metrizamide isopycnic density gradient centrifugation. The electrophoretic patterns of these proteins were very similar to those prepared by sucrose density gradient centrifugation. Therefore, we concluded that the proteins of RNP particles constituted part of the chromatin proteins.
Histochemical and cytochemical localization of acetylcholinesterase in retina and optic tectum of teleost fish
Authors: Drujan Boris D. , Borges J. M. Díaz , Brzin M.
Published: December 2009

The activity of cholinesterase and its cellular and subcellular localization were investigated in the retina and optic tectum of Eugenes plumieri and in the retina of Carassius carassius by means of radiometric, histochemical, and cytochemical procedures. In both fishes only the presence of acetylcholinesterase could be demonstrated.This study, besides confirming previous findings that acetylcholinesterase is located in the ganglion and amacrine cells of the retina as well as in the inner plexiform layer, in addition provides evidence that the enzyme is also present at the region of photoreceptor synapses and between the cell bodies and apposing extensions of the horizontal cells of the same layer. The latter localization may indicate the involvement of a cholinergic mechanism at the functional contacts (transferapses) between the horizontal cells.In the optic tectum of Eugerres plumieri, histochemistry reveals fine distinguishable bands of acetylcholinesterase activity; two of the bands are quite sharply defined, whereas three others have rather a more diffuse appearance. The presence of these bands and their distribution may suggest a widespread distribution of cholinergic elements in the optic tectum.
Effect of phalloidin on actin proteolysis as measured by viscometry and fluorimetry
Authors: Pollender Jean-Marc , Gruda Julian
Published: December 2009

Even at low concentration, phalloidin shows a marked protection of F-actin against the action of trypsin or pronase. G-actin is not protected at any concentration of phalloidin. The kinetics of the proteolysis show that a change in the environment of tryptophan residues is preceded by disruption of the filamentous structure of F-actin.
Effect of lysolecithin in solubilizing membrane-bound glycosyltransferases
Authors: Mookerjea Sailen
Published: December 2009

Microsomal membranes were solubilized by incubation with lysolecithin which caused considerable release of galactosyl- and N-acetylglucosaminyl-transferase into a high-speed supernatant fraction. With a critical concentration of lysolecithin (2.5 mg/10 mg protein in 1 mL microsome suspension), there was a maximal binding of radioactive lysolecithin to the sediment fraction obtained after high-speed centrifugation. Increase of lysolecithin concentration (above 2.5 mg/mL) in the incubation mixture caused a progressive release of the enzymes into the supernatant fraction.Lysolecithin binding to the membrane was greatly inhibited by 1 M NaCl, and high salt concentration also inactivated galactosyltransferase in the sediment, suggesting an electrostatic interaction between lysolecithin and membrane enzyme. In contrast, high NaCl concentration had no inhibitory effect on the enzyme activity in the sediment when the fraction was prepared by treatment with Triton X-100.Lysolecithin-treated microsomal sediment and supernatant galactosyltransferase was inactivated by oleoyllysophosphatidic acid but not by palmitoyllysophosphatidic acid or egg yolk lysophosphatidic acid. Triton X-100 treated microsomal fractions were also similarly affected by different species of lysophosphatidic acid. The results suggested a similarity of interactions of lysophosphatidic fatty acyl species with lysolecithin and Triton-treated galactosyltransferase.
Identification of lipoprotein X-like particles in rat plasma following Intralipid infusion
Authors: Breckenridge W. C. , Kakis G. , Kuksis A.
Published: December 2009

Fasting rats were infused with 10% Intralipid for 24 h (0.33 mL/h per 100 g body weight) and the plasma lipoproteins isolated and compared with those of fed animals and animals with bile duct ligatures as controls. There was a 6- to 10-fold increase in the free cholesterol and phospholipid content of total plasma in animals infused with Intralipid or with ligated bile ducts. The changes were largely restricted to the low density lipoproteins (d = 1.019–1.063 g/mL) where free cholesterol and phospholipid increased 30- to 60-fold compared with fed control animals. Hydroxylapatite chromatography of the low density lipoprotein fractions of both Intralipid-infused and bile duct ligated animals yielded a subfraction which was rich in free cholesterol (27%), phosphatidylcholine (66%), and protein (6%); the latter was composed primarily of albumin and apo C proteins. The electrophoretic mobility and polyanionic precipitation properties of the abnormal lipoprotein were indistinguishable from those of lipoprotein X isolated from the animals with bile duct ligatures. The albumin in the abnormal lipoprotein from both groups of experimental animals was detected immunochemically only after delipidation of the lipoprotein. Twice as much of the lipoprotein X accumulated in Intralipid-infused than in the bile duct ligated animals. On rechromatography of the residual low density lipoprotein other subfractions could be isolated which possessed lipid and protein proportions intermediate between those of the lipoprotein X and of normal rat plasma low density lipoprotein. The activity of lecithin cholesterol acyl transferase was increased twofold in the Intralipid-infused animals when compared with control animals, but it decreased by 50% in the animals with bile duct ligatures. It is concluded that the unusual lipoprotein X accumulates in the plasma of Intralipid-infused animals owing to incomplete clearance of the exogenous phospholipid, which mobilizes tissue cholesterol and in the form of vesicular particles serves as a lipid phase for apo C proteins. A comparable mechanism is suggested for the formation of lipoprotein X in the animals with bile duct ligature.
A structural basis for four distinct elution profiles on concanavalin A – Sepharose affinity chromatography of glycopeptides
Authors: Narasimhan Saroja , Wilson James R. , Martin Eva , Schachter Harry
Published: December 2009

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanavalin A (Con A) – Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains:[Formula: see text]Such glycopeptides have only a single mannose residue capable of interacting with Con A – Sepharose; an interacting mannose residue is either an α-linked nonreducing terminal residue or an α-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl α-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures:[Formula: see text]where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A – Sepharose and elution as a sharp peak with 0.1 M methyl α-D-glucopyranoside; glycopeptides giving this pattern had the structures:[Formula: see text]where R2 is either H, GlcNAc, Gal-β1,4-GlcNAc, or sialyl-Gal-β1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the minimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl α-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures:[Formula: see text]where R3 is either GlcNAc, Gal-β1,4-GlcNAc, or siaryl-Gal-β1,4-GlcNAc. Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A – Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.
The biosynthesis of the free sterols and sterol esters of Neurospora crassa
Authors: Pierce A. M. , Pierce Jr. H. D. , Unrau A. M. , Oehlschlager A. C. , Subden R. E. , Renaud R. L.
Published: December 2009

The composition of the free and esterified sterols in Neurospora crassa was examined as a function of incubation time in starvation medium containing [2-14C]mevalonic acid. The 14C incorporation was monitored in nuclear methylated and 4, 14-desmethyl sterol fractions. After 7 h incubation, sterol esterification had increased from an initial 5% in the log phase culture to 48% of the total sterol pool, with a concomitant decrease in free sterols. The relationship of the free and esterified sterol components in ergosterol biosynthesis is discussed.
Stereospecific analysis of triacylglycerols via racemic phosphatidylcholines and phospholipase C
Authors: Myher J. J. , Kuksis A.
Published: December 2009

A method of simultaneous determination of stereospecific distribution and molecular association of acyl groups in triacylglycerols has been developed. The analysis is based on a random generation of rac-1, 2-diacylglycerols by Grignard degradation, synthesis of rac-phosphatidylcholines, and a stereospecific stepwise release of 1, 2-sn- and 2, 3-sn-diacylglycerols by phospholipase C. The exact structure of the original triacylglycerols is reconstituted on the basis of complete analysis of the molecular species of the 1, 2-sn- and 2, 3-sn-diacylglycerols as the tertiary-butyldimethylsilyl ethers by gas chromatography with mass spectrometry. The validity of the method is demonstrated by analyses of synthetic triacylglycerols of known structure. A practical application is illustrated by determination of the fatty acid distribution in lard.
Purification and some properties of five endo-1,4-β-D-xylanases and β-D-xylosidase produced by a strain of Aspergillus niger
Authors: John Michael , Schmidt Bernd , Schmidt Jurgen
Published: December 2009

Five different xylanases and a β-D-xylosidase in the culture medium of Aspergillus niger have been purified to homogeneity from 13- to 52-fold by a procedure of gel and hydroxylapatite chromatography. The strain was isolated from soil of the African equatorial forest. Gel chromatography of the purified enzymes indicated that three of the xylanases have molecular weights of 31 000 and the other two xylanases have molecular weights of 50 000.β-D-Xylosidase has a molecular weight of 78 000. The pH curves of the xylanases were quite diverse and showed pH optima ranging from 4.0 to 6.5. Characteristic action patterns were obtained for each of the purified xylanases by gel chromatography of the xylan digests on Bio-Gel P-2. The enzymes degraded arabinoxylan by an endomechanism, producing L-arabinose, D-xylose, xylobiose, and a mixture of branched arabinose–xylose and D-xylose oligosaccharides. All xylanases seemed to be capable of liberating L-arabinose from either arabinoxylan or the arabinose–xylose oligosaccharides. Branched arabinose-containing D-xylose oligosaccharides were slowly hydrolyzed, so that these sugars accumulate in the digest. Two xylanases showed relatively broad substrate specificity and were able to degrade also crystalline cellulose. β-D-Xylosidase showed optimal activity at pH 6.7 to 7.0 and at 42 °C. The Km for o-nitrophenyl-β-D-xylopyranoside was 0.22 mM and xylotriose was hydrolyzed more rapidly than xylobiose.
The 2.8 Å resolution structure of Streptomyces griseus protease B and its homology with α-chymotrypsin and Streptomyces griseus protease A
Authors: Delbaere Louis T. J. , Brayer Gary D. , James Michael N. G.
Published: December 2009

The 2.8 Å (1 Å = 0.1 nm) resolution structure of the crystalline orthorhombic form of the microbial serine protease Streptomyces griseus protease B (SGPB) has been solved by the method of multiple isomorphous replacement using five heavy-atom derivatives. The geometrical arrangement of the active site quartet, Ser-214, Asp-102, His-57, and Ser-195, is similar to that found for pancreatic α-chymotrypsin. SGPB and α-chymotrypsin have only 18% identity of primary structure but their tertiary structures are 63% topologically equivalent within a root mean square deviation of 2.07 Å. The major tertiary structural differences between the bacterial enzyme SGPB and the pancreatic enzymes is due to the zymogen requirement of the multicellular organisms in order to protect themselves against autolytic degradation. The two pronase enzymes, SGPB and Streptomyces griseus protease A (SGPA), have 61% identity of sequence and their tertiary structures are 85% topologically equivalent within a root mean square deviation of 1.46 Å. The active site regions of SGPA and SGPB are similar and their tertiary structures differ only in three minor regions of surface loops.
Preparation and mass spectral behaviour of some 5β-cholenoic acids
Authors: Child P. , Kuksis A. , Marai L.
Published: December 2009

Monounsaturated 5β-cholanoic acids with double bonds in rings A, B, and C were prepared by POCl3 and ZnCl2 dehydration from natural bile acids with selectively blocked hydroxyl functions. The yields ranged from 15 to 100%. The products were purified by thin-layer and AgNO3 thin-layer chromatography and the structures were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. The methyl ester acetates of the unsaturated 5β-cholanoic acids possessed chromatographic properties closely similar to those of the corresponding saturated bile acids. Several characteristic fragments were seen in the mass spectra which, in conjunction with the chromatographic properties, permitted an unambiguous distinction between different monounsaturated acids, and between saturated and unsaturated bile acids of the same number and configuration of functional groups. The 20 5β-cholenoic acids examined represent all of the simple chemical and enzymatic dehydration products of natural bile acids and can be completely identified by their combined chromatographic and mass spectral properties.
Characterization of an octopamine-sensitive adenylate cyclase from insect brain (Mamestra configurata Wlk.)5
Authors: Bodnaryk Robert P.
Published: December 2009

An adenylate cyclase present in the brain of the moth Mamestra configurata Wlk. that is stimulated selectively by low (micromolar) concentrations of octopamine has been characterized with respect to several properties. The optimum pH, optimum ATP:Mg2+ ratio, the concentration of ATP required for half-maximal and maximal reaction velocity, metal ion specificity, effect of NaF, and effects of GTP and 5′-guanylylimidodiphosphate were in general similar to those of catecholamine-sensitive adenylate cyclases from various regions of mammalian brain. However, ethylene glycol bis-(β-aminoethyl ether)-N,N-tetraacetic acid (EGTA), a calcium chelator, stimulated both basal and octopamine-sensitive enzyme activity in the insect brain, whereas in mammalian brain EGTA is usually observed to inhibit basal activity but not catecholamine-stimulated activity.Adenylate cyclase activity of the 47 000 g particulate fraction of the insect brain was almost undetectable in the absence of added GTP. Addition of saturating concentrations (100 μM) of GTP to the particles restored about 30% of the basal and octopamine-sensitive enzyme activity present in the homogenate. Addition of 100 000 g supernatant to the particles doubled both basal and octopamine-sensitive enzyme activity in the presence of saturating concentrations of GTP, indicating that in addition to GTP, a cytosolic factor(s) is necessary for enhanced adenylate cyclase activity.
The molecular mechanisms of substrate transport in gram-negative bacteria
Authors: Lo Theodore C. Y.
Published: December 2009

not available
Products of mitochondrial protein synthesis in Tetrahymena
Authors: Young Paul G. , Hunter Neil P.
Published: December 2009

The products of mitochondrial protein synthesis have been investigated in Tetrahymena after labelling with [35S]methionine in the presence of cycloheximide. The labelled proteins were analyzed by sodium dodecyl sulfate slab polyacrylamide gel electrophoresis. We have identified 13 electrophoretically discrete bands as well as 4 other bands with a more variable occurrence. These proteins ranged in apparent molecular weight from 8100 to 57 500. The cycloheximide-resistant incorporation could be blocked with chloramphenicol. The mitochondrial proteins appeared to be in a disaggregated state and were stable to agents such as trichloroacetic acid (hot or cold) and chloroform–methanol. The pattern of proteins was similar following labelling times ranging from 30 min to 3 h.
Partial characterization of the mode of inhibition of Escherichia coli RNA polymerase by the mixed disulfide, CoASSG
Authors: Bees William C. H. , Loewen Peter C.
Published: December 2009

The coenzyme A – glutathione mixed disulfide (CoASSG), when complexed with iron, is capable of inhibiting the RNA polymerase of Escherichia coli. A modified procedure involving a short time of exposure to high salt allowed the reliable preparation of CoASSG–Fe which was active in inhibiting RNA polymerase. The CoASSG–Fe complex acted as a noncompetitive inhibitor for the incorporation of all four nucleoside triphosphates but had a greater effect on GMP and CMP incorporation than AMP and UMP incorporation. Neither temperature nor ionic-strength changes affected CoASSG–Fe inhibition, and the use of rifampicin showed that CoASSG–Fe did not inhibit either the initiation or elongation processes of the polymerase. CoASSG–Fe was a more effective inhibitor at low DNA-template concentrations and it was more effective in inhibiting the incorporation of CMP and GMP on simple dG-dC containing templates and the asymmetric polymer poly d(T-C)∙poly d(G-A). The inhibition of transcription of poly d(I-C) was less effective than the inhibition of transcription of poly d(G-C). Equilibrium dialysis in microdialysis cells showed that CoASSG–Fe could associate with DNA in the absence of RNA polymerase.
The effect of binding ions on the oxidation of horse heart ferrocytochrome c
Authors: Peterman B. F. , Morton R. A.
Published: December 2009

The effect of various specific binding ions on the rate of oxidation by potassium ferricyanide of electrodialyzed horse heart ferroeytochrome c was studied. The ionic strength was kept constant using Tris–cacodylate buffer, pH 7.0. Either the Tris or cacodylate ion was replaced by the binding ion studied. At an ionic strength of 0.194 M (24 °C), replacing cacodylate by chloride decreased the bimolecular oxidation rate constant from about 13.3 × 106 (Tris–cacodylate) to about 8.8 × 106 M−1 s−1 (Tris–chloride). Comparable decreases were found when cacodylate was replaced by phosphate or when Tris was replaced by potassium. When picrate replaced cacodylate (ionic strength 0.0485 M) a larger decrease was found, from about 5.2 × 107 to about 0.8 × 107 M−1 s−1. Data at intermediate ion concentrations were consistent with a simple cooperative binding model. The calculated association constants for chloride, potassium, and phosphate were in the range of 2–20 M−1, while for picrate it was 500 M−1. The data were consistent with one bound ion per ferroeytochrome c molecule, except for picrate, for which two binding sites were suggested. The results were interpreted by the hypothesis that the picrate ion binds near the solvent-exposed heme edge. The electron transfer reaction of ferricyanide was also presumed to take place through this region. The other ions probably bind at some distance from the heme edge and were suggested to exert their effect by perturbing the proteins' solvent shell. Consistent with this was the effect of replacing H2O by D2O which decreased the oxidation rate constant by about 50%.
Positional distribution of isomeric spin-labelled stearic acids and microsomal fatty acids in biosynthetic phosphatidylcholines
Authors: Stuhne-Sekalec L. , Stanacev N. Z. , Marai L. , Kuksis A.
Published: December 2009

Spin-labelled radioactive sn-1,2-diacylglycerols and sn-3-phosphatidylcholines obtained by acylation of sn-3-[U-14C]glycerophosphate with 5-, 12-, and 16-doxyl stearic acids in the presence of microsomal membranes of guinea pig liver were isolated and purified by column and thin-layer chromatography. Positional distribution of isomeric doxyl stearic acids in the glycerolipids was established by hydrolysis of the phosphatidylcholines with phospholipase A2, while the molecular association of the endogenous and doxyl fatty acids was determined by mass spectrometry of the sn-1,2-diacylglycerols as the tertiary-butyl-dimethylsilyl ethers. It was found that the 5-doxyl stearic acid predominantly occupied the secondary hydroxyl position, but that the 12- and 16-doxyl stearic acids were approximately equally distributed between the primary and secondary hydroxyl groups. Furthermore, the 5-doxyl stearic acid was preferentially associated with saturated endogenous microsomal fatty acids, while the 12- and 16-doxyl stearic acids were associated with both saturated and unsaturated fatty acids. In these respects, the positional distribution and molecular association of the 5-doxyl stearic acid approximated that of linoleic acid, while the 12- and 16-doxyl stearic acids were incorporated into the glycerolipids very much like oleic acid. The doxyl stearic acid containing diacylglycerols were relatively low in stearic acid. This finding agrees with previously established relative absence of stearic acid from newly formed phosphatidic acids. The relevance of these findings to the use of biosynthetic spin-labelled phosphatidylcholines in studies of lipid organization in biological membranes is discussed.
Respiration-driven accumulation of C4 dicarboxylic acids by isolated membrane vesicles of Paracoccus denitrificans
Authors: Pik Jeanette R. , Lawford Hugh G.
Published: December 2009

Membrane vesicles derived by osmotic lysis of spheroplasts of Paracoccus denitrificans (ATCC 13543) grown aerobically in continuous culture under conditions of carbon limitation with succinate as sole carbon and energy source accumulate radioactivity in an uncoupler-sensitive respiration-dependent manner when incubated in the presence of [14C]succinate or L-[14C]malate. Membranes prepared from cells grown under conditions of sulphate limitation with succinate as the sole carbon source do not accumulate L-[14C]malate during ascorbate + N,N,N′,N′-tetramethyl-p-phenylene diamine (TMPD) oxidation. The apparent Km for succinate and L-malate uptake is 6.7 and 10 μM respectively with a Vmax for uptake of either substrate being 8.3 nmol/min per milligram of membrane protein. Intravesicular radioactivity was largely confined to C4 dicarboxylic acids, succinate, fumarate, and malate and was freely exchangeable with external unlabeiled C4 dicarboxylic acids but not aspartate. Both ubiquinol1 and ascorbate (+ TMPD) oxidation supported accumulative uptake of succinate or L-malate but NADH did not. Since energization of the dicarboxylate transport system is accomplished by the oxidation of ascorbate + TMPD in the presence of antimycin A, it is concluded that heterotrophically grown P. denitrificans containing cytochrome a3 possess a functional energy-coupling site 3 (terminal energy-transducing region of the respiratory chain) despite claims to the contrary.
Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells
Authors: Terris S. , Hofmann C. , Steiner D. F.
Published: December 2009

The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine, inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and anti-microtubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 °C, insulin degradation was negligible but rose rapidly between 20 and 37 °C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.