bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of steroid synthesis in human fetal adrenals in monolayer culture
Authors: Baird Andrew C. , Brisson Gilles , Kan Kenneth W. , Duguid W. C. , Solomon Samuel
Published: December 2009

The actions of adrenocorticotrophin 1-39 (ACTH), N6, O2-dibutyryl adenosine-3′, 5′-cyclic phosphate (dbcAMP), and human chorionic gonadotrophin (hCG) on the sulfation of DHA and the synthesis of cortisol by human fetal adrenal cells in monolayer culture have been described. Electron microscopic studies indicate that the cells present in our cultures at the end of 5 days are intact and have many of the characteristics of secretory cells. Sulfation of DHA was strongly inhibited by 11-deoxycorticosterone, testosterone, androsterone, and dihydrotestosterone. Very small effects were observed with progesterone and corticosterone and insignificant effects with estrone, estradiol, and estriol. Androstenedione as well as 17α-hydroxylated corticosteroids showed no inhibition. When used as substrate, C21 Δ4-3-ketosteroids were good precursors of cortisol with 11-deoxycortisol being the best one followed by 17α-hydroxyprogesterone and progesterone. The conversion of progesterone (4 μg/2 ml) to cortisol was fivefold greater than under conditions where no substrate was present (endogenous production). ACTH in the concentration of 5 × 10−4 U/ml added twice daily to the cell cultures for 8 days increased eightfold the sulfation of DHA and increased cortisol synthesis from progesterone 35-fold in 5 days. The addition of dbcAMP (5 × 10−5 M) or hCG used at 2.0 or 0.2 U/ml did not stimulate the sulfation of DHA. A lag period of at least 36 h is required before the stimulation of DHA sulfation or cortisol synthesis from progesterone can be observed. dbcAMP can stimulate the conversion of progesterone, 17α-hydroxyprogesterone, and 11-deoxycortisol to cortisol by the adrenal cells. These data suggest that 11β- and 21 -hydroxylation are stimulated by dbcAMP but 17α-hydroxylation is not altered.
Biochemical and genetic approaches to the study of mammalian mitochondrial tRNAs
Authors: Aujame Luke , Yatscoff Randall W. , Freeman Karl B.
Published: December 2009

The possible existence of mammalian mitochondrial asparaginyl-tRNA has been examined using a variety of approaches. [3H]Asparagine was incorporated into protein by mitochondria of the Chinese hamster ovary (CHO) cell line Asn-7, which has a temperature-sensitive cytosolic asparaginyl-tRNA synthetase, either in the presence of cycloheximide or at a nonpermissive temperature. Isolated mitochondria of CHO thymidine kinase minus (TK−) cells also incorporated the amino acid into protein. In each case, the number and electrophoretic mobility of the proteins was the same as mitochondrially synthesized proteins of CHO TK− ceils labelled with [35S]methionine. A tRNAAsn could be charged in isolated CHO TK cell mitochondria and the asparaginyl-tRNA was found to elute before its cytosolic counterpart on an RPC-5 column and to have a higher mobility on polyacryiamide slab gels run under denaturing conditions. This is the first demonstration of a unique mitochondrial asparaginyl-tRNA.
The control of pyruvate kinases of Escherichia coli: Further studies of the enzyme activated by ribose-5-phosphate
Authors: Mort John S. , Sanwal B. D.
Published: December 2009

The pyruvate kinase activated by ribose-5-phosphate from Escherichia coli has been purified to homogeneity, taking advantage of the stabilization of the enzyme by its inhibitor phosphate and by thiol reagents. The native enzyme has a tetrameric quaternary structure which, while prone to dissociation under many conditions, remains intact in the presence of the above reagents.The enzyme was found to reactivate on dilution out of 8 M urea. Interestingly, the recovery of activity is greatly increased by phosphate, an allosteric inhibitor, but markedly reduced by the allosteric activator, ribose-5-phosphate, implying that it is harder for the enzyme to refold to a 'relaxed state.' Proteolysis studies indicate a more open structure in the presence of the activator.
Aminooxyacetic acid induced changes in γ-aminobutyrate metabolism at the subcellular level
Authors: Wood J. D. , Kurylo E. , Newstead J. D.
Published: December 2009

Aminooxyacetic acid (AOAA) (0.1 or 0.23 mmol/kg) was administered to mice which were killed 1.5 and 6 h after treatment. Synaptosomal- and mitochondrial-enriched fractions were obtained by conventional ultracentrifugation procedures. The γ-aminobutyric acid (GABA) content of the synaptosomal fraction was elevated by previous treatment of the mice with AOAA, the increase being greater at 6 h than at 1.5 h posttreatment. This finding suggested that a transient elevation of the GABA content of nerve endings was not responsible for the fast-developing anticonvulsant action of AOAA. The activity of aminobutyrate aminotransferase (GABA-T) was inhibited after AOAA treatment, the degree of inhibition being greater in the mitochondrial-enriched than in the synaptosomal-enriched fraction. Evidence for circadian changes in GABA-T activity was obtained. The limitations and advantages of subcellular fractionation techniques in determining the effects of drugs on GABA metabolism were assessed.
The development of hepatic drug-metabolizing enzymes in perinatal guinea pigs: A biochemical and morphological study
Authors: Ecobichon D. J. , Dykeman R. W. , Hansell M. M.
Published: December 2009

The development of four functionally diverse, hepatic enzymes (p-nitroanisole O-demethylase, aniline hydroxylase, carboxylesterase, and glucuronyltransferase (with α-naphthol as the aglycone acceptor)) was studied in perinatal Hartley guinea pigs from 8 days prepartum to 28 days postpartum. A good correlation was observed between the activities measured in resuspended Ca2+-aggregated microsomes and the quantities of hepatic smooth endoplasmic reticulum visible by electron microscopic examination at the different stages of development. The study demonstrated that, postnatally, the guinea pig developed competent enzymatic systems as rapidly as did other laboratory species but that, prenatally, these same enzyme(s) systems were much further advanced than those in other species.
Probes of the structure of phosphoenolpyruvate synthetase: Effects of a transition state analogue on enzyme conformation
Authors: Narindrasorasak Suree , Bridger William A.
Published: December 2009

Phosphoenolpyruvate synthetase of Escherichia coli is strongly inhibited by oxalate. The magnitude of the inhibition constant for oxalate suggests that this compound acts to produce a transition state analogue, in keeping with the suggestion of others that oxalate mimics the structure of enolpyruvate, a presumed catalytic intermediate in the enzymatic reaction. The addition of oxalate together with ATP results in a dramatic shielding of sensitive amino acid residues from reaction with both N-ethyl maleimide and phenylglyoxal. Thus, under conditions otherwise giving rise to almost complete inactivation by either reagent, no loss of activity is detectable in the presence of oxalate and ATP. These results indicate the formation of an enclosed structure during catalysis in which reactive groups are rendered quite inaccessible to solvent.
A rapid method for the determination of CTP and phosphocholine in rat liver and baby hamster kidney 21 cells
Authors: Choy Patrick C. , Whitehead Frederick W. , Vance Dennis E.
Published: December 2009

A rapid and sensitive assay for CTP and phosphocholine was required for us to determine the concentration of these compounds in tissues and cell cultures. Such a procedure was devised with CTP:phosphocholine cytidylyltransferase, an enzyme which is highly specific for CTP and phosphocholine. The 0–22% ammonium sulfate precipitate of a cytosolic extract from rat liver was used as the source of the enzyme. The amount of CTP in an extract was estimated by the conversion of [3H]phosphochoiine to 3H-labelled CDP-choline. Similarly, the concentration of phosphocholine was estimated by the formation of 3H-labelled CDP-choline from 3H-labelled CTP. The conversion of CTP and phosphocholine to CDP-choline was 90% when inorganic pyrophosphatase was added to the incubations. The formation of CDP-choline was linear between 1 and 10 nmol of CTP or phosphocholine. The concentration of CTP was determined in rat liver (62 nmol/g wet weight) and baby hamster kidney 21 (BHK-21) cells (161 nmol/g wet weight). The concentration of phosphocholine in rat liver was 1.16 μmol/g wet weight whereas in BHK-21 cells it was much less (69 nmol/g wet weight). By this procedure, it may be possible to establish the importance of CTP and phosphocholine in the control of phosphatidylcholine biosynthesis.
Plasma membrane associated proteins and glycoproteins of a temperature-sensitive hamster cell line (ts 3)
Authors: Nigam Vijai N. , Brailovsky Carlos A.
Published: December 2009

Hamster ts 3 cells grown at 32 and at 39 °C were compared for (a) cell phenotype and growth rates, (b) sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) patterns of plasma membrane polypeptides, and (c) distribution of radioactivity in SDS–PAGE of plasma membrane glycopeptides metabolically labeled with [3H]glucosamine and [3H]fucose. It was observed that ts 3 cells growing at 39 °C and having a normal phenotype contained increased amounts of a glycopeptide of about 250 000 daltons and a polypeptide of about 200 000 daltons, as compared with ts 3 cells grown at 32 °C and having a transformed phenotype. No other alterations of major significance were observed.
Growth temperature-dependent stearoyl coenzyme A desaturase activity of Fusarium oxysporum microsomes
Authors: Wilson Alan C. , Miller Richard W.
Published: December 2009

The characteristics of the microsomal stearoyl CoA desaturase (EC 1.14.99.5) of vegetative Fusarium oxysporum cells grown at different temperatures were studied. The enzyme had an unusual preference for NADPH (Km = 38 μM) over NADH (Km = 89 μM) as electron donor, and a relatively high optimum pH of 8.3. Enzyme activity was highest in microsomes from cells grown at 37 °C and lowest in cells grown at 15 °C. This result correlated well with the observed changes in oleic acid content of the microsomal lipids.Both NADPH-linked reductase activities and hemoprotein content were lowest in cells grown at 37 °C. Spectrophotometric analysis of the microsomal hemoproteins indicated the absence of cytochrome b5 and the presence of a b-type heme with a pyridine hemochrome α band absorption maximum at 565 nm. Labile sulfide analysis and inhibitor studies with thenoyltrifluoroacetone suggested a role for an iron–sulfur protein in the electron transfer system associated with the desaturase.
Synthesis of 1-sn-lysophosphatidylcholine and mixed acid 1-sn-phosphatidylcholine
Authors: Smith Norman B. , Kuksis Arnis
Published: December 2009

An investigation of the biochemical effects of L-lysophosphatidylcholine (1-acyl-sn-glycerol-3-phosphorylcholine) requires as a control for its detergent action the metabolically inert, unnatural enantiomer, D-lysophosphatidylcholine (3-acyl-sn-glycerol-1-phosphorylcholine). The latter can be easily prepared in milligram quantities from the commercially available DL-phosphatidylcholine. For this purpose rac-1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine is solubilized with the aid of one-half its weight of taurocholate and hydrolyzed with phospholipase A2 to completion and the intact residual 2,3-dipalmitoyl-sn-glycerol-1-phosphorylcholine recovered by chromatography. The pure D-phosphatidylcholine is then subjected to methanolysis in the presence of octylamine and the product isomerized with glacial acetic acid. The 3-palmitoyl-sn-glycerol-1-phosphorylcholine thus obtained is chromatographically pure, possesses correct optical rotation and NMR spectrum, and is resistant to hydrolysis by phospholipase A2 following reacylation to the diacylphosphatide. Acylation of the 1-sn-lysophosphatide provides a convenient method for preparation of mixed acid 1-sn-phosphatidylcholines which are not otherwise available.
Erratum: S-Adenosylhomocysteine metabolism in various species
Authors: Walker R. D. , Duerre J. A.
Published: December 2009

not available
Changes in the net charge and subunit properties of ribulose bisphosphate carboxylase–oxygenase during cold hardening of Puma rye
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. The specific activity of the hardened form was twice that of the unhardened form. A difference in charge between the two forms of this enzyme was proved by gel electrofocussing. The estimated isoelectric point (pI) values were 6.4 and 6.3 for the enzyme from the hardened and unhardened source respectively. The large subunit (55 000 molecular weight) of the enzyme from only the unhardened source formed an apparent dimer during sodium dodecyl sulfate (SDS) gel electrophoresis. At pH 6.8 it was also the source of an anomalous polypeptide with an apparent molecular weight of 47 000. This anomalous polypeptide appeared in both hardened and unhardened preparations after irreversible inactivation of RUBPCase activity by NaCl. It also appeared after preparation of the purified enzymes for SDS–PAGE in the absence of β-mercaptoethanol, but this was reversible. The enzyme from the hardened source was less affected in the absence of reducing agent. Structural evidence was obtained for the previously reported cold hardening of the enzyme against freeze inactivation. A freeze–thaw cycle applied to the enzyme in vitro caused some polymerization of the large subunit and its anomalous polypeptide, in the absence of reducing agent, especially in the unhardened case. This increased with repeated cycles until the fifth cycle when the large subunit monomer and its satellite were abolished only in preparations from the unhardened source. These data indicate that the large subunit is a probable site of change that occurred in this enzyme during cold hardening.
A magnetic resonance study of the coenzyme A – manganese(II) complex in solution
Authors: Theriault Yvon , Morio Yuri , Kotowycz George
Published: December 2009

Proton and 31P magnetic resonance experiments were employed in the study of the weak, stoichiometric, one-to-one complex that is formed between coenzyme A and manganese-(II). The distances in solution between the three phosphorus atoms and the manganese(II) ion are obtained at 287 K and at 298 K. The experiments are carried out over a pH range of 6.3 to 7.7. The correlation time τc of the water molecules bound to the Mn(II) ion in the CoA–Mn(II) complex was calculated using the Solomon–Bloembergen equation from the T1M value obtained for the water protons for solutions where most of the Mn(II) ions are complexed. Paramagnetic spin-lattice relaxation rates for all three phosphorus atoms in the CoA molecule were obtained using proton decoupled Fourier transform 31P nmr techniques. For most probable values of two or three water molecules in the CoA–Mn(II) complex, the distances between the manganese (II) ion and the three phosphorus nuclei are 4.0 ± 0.4 Å (1 Å = 0.1 nm) or 3.7 ± 0.4 Å respectively for 0.062 M CoA solutions. For experiments carried out on 0.010 M CoA, a correlation time which has been determined previously for propionyl CoA was used for the distance calculations and the distances remain unchanged within experimental error. These results indicate that the Mn(II) ion coordinates equally with the oxygens of the three phosphate groups in the CoA–Mn(II) complex.
Proton translocation coupled to ubiquinol oxidation in Paracoccus denitrificans
Authors: Lawford Hugh G.
Published: December 2009

Measurements were made of the stoichiometry of proton translocation associated with electron flow through the cytochrome-dependent region of the aerobically induced respiratory chain of Paracoccus denitrificans using the pulse oxidant method and rotenone to inhibit NADH dehydrogenase activity. Paracoccus denitrificans (ATCC 13543) was grown aerobically in carbon-limited continuous culture D = 0.27 h−1D, dilution rate per hour) with succinate as sole carbon and energy source. Oxidation of exogenous ubiquinol1 by starved cells was significantly accelerated by treatment of the cells with lysozyme. Spectrophotometric assay of ubiquinol1 oxidase activity in rotenone-poisoned spheroplasts revealed an apparent Km of 25 μM and a Vmax of 217 nmol/min per milligram protein. In site 1 poisoned spheroplasts proton translocation was dependent on the presence of added respiratory substrate, either succinate or ubiquinol1, where the observed ratios of protons ejected from the membrane per atom of oxygen consumed (← H+: O) were 5.3 and 5.2 respectively. The rate of proton translocation associated with succinate and ubiquinol1 oxidation was significantly decreased in the presence of antimycin A and completely abolished with cyanide. Net proton translocation was not observed after the addition of uncoupler (CCCP). Assuming there are two potential sites of energy conservation associated with the region of the respiratory chain from ubiquinone to cytochrome oxidase (i.e., ubiquinol oxidase activity), then the number of protons ejected during the transfer of one pair of reducing equivalents along a region of the electron transport chain equivalent to a single energy-coupling or conservation site (← H+: site) ratio in P. denitrificans is closer to 3 than 2 as predicted by Mitchell's chemiosmotic coupling hypothesis.
Double-stranded protamine cDNA: synthesis and characterization
Authors: Davies Peter L. , Dixon Gordon H. , Dugaiczyk Achilles
Published: December 2009

Double-stranded protamine complementary DNA (cDNA) was synthesized from a protamine mRNA template via the single-stranded cDNA intermediate using avian myeloblastosis virus reverse transcriptase. Synthesis at 37 and 46 °C resulted in similar overall yields (≥ 18%), although the initial rate of synthesis was higher at 46 °C than at 37 °C. The DNA of the second strand of the double-stranded cDNA product was 84% resistant to prolonged digestion with excess S1 nuclease. The S1 nuclease resistant material ranged in size from 235 to 100 base pairs (bp) with an average length of 185 bp. Analysis of the products released from double-stranded protamine cDNA by depurination indicated that there were a number of cytosine-rich oligopyrimidine tracts in protamine mRNA, namely C4, C4U1, C5U1, C6U1, C6U4, and C7U1. On the basis of the amino acid sequences for rainbow trout protamines, C5U1, C6U1 and C7U1 must be located within the noncoding regions. Double-stranded protamine cDNA was cleaved at least once by the restriction endonucleases HaeIII and HhaI and in several places by HpaII. These restriction endonucleases cleave at sequences which have a high probability of occurring within the coding region of protamine mRNA, again based on the known amino acid sequences of the rainbow trout protamines.
Conformation of exocyclic amino groups in purines and pyrimidines: crystal structure and conformation of 1-methyl-N4-hydroxycytosine hydrochloride
Authors: Birnbaum George I. , Kulikowski Tadeusz , Shugar David
Published: December 2009

The hydrochloride salt of 1-methyl-N4-hydroxycytosine crystallizes in the triclinic space group [Formula: see text] with cell dimensions: a = 8.232(1), b = 9.293(1), c = 5.416(1) Å (1 Å = 0.1 nm); α = 91.95(1), β = 91.72(2), γ = 71.56(1)° (SE in parentheses). The structure was solved by direct methods and refined to R = 3.7% for 1514 reflections. Despite the absence of an intramolecular hydrogen bond in the solid state, the N4-hydroxy substituent is syn to the ring N(3). This conformation, which probably prevails also in the neutral form, is of relevance to the mechanism of attack of cytosine by hydroxylamine, known to involve predominantly the cytosine cation, as well as to the mechanism of hydroxylamine mutagenesis. Such conformational aspects are also relevant to other phenomena, including translation and restriction.
The fatty acid composition of phosphoglycerides of nerve cell bodies isolated in bulk from rabbit cerebral cortex: changes during development and positional distribution
Authors: Baker R. Roy
Published: December 2009

Analyses were made of the component fatty acids of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) of nerve cell bodies isolated in bulk from cerebral cortex of rabbits of 8, 15, and 22 days and 9 weeks of age. PC has palmitate as its major saturated fatty acid and this showed a decrease with age (8 days to 9 weeks) from 46 to 39% of the total fatty acid. Stearate, the dominant saturate in PE, (22–23%), PI (35–37%), and PS (42–44%), remained relatively constant with age, although there was an increase in stearate in PC (6 to 10%) over this time period. Of the four phosphatides, PC had the highest levels of monoenoic fatty acids, showing progressive increases in oleate (23 to 28%) and declines in palmitoleate (6 to 2%). During development, the level of arachidonate (20:4) remained relatively constant in PC (10–11%) and PI (47–48%), but declined in PE (26 to 21%) and PS (8 to 5%). Conversely, the levels of docosahexaenoate (22:6) rose in PE (11 to 17%) and PS (16 to 21%) over the same time period. Phospholipase A2 hydrolyses of these phosphoglycerides showed stearate principally at position 1 and polyunsaturates (e.g., 20:4, 22:6) almost exclusively at position 2. Palmitate and oleate were generally found in greater abundance at position 1, with the exception of PC where both these fatty acids were almost equally distributed between positions 1 and 2. The existence of dipalmitoyl and 1-oleoyl-2-palmitoyl lecithins in nerve cell bodies may thus be suggested.
Distribution of the multiple molecular forms of glucose-6-phosphate dehydrogenase in different physiological states
Authors: Chang Hsiao-Lin , Holten Darold , Karin Rom
Published: December 2009

The distribution of the multiple molecular forms of rat liver and mammary gland glucose-6-phosphate dehydrogenase was determined by electrophoresis on 5% polyacrylamide gels. In both of these organs, changes in the distribution of enzyme activity among the several forms was slight even when approximately 20- to 40-fold changes in enzyme specific activity were achieved by fasting-refeeding experiments (for liver) or during pregnancy and lactation (for mammary gland), it was concluded that the induction of glucose-6-phosphate dehydrogenase in these two organs occurs without any major redistribution among the multiple molecular forms of this enzyme.
The nature of the coenzyme A – manganese(II) complex in solution as studied by proton magnetic resonance
Authors: Theriault Yvon , Kotowycz George
Published: December 2009

The longitudinal relaxation rates of the adenosine AH(8), AH(2), AH(1′), and the pantetheine PH(3′) protons of coenzyme A (CoA) were measured in solutions of free CoA (0.062 M) and for the CoA-Mn(II) complex. The experiments were carried out in D2O at slightly basic values of the pH at 100.0 MHz and at 298 K. By observing the paramagnetic effects of the weakly bound metal ion on the proton longitudinal rates, distances were calculated between the Mn(II) ion and these four protons. For the complex containing two water molecules, the four distances are 4.8 ± 0.5 Å (1 Å = 0.1 nm) to the AH(8) proton, 6.5 ± 0.6 Å to the AH(2) proton, 6.4 ± 0.6 Å to the AH(1′) proton, and 6.0 ± 0.6 Å to the pantetheine PH(3′) proton. For three water molecules, the four distances are 4.5 ± 0.5, 6.1 ± 0.6, 5.9 ± 0.6, and 5.6 ± 0.6 Å, respectively. The correlation times have been determined previously, based on water proton longitudinal relaxation measurements, and these are 6.6 × 10−10 s for the first case and 4.0 × 10−10 s for the latter case. Experiments were also carried out on 0.010 M CoA solutions and, within experimental error, the distances remained unchanged.These distances, combined with the Mn(II) ion to the phosphorus nuclei distances, together with a knowledge of the conformation of the free CoA in solution are used to construct models for the conformation of the CoA–Mn(II) complex in solution. These results are compared with the available data for the Mn(II)–ATP complex, with the lanthanide ion complexes with dephospho CoA, and with the Co(II) ion complexes with propionyl CoA and the enzyme transcarboxylase.
Preparation of β-cells from fetal bovine pancreas: characterization of insulin biosynthetic activity
Authors: Rosenzweig Steven A. , Yip Cecil C.
Published: December 2009

Dispersed cell preparations enriched in β-cells were obtained by collagenase digestion of fetal bovine pancreas and separation by Ficoll gradient centrifugation. These cells actively incorporated [3H]leucine into proinsulin and insulin. Incubation of these cells in the presence of the arginine analogue, L-canavanine, resulted in the inhibition of conversion of newly formed proinsulin to insulin and the appearance of a radioactive component of molecular weight 11 000–12 000. Incorporation of [35S]methionine into this component was detected in the presence of canavanine, an event not observed in control incubation. Canavanine thus induced the formation of a component possessing molecular weight and compositional properties expected for preproinsulin. Further characterization of cellular products by polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate showed a highly labeled band corresponding to molecular weight 18 000–20 000 which might be involved in insulin biosynthesis.
Effect of nucleotides on UDP-N-acetylglucosamine pyrophosphatase and N-acetylglucosaminyltransferase activities in microsomal membranes
Authors: Mookerjea Sailen
Published: December 2009

Rat liver microsomes solubilized by incubating with lysolecithin or Triton X-100 showed very active UDP-N-acetylglucosamine pyrophosphatase activity leading to the hydrolysis of the substrate into N-acetylglucosamine-1-P and N-acetylglucosamine. ATP, GTP, CDPcholine, and CDPglucose exerted a considerable inhibitory effect on the solubilized membrane pyrophosphatase activity. CDPcholine and CDPglucose, in addition, appeared to stimulate the transfer of N-acetylglucosamine into endogenous and exogenous acceptor proteins. Evidence is also presented of an inhibitory effect of ATP (and to some extent GTP) on N-acetylglucosaminyltransferase activity. This inhibitory effect of ATP and GTP became clearly evident when the pyrophosphatase activity in the membranes was virtually eliminated in the presence of CDPcholine and CDPglucose. The effect of ATP and GTP on the solubilized membrane enzymes indicated that the inhibition of pyrophosphatase activity alone did not determine the rate of transfer of sugar to protein. The results also suggested that the UDP-N-acetylglucosamine pyrophosphatase and N-acetyiglucosaminyltransferase activities were controlled independently and the effect of each nucleotide on these enzymes should, therefore, be carefully evaluated to understand its role in glycopolymer biosynthesis. Also, a possible role of choline and its derivatives in glycoprotein synthesis is discussed.
Effects of dietary carbohydrate on cholesterol metabolism in rats and rabbits
Authors: Falconer A. D. , Carroll K. K.
Published: December 2009

Semipurified diets produce a marked hypercholesterolemia in rabbits and tend to elevate the level of plasma cholesterol in rats. They also decrease rates of oxidation of [26-14C]cholesterol to respiratory 14CO2 and excretion of label in fecal lipid, compared with commercial feed. In both species, the hypercholesterolemia was prevented and the rate of oxidation of [26-14C]-cholesterol increased by using unmodified potato starch as the carbohydrate component of the semipurified diets. Potato starch was poorly digested by rats but appeared to be well utilized by rabbits. A semipurified diet containing cooked potato starch gave results in rats comparable to those obtained with diets containing other types of carbohydrate. Cooked potato starch produced diarrhea in rabbits, thus complicating interpretation of the results. When the diarrhea was treated with antibiotics, the results were similar to those obtained with other carbohydrates. Rats fed commercial diet which had been heated in an oven or autoclaved had higher plasma levels than those fed untreated commercial diet but no significant differences in rates of oxidation or excretion of cholesterol were observed.
Binding of mitochondrial malate dehydrogenase to mitoplasts
Authors: Strasberg Paula M. , Webster Keith A. , Patel Hasmukh V. , Freeman Karl B.
Published: December 2009

The binding of 14C-labelled bovine and porcine malate dehydrogenase (EC 1.1.1.37) to rat liver mitochondria and mitoplasts was examined. The bovine enzyme was found to associate nonspecifically with isolated mitochondria and sonicated mitoplasts. Scatchard plot analysis suggested a specific binding to mitoplasts of the order of 5 pmol malate dehydrogenase per milligram of mitoplast protein. Porcine malate dehydrogenase dimer but not monomer exhibited a similar binding. The results are discussed in relation to the mechanism of uptake of the enzyme by mitochondria after synthesis on cytosolic ribosomes.
The amino acid sequences of the three heavy chain constant region domains of a human IgG2 myeloma protein
Authors: Connell George E. , Parr Dorothy M. , Hofmann Theo
Published: December 2009

The amino acid sequences of most of the CH1, CH2 and CH3 domains of IgG Zie, a myeloma protein belonging to the IgG2 subclass, are presented. These data make possible a comparison of the sequences of residues 253–446 of all four subclasses of immunoglobulins; these residues make up almost the entire Fc regions. A comparison can also be made of the CH1 domain of IgG1 Eu and the CH1 domain of IgG2 Zie. Earlier sequence analyses of the Fc regions of subclass 1 and 3 proteins, and parts of the Fc regions of subclass 2 and 4 proteins showed that about 95% of these sequences were identical. The extended comparisons made possible by the data presented here show that this very high degree of identity is maintained throughout the four subclasses. Similarly, the CH1 domains of γ1 and γ2 chains were found to have about 93% sequence identity. It is unlikely that the few single amino acid changes within the constant region domains can account for the marked differences between subclasses observed in the biological effector functions of immunoglobulin Fc regions, especially since most of the changes are highly conservative. Rather, it seems probable that these functional differences are caused by conformational differences between the subgroups, which result from sequence differences in the hinge regions.
Elucidation of the quaternary structure of reversibly immobilized alkaline phosphatase derivatives
Authors: McCracken Susan , Meighen Edward
Published: December 2009

Escherichia coli alkaline phosphatase has been reversibly immobilized on Sepharose CL-4B through two different methods, both based on a disulfide linkage, under conditions selected to favour the coupling of the enzyme to the solid support through one covalent linkage. The quaternary structure of the reversibly immobilized subunit, produced by dissociation of the matrix-bound dimer, was examined by cross-linking with the bifunctional reagent dimethyl suberimidate. Following release from the solid support, the protein was analysed by sodium dodecyl sulfate gel electrophoresis demonstrating the presence of a sufficient amount of dimeric structures in the immobilized subunit preparation to account for all the enzyme activity observed in this sample. These results suggest that the subunit of alkaline phosphatase may be catalytically inactive. This approach to studying the quaternary structure of immobilized subunit derivatives offers the opportunity to directly determine the homogeneity and structure of matrix-bound 'monomer' preparations and is particularly useful in determining if low levels of catalytic activity observed in some immobilized subunit populations are due to the presence of contaminating oligomeric structures.
Relationships among purine nucleoside metabolism, adenosine triphosphate catabolism, and glycolysis in human erythrocytes
Authors: Henderson J. Frank , Zombor George , Burridge Phillip W. , Barankiewicz George , Smith Camilla M.
Published: December 2009

In human erythrocytes incubated with both naturally occurring purine nucleosides and with a variety of purine nucleoside analogs, ATP catabolism was accelerated and lactate accumulation was increased. Tubercidin was a particularly potent inducer of ATP catabolism. In cells incubated with tubercidin, the major route of adenylate metabolism was deamination, whereas in cells incubated with deoxyglucose, the major pathway was dephosphorylation.
Myoinositol biosynthesis by Sertoli cells, and levels of myoinositol biosynthetic enzymes in testis and epididymis
Authors: Robinson Ranga , Fritz Irving B.
Published: December 2009

Levels of glucose-6-phosphate cyclase (myoinositol-1-phosphate synthase, EC 5.5.1.4) and myoinositol-1-phosphate phosphatase (myoinositol-1-phosphatase, EC 3.1.3.25) were determined in extracts of testes from 10-, 20-, and 30-day-old rats, and in extracts of Sertoli cells, germinal cells, and epididymides. The specific activity of the cyclase was approximately [Formula: see text] that of the phosphatase in all extracts found to contain either enzyme. Among cells in the testis examined, Sertoli cells had highest levels of enzymes required for inositol biosynthesis from glucose, while spermatocytes and round spermatids did not have detectable activity. Spermatozoa from the epididymis also had no detectable cyclase or phosphatase activity. In contrast, extracts of washed epididymides contained exceedingly high specific activities of these enzymes. Primary cultures of Sertoli cells, maintained in a chemically defined medium without added inositol, released inositol into the medium during three successive 24-h periods. The amounts released were greater in cells stimulated by dibutyryl cyclic AMP. Results were interpreted to indicate that inositol in the fluid of seminiferous tubules most probably originates from Sertoli cells, which synthesize inositol from glucose. Additional inositol in the fluid of epididymal tubules could readily be provided by metabolism of glucose by epididymal epithelial cells.
Inhibition of rat and human adipocyte adenylate cyclase in the antilipolytic action of insulin, clofibrate, and nicotinic acid
Authors: D'Costa Mario A. , Asico Winifreda , Angel Aubie
Published: December 2009

Clofibrate (Atromid-S®), nicotinic acid, and insulin are known to be potent hypolipidemic and antilipolytic agents. The present study was undertaken to define the mechanism of action of this latter effect on isolated rat and human fat cells. Sodium clofibrate (0.42 mM), nicotinic acid (0.42 mM), and insulin (100 μU/mL) were shown to inhibit norepinephrine-stimulated lipolysis in rat and human adipose cells and this inhibition was associated with a reduction in intracellular 3′,5′-cyclic AMP levels. A similar cyclic AMP lowering effect was demonstrated with insulin in the presence of procaine–HCl, which uncouples the adenylate cyclase system from lipolysis. This insulin effect was attributed to inhibition of adenylate cyclase. A direct and significant inhibition of adenylate cyclase in membrane fractions obtained from isolated human adipocytes was demonstrated for all three antilipolytic agents. The common membrane site of action of these agents whereby adenylate cyclase activity is depressed, thus decreasing cyclic AMP production and free fatty acid (FFA) mobilization from adipose stores, implies a central role for the adenylate cyclase system. These findings are consistent with the view that the hypotriglyceridemic effects of clofibrate, nicotinic acid, and insulin may be partly explained by deprivation of FFA substrate for hepatic very low density lipoprotein synthesis.
A kinetic study of hydrophobic interactions at the S1 and S2 sites of papain
Authors: Brubacher Lewis J. , Zaher M. R.
Published: December 2009

Two series of peptide substrates of papain, N-benzyloxycarbonyl-Y-X-OCH3 and N-benzyloxycarbonyl-L-alanyl-L-phenylalanyl-X-OCH3 in which X and Y represent L-alanine, L-leucine, and L-phenylalanine, were synthesized. All hydrolyze at the ester linkage in the presence of papain. The kinetic parameters kcat and Km were determined and interpreted in terms of binding interactions at the S1 and S2 sites of the enzyme. Significant kinetic specificity is seen in k3 (= kcat) as well as in kcat/Km. At the S1 site, deacylation reflects a preference for L-alanine by factors of 4- to 9-fold over L-leucine and 10-fold over L-phenylalanine provided the substrate has L-phenylalanine in the P2 position. There is no significant specificity at the S1 site reflected in the parameter kcat/Km. At the S2 site the well-known preference for L-phenylalanine is always more pronounced in the parameter kcat/Km than in k3. Indeed, deacylation is sometimes slower when L-phenylalanine rather than L-alanine or L-leucine interacts at the S2 site. A possible correlation between the effects of amino acid replacements at the S1 site on deacylation rates for substrates and inhibition constants for peptide inhibitors is discussed. Five dipeptide amide substrates were synthesized and their kinetic parameters were determined at pH 8 and pH 9. All hydrolyze at the amide linkage except N-benzyloxycarbonyl-L-alanyl-L-phenylalanine amide which hydrolyzes at the peptide linkage (55%) and the amide linkage (45%). Each of three amide substrates has nearly identical acylation and deacylation rates which may be a sign that the active site of papain has attained evolutionary perfection. The results are discussed in terms of the three-dimensional structure of papain.
Synthesis of peptides by the solid-phase method. IV. Des-Arg9-bradykinin and analogs
Authors: St-Pierre S. , Gaudreau P. , Drouin J. N. , Regoli D. , Lemaire S.
Published: December 2009

We have synthesized a series of 19 analogs of the octapeptide fragment of bradykinin (BK), des-Arg9-bradykinin, in order to perform a structure–activity study of this peptide on the newly discovered B1 receptor of bradykinin. The first time, each residue of the octapeptide was replaced by L-alanine to pinpoint biologically important residues. Thereafter, both phenylalanine residues in positions 5 and 8 were substituted by L-tyrosine methyl ether, L-cyclohexylalanine, D-phenylalanine, and L-leucine. This paper describes the synthesis of the analogs by the solid phase method. A Beckman peptide synthesizer was used to assemble the peptides on the resin support. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by ion-exchange chromatography on carboxymethylcellulose and by gel filtration on Bio-Gel P2 resin. The purity of the octapeptides was then checked by tlc, paper electrophoresis, amino acid analysis, and elemental analysis.The new peptides were tested on the rabbit aorta in order to evaluate their kinin-like activities and to see if they act as antagonists. The results of the biological assays are discussed in terms of structure–activity relationships.
Temperature-dependent interactions between poly-L-lysine and phosphatidylcholine vesicles
Authors: Campbell Ian M. , Pawagi Asha B.
Published: December 2009

Studies on the effect of temperature on the optical density of suspensions of dipalmitoyl phosphatidylcholine vesicles, in the presence of polylysine, indicate that the polypeptide can undergo two different kinds of interactions with the vesicles. At temperatures below the phase transition there seems to be a small surface–polar interaction, which maximizes at about 36 °C (i.e., about 5 °C below the phase transition). Upon transition of the lipid membranes to the liquid crystalline phase, there is a much larger rapid interaction, which seems to be hydrophobic. Whereas the first interaction is not temperature reversible, the second interaction rapidly reverses when the temperature is decreased below the phase transition of dipalmitoyl phosphatidylcholine. Incorporation of as little as 5% unsaturated lecithin, either dioleoyl or dilinoleoyl, into the dipalmitoyl vesicles, stabilizes the liquid phase interaction, so that it does not readily reverse when the temperature is decreased below that of the phase transition. Whereas dioleoyl only partially and temporarily interferes with the temperature reversal, dilinoleoyl almost completely stabilizes the interaction. Associated studies using dilatometry and electron microscopy show that the liquid phase interaction between polylysine and phosphatidylcholine vesicles results in the formation of a complex which is of high molecular and electron density.
Characterization of an inducible porter required for L-proline catabolism by Escherichia coli K12
Authors: Wood Janet M. , Zadworny David
Published: December 2009

L-Proline can serve as sole source of carbon and nitrogen for the growth of Escherichia coli K12 and other Enterobacteria. L-Proline uptake and L-proline oxidase are suoject both to catabolite repression and to specific induction by L-proline or glycyl-L-proline, although their regulation is not strictly coordinate. A strain defective for L-proline uptake due to a lesion at the locus putP does not show elevated uptake activity either on relief of catabolite repression or when grown on glycyl-L-proline as nitrogen source. The apparent Km for L-proline uptake decreases up to 14-fold as uptake Vm increases when cells are induced for both L-proline uptake and L-proline oxidase; cells with increased uptake activity, alone, do not show an altered Km. Although L-proline is metabolized during the uptake measurements, uptake is always active. The observed variations in uptake Km are unlikely to result from the escape of radioactive L-proline metabolites or from reversal of the transport reaction during the uptake measurements. We conclude that the L-proline porter encoded in putP is responsible for 80 to 90% of the constitutive and for the inducible L-proline uptake activity of wild-type bacteria. Although this porter is amplified in cells induced for L-proline catabolism, the observed values for uptake Vm may not be taken as a direct indicator of porter concentration.
Enzymatic degradation and partial biosynthetic reconstitution of microsomal and mitochondrial membranes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

Guinea pig liver microsomal and mitochondrial membranes were degraded with phospholipase C and D followed by partial biosynthetic reconstitution. Activities of phosphatidylinositol synthetase in microsomal membranes and NADPH – cytochrome c reductase were almost completely lost after phospholipase C and D treatment; almost complete restoration of the original activity was achieved after biosynthesis of phosphatidylcholine in degraded microsomes, but was not reparable after biosynthesis of cytidinediphosphodiglycerides (CDP-diglycerides). The mitochondrial biosynthesis of polyglycerophosphatides was completely retained after degradation of these membranes with phospholipase C, but after similar treatment with phospholipase D, only about one-quarter of the original activity remained, the relative composition of polyglycerophosphatides being significantly different. The activity of NADPH – cytochrome c reductase of microsomes represented about 76% of the originalactivity after phospholipase C treatment, but only ~1% after treatment with phospholipase D. Although this activity could not be restored with CDP-diglyceride synthesis, it was restored to about 75% of the original activity after the biosynthesis of phosphatidylcholine in these fragments. These and additional experimental findings are discussed in terms of the relation between structural organization of lipids and proteins and enzymatic activities of membrane-bound phospholipid-synthesizing enzymes in microsomal and mitochondrial membranes isolated from guinea pig liver.
Additional properties of a soluble polymer of glucose from cultures of Acetobacter xylinum
Authors: Colvin J. Ross , Sowden L. C. , Daoust V. , Perry Malcolm
Published: December 2009

The results of differential, thermal analysis of a soluble, β(1 → 2)-branched, β(1 → 4)-D-glucan isolated from cultures of Acetobacter xylinum are consistent with previous conclusions about its structure. The O-acetyl content of the polymer is 8.3% which corresponds to a maximum substitution of one acetyl group per three glucose residues. Proton nuclear magnetic resonance spectra confirm that all the glycosidic bonds are β linkages. Some preparations of the polymer are contaminated by another polymer containing mannose and rhamnose. No evidence was obtained to support a previous suggestion that the branched D-glucan is a precursor of bacterial cellulose and this suggestion is now withdrawn.
Kinetics of rat liver GM2 ganglioside-β-D-N-acetylhexosaminidase
Authors: Novak Anton , Lowden J. Alexander
Published: December 2009

The kinetics of β-D-N-acetylhexosaminidase against GM2 ganglioside were examined. We used a crude preparation of rat liver as the enzyme source because purification of β-D-N-acetylhexosaminidase results in a decrease in specific activity against GM2 ganglioside. Kinetic plots were not linear but showed a break. At substrate concentrations less than 50 μM the Vmax was 6 pmol GM2 hydrolyzed per hour per micromole 4-MU-GlcNAc hydrolyzed per hour (pmol GM2/μmol 4-MU-GlcNAc) and the Km was 5 μM. At substrate concentrations greater than 50 μM, the Vmax was 7 pmol GM2/μmol 4-MU-GlcNAc and the Km was 14 μM. The critical micelle concentration of GM2 ganglioside was 20–25 μM as determined by spectral shifts of the dye pinacyanol chloride in association with GM2, and 10–15 μM from electrical conductivity measurements which also showed the end of the monomer–micelle transition to occur at 40–50 μM GM2. The increasing excess of micellar substrate at greater than 50 μM GM2 explains the discontinuity in the kinetic plots. Sodium taurocholate had a critical micelle concentration of 9–11 mM using pinacyanol chloride and 2.5–3 mM using electrical conductivity. When included in the assay mixture at a concentration of 10 mM, sodium taurocholate produced a linear kinetic plot. This is probably due to the formation of mixed micelles of detergent and GM2 ganglioside. The Vmax was 200 pmol GM2/μmol 4-MU-GlcNAc and the Km was 93 μM. The data suggest that ganglioside hydrolysis occurs more readily when the substrate is incorporated into a membrane-like environment.
Cross-linking of cytochrome oxidase subunits with difluorodinitrobenzene
Authors: Kornblatt Jack A. , Lake Diane F.
Published: December 2009

Cytochrome c oxidase was treated with 1,5-difluoro-2,4-dinitrobenzene at molar ratios (DFDNB:oxidase) varying from 5 to 625. At the lowest ratio, there was virtually no effect of the probe on oxidase activity or on migration of oxidase subunits on sodium dodecyl sulfate–polyacrylamide disc gel electrophoresis. At ratios of 25 and greater, there was loss of oxidase activity and a change of the pattern of subunit migration on sodium dodecyl sulfate electrophoresis. (i) Activity loss was probably a result of severely perturbing the cytochrome c binding site since oxidase activity with a low molecular weight reductant (N,N,N′,N′-tetramethyl-phenylenediamine) was unaltered. Also unaltered were the oxidized, reduced, and carbon monoxide binding spectra of the treated oxidase. (ii) The staining pattern on sodium dodecyl sulfate electrophoresis showed that subunits III and VI disappeared from their normal positions on the gel. A new band of higher molecular weight accompanied their loss from the gel indicating that the two subunits were being cross-linked. Subunits III and VI are thus shown to have two reactive groups within 4.8 Å (1 Å = 0.1 nm) of one another. This proximity has not been detected with other probes that react with the same groups.