bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Enzymatic Properties of Avian Liver and Skeletal Muscle D-Fructose-1,6-bisphosphate 1-Phosphohydrolase
Authors: Marquardt R. R. , Olson J. P.
Published: December 2009

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme.Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 × 10−9 and 1 × 10−6 M, respectively.EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 × 10−4 M) and muscle (Ki, 1 × 10−5 M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.
Further Studies of the Kinetics of Oxygenation of Arachidonic Acid by Soybean Lipoxygenase
Authors: Cook H. W. , Lands W. E. M.
Published: December 2009

A kinetic model for soybean lipoxygenase (EC 1.13.11.12) has been examined by comparing results from extensive experimental data with theoretical data generated from a computer program. Kinetic constants have been established by closely fitting experimental and computer-generated data with both product formation versus time, and the more complex accelerative and decelerative relationships of velocity changes with time.It has been confirmed that activation of lipoxygenase by its hydroperoxide product is necessary for activity, and product removal gives inhibition in a manner quantitatively predicted by the model. The earliest accurate measurement of velocity (at 9 s) is a convenient index of the amount of product–activator present in reaction mixtures, and can be used to assay quantitatively the amount of product–activator.The results confirm that soybean lipoxygenase catalyzes a product-activated, substrate-inhibited oxygenation accompanied by a self-catalyzed destruction of its activity.
Metabolism of Estrogens and Their Sulfates by Mouse Kidney Tissue in Vitro
Authors: Hobkirk R. , Nilsen Mona
Published: December 2009

Whole kidney tissue from mouse was incubated with labelled estrone 3-sulfate (E13S), 17β-estradiol 3-sulfate (17βE23S), estrone (E1), and 17β-estradiol (17βE2). Considerable reduction of E13S and E1 occurred. E13S gave rise primarily to 17α-estradiol 3-sulfate (17αE23S) together with lesser amounts of 17βE23S. By incubating [3H-35S]E13S with the tissue it was confirmed that formation of the diol sulfates was direct, without accompanying hydrolysis and reconjugation. Conversion of E1 was mainly to 17βE2 with, on the average, lesser amounts of 17αE2. A small degree of direct conversion of 17βE23S to E13S was found. 17β-Estradiol was converted to a limited extent to E1 and to much smaller amounts of 17αE2.
Assembly of Complex III into Newly Developing Mitochondrial Membranes
Authors: Aithal H. N. , Tustanoff E. Reno
Published: December 2009

Yeast cells grown anaerobically in 0.02% linoleic acid were transferred to air in the presence of 0.02% elaidic acid. At varying times Arrhenius plots were made of QH2-cytochrome c reductase activities in isolated mitochondria. A transition temperature of 8.2 °C at 0.5 h was characteristic of linoleate; at 3 h the transition temperature was increased to 24 °C characteristic of elaidate. At early times the enzyme was associated with anaerobic promitochondrial membranes; at later stages the newly synthesized enzyme was associated with newly developed elaidate membranes.
A High Molecular Weight Dihydro-orotate Dehydrogenase of Neurosporu crassa. Purification and Properties of the Enzyme
Authors: Miller R. W.
Published: December 2009

Some of the unusual molecular and catalytic properties of a high molecular weight dihydro-orotate dehydrogenase (DHOD) from Neurospora crassa have been determined. Comparison of the properties of this enzyme with the properties of the soluble biosynthetic enzyme of prokaryotes has revealed several important differences. The fungal enzyme is located in a mitochondrial membrane in a position consistent with linkage with the respiratory chain through ubiquinone (Miller, R. W.: Arch. Biochem. Biophys. 146, 256–270 (1971)). Release of the enzyme from the membrane results in a solubilized protein complex containing bound lipids and inactive hydrophobic proteins. Non-specific protein aggregation is minimized during purification by Triton-X-100 and phospholipase treatments. The catalytically active enzyme has an apparent molecular weight of 210 000.In contrast to soluble DHOD preparations the high molecular weight enzyme has no endogenous dihydro-orotate oxidase (EC 1.3.3.1) activity and is relatively insensitive to inactivation by sulfhydryl-reactive reagents in the presence of dihydro-orotate (DHO). The enzyme activity is highly sensitive to conditions causing oxidation of flavin mononucleotide (FMN). The activity cannot be restored by cysteine or other means. FMN is present in all purified preparations in a bound, non-fluorescent (reduced) form until dihydro-orotic acid is removed or oxidized. Catalytic efficiency of the purified enzyme was 12 000 mol DHO oxidized per minute per mole FMN. This high turnover rate is due in part to the small flavin content of the purified enzyme, equivalent to 1 mol FMN per 120 000 g of catalytically active protein. Iron was detected in the purified enzyme by atomic absorption spectroscopy but labile sulfide was absent. Thenoyltrifluoroacetone, an iron chelator, only partially inhibited DHO oxidation regardless of electron acceptor.Fatty acids interact with a hydrophobic site of the enzyme in non-competitive fashion but under certain conditions appear to significantly alter the Km for ubiquinone. Orotate, by comparison, is a purely competitive inhibitor. Both types of inhibitor may function to regulate the biosynthesis of orotate in vivo.Superoxide anion is not produced in significant quantities by the DHO-reduced enzyme unless both ubiquinone and a suitable single electron carrier such as phenazine metho-sulfate are present. DHOD has been proposed as a source of superoxide anion in mammalian mitochondria (Forman, H. J. &Kennedy, J. A.: J. Biol. Chem. 250, 4322–4326(1975)).
3′:5′-Cyclic-AMP Phosphodiesterase Activities in White and Brown Adipose Tissues of Cold-Acclimated Rats
Authors: Bertin R. , Portet R.
Published: December 2009

3′:5′-Cyclic-AMP phosphodiesterase (PDE) (EC 3.1.4.17) activity was measured in interscapular brown adipose tissue (BAT) and in white epididymal adipose tissue of rats acclimated to constant or fluctuating cold. Experiments were carried out on isolated adipocytes or tissue homogenates. In brown or white adipose tissue or isolated adipocyte homogenates, two different apparent Km values were found according to the substrate (cAMP) concentration. The low Km was at about 10−6 M and the high one at about 10−4 M. The apparent V of the high Km enzyme was about 10-fold higher than the V of the low Km enzyme. Cold acclimation to constant or fluctuating cold did not modify appreciably the Km or V values. For low substrate concentrations (10−6–10−8 M), the specific activity of PDE expressed per milligram of protein was decreased in BAT adipocytes of the two groups of cold-acclimated rats, compared to controls. Inversely, it was increased in total tissue homogenates. These variations were smaller in fluctuating cold than in constant cold-acclimated rats. They could, in part, induce the increases in lipolysis and in blood flow observed in the BAT of cold-acclimated rats.
The Conservation of Amino Acids in the N-Terminal Position of Ribosomal and Cytosol Proteins from Escherichia coli, Bacillus stearothermophilus, and Halobacterium cutirubrum
Authors: Matheson Alastair T. , Yaguchi Makoto , Visentin Louis P.
Published: December 2009

Alanine, methionine, and serine are the predominant N-terminal residues in the cytosol and ribosomal protein fractions from the thermophile Bacillus stearothermophilus and the extreme halophile Halobacterium cutirubrum, a similar situation to that previously found in Escherichia coli. In all three bacteria the N-terminal residues of the 30S ribosomal proteins are mainly alanine [Formula: see text] methionine > serine whereas in the 50S ribosomal proteins from E. coli and B. stearothermophilus the predominant residues are methionine > alanine > serine suggesting conservation of specific N-terminal residues in these ribosomal proteins. However, the 50S ribosomal proteins from H. cutirubrum showed serine as the major N-terminal residue.
Structural determination of the polysaccharide antigens of Neisseria meningitidis serogroups Y, W-135, and BO
Authors: Bhattacharjee Apurba K. , Jennings Harold J. , Kenny C. Paul , Martin Adèle , Smith Ian C. P.
Published: December 2009

The purified high molecular weight serogoup Y meningococcal polysaccharide contains equimolar proportions of D-glucose and N-acetylneuraminic acid and is partially O-acetylated. Carbon-13 nuclear magnetic resonance (NMR) studies, together with other chemical data, have indicated that the polysaccharide is linked only at C-6 of the D-glucose and C-4 of the sialic acid residues, all the linkages being in the α-configuration. The 13C NMR data also indicated that the Y polysaccharide is composed of an alternating sequence of these two different residues, and this was confirmed by its autohydrolysis where the major product was 4-O-α-D-glucopyranosyl-β-D-N-acetylneuraminic acid. The W-135 polysaccharide differs from that of Y only in the absence of O-acetylation and in the configuration of one hydroxyl group of the disaccharide repeating unit. In this case autohydrolysis yielded 4-O-α-D-galactopyranosyl-β-D-N-acetylneuraminic acid as the major product. Structural evidence indicates that the BO and Y polysaccharides are identical.Methanolysis of the Y polysaccharide yielded in addition to the methyl glycosides of glucose and sialic acid, a 9-O-acetyl derivative of the latter. This derivative was formed during the re-N-acetylation process and its formation was mainly due to the presence of sodium ions in the original polysaccharide.
The carbohydrate composition of human apo low density lipoprotein from normal and type II hyperlipoproteinemic subjects
Authors: Lee P. , Breckenridge W. Carl
Published: December 2009

The carbohydrate composition of the apoprotein from low density lipoprotein (LDL) of normal (average LDL cholesterol, 122 mg/100 ml) and type II hyperlipoproteinemic (average LDL cholesterol, 236 mg/100 ml) males was studied using gas–liquid chromatographic analysis of the methyl glycoside derivatives. All samples containing detectable sinking pre-beta-lipoprotein were excluded from the study.The apo LDL from both groups of subjects contained mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid. Glucose and fucose were not found while trace quantities of galactosamine were detected. Although the quantities of galactose and N-acetylglucosamine were the same in the two groups, lower quantities of mannose [Formula: see text] and N-acetylneuraminic acid [Formula: see text] were found in the type II patients as opposed to normal subjects.
Androgen and estrogen binding in rat skeletal and perineal muscles
Authors: Dubé Jean Y. , Lesage Renée , Tremblay Roland R.
Published: December 2009

Specific in vitro binding of [3H]testosterone (T), 5α-[3H]dihydrotestosterone (DHT), and [3H]estradiol (E2) was demonstrated in the 30 000 × g supernatant (cytosol) of thigh muscles (TM) and of the levator ani – bulbocavernosus muscle complex (LA–BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity (Ka = 1.1 × 109 M−1 and 2.3 × 109 M−1, respectively) whereas DHT binding is of lower affinity (Ka = 5.0 × 107 M−1).] In LA–BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 × 109 M−1, 0.3 × 109 M−1 and 0.5 × 109 M−1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA–BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.
Amino acid composition of sperm histones in the house cricket Acheta domesticus
Authors: Pallotta Dominick , Tessier Anne
Published: December 2009

Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide–urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L. L.: Anal. Biochem. 44, 81–88 (1971)). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2a1. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine–arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is an arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.
Reagentless determination of L-asparagine and L-arginine via the combined use of immobilized enzymes and an ion-selective electrode
Authors: Ngo That Tjien
Published: December 2009

New methods for the determination of L-asparagine and arginine are described. Solutions containing L-asparagine were pumped through an asparaginase tube, which catalyzed the hydrolysis of L-asparagine to L-aspartic acid and ammonium ion. For L-arginine determination, solutions containing L-arginine were pumped through an arginase–urease tube. This dual enzyme tube catalyzed the conversion of L-arginine to L-ornithine, carbon dioxide, and ammonium ion. The ammonium ion concentrations in the effluent of the enzyme tubes were determined quantitatively by an ammonium-ion-selective electrode. The potentiometric response of the electrode was directly proportional to the logarithm of the concentration of L-asparagine and L-arginine in the range of 0.1–50 mM. An equation relating the electrode response and the substrate concentration is derived.
Physodes and the phenolic compounds of brown algae. Isolation and characterization of phloroglucinol polymers from Fucus vesiculosus (L.)
Authors: Ragan Mark A. , Craigie J. S.
Published: December 2009

A series of vanillin-reactive compounds has been isolated from extracts of the brown alga Fucus vesiculosus. The first three members of this series have been examined by mass spectrometry, nuclear magnetic resonance spectrometry, and chemical methods, and were shown to be phloroglucinol, its phenyl-linked dimer, and a trimer also composed of phloroglucinol units. Evidence is presented for the presence of tetrameric, pentameric, and hexameric phloroglucinol derivatives. Polymeric vanillin-reactive compounds were isolated and separated into two fractions. Oxidative hydrolysis of the major one produced phloroglucinol and related oligomers in low yields. The classical vanillin reactivity of physodes may thus be attributed to phloroglucinol-containing derivatives, as originally proposed by Crato (Crato, E.: Ber. Dtsch. Bot. Ges. 10, 295–302 (1892)).
Studies related to antitumor antibiotics. Part V. Reactions of mitomycin C with DNA examined by ethidium fluorescence assay
Authors: Lown J. Wlliam , Begleiter Asher , Johnson Douglas , Morgan A. Richard
Published: December 2009

The cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, three aspects of mitomycin C action on DNA have been studied: (a) cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. Cross-linking of DNA is determined by the return of fluorescence after a heat denaturation step at alkaline pH's. Under these conditions denatured DNA gives no fluorescence. The cross-linking was independently confirmed by St-endonuclease (EC 3.1.4.–) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was shown not to be due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship exists between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation is due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggests that the cross-linking event, to which the primary cytotoxic action has been linked, occurs sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, was shown to induce single strand cleavage as well as monoalkylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhibition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggests that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C is largely due to the free radical [Formula: see text]. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C does not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C is able to cross-link DNA in the absence of reduction at pH 4. This is consistent with the postulated cross-linking mechanisms.
Biosynthesis of diacylglycerols by rat intestinal mucosa in vivo
Authors: Breckenridge W. C. , Yeung S. K. F. , Kuksis A. , Myher J. J. , Chan M.
Published: December 2009

The biosynthesis of diacylglycerols was studied in rat intestinal mucosa during in vivo absorption of a low molecular weight fraction of butter oil and of the corresponding medium and long chain fatty acids. The experimental fat solutions were given by stomach tube to the animals after a 24-h fast and mucosal scrapings were collected 3 h later. The lipids were isolated and the acylglycerols determined by combined thin-layer chromatography gas–liquid chromatography techniques and stereospecific analyses. Free fatty acid feeding led mainly to, sn-1,2-diacylglycerols, which contained exogenous and endogenous fatty acids. During triacylglycerol feeding, both.sn-1,2- and sn-2,3-diacylglycerols were recovered in significant amounts from the intestinal mucosa. The composition of the sn-2,3-diacylglycerols corresponded to that with exogenous fatty acids but the sn-1,2-diacylglycerols clearly contained both exogenous and endogenous fatty acids. In all cases it was possible to isolate endogenous sn-1,2-diacylglycerols made up largely of species with linoleic and arachidonic acids in the 2 position and palmitic and stearic acids in the 1 position, which apparently were not converted to triacylglycerols. The in vivo reacylation of 2-monoacylglycerols via both sn-1,2- and sn-2,3-diacylglycerols is in agreement with similar findings in vitro with everted sacs of rat intestinal mucosa.
Biosynthesis of triacylglycerols by rat intestinal mucosa in vivo
Authors: Breckenridge W. C. , Yeung S. K. F. , Kuksis A.
Published: December 2009

The structure of mucosal triacylglycerols was studied in rat intestinal mucosa in vivo during the absorption of a low molecular weight fraction of butter oil and of the corresponding free fatty acids of medium and long chain length. The mucosal lipids were isolated by solvent extraction and the acylglycerol structures were determined by combined AgNO3 – thin-layer chromatography and gas–liquid chromatography techniques and stereospecific analysis. Evidence was obtained for a rapid biosynthesis of triacylglycerols from diacylglycerols arising from the operation of both the monoacylglycerol and the phosphatidic acid biosynthetic pathways. Both sn-1,2- and sn-2,3-diacylglycerols appeared to be converted to triacylglycerols at significant rates, but a preferential utilization of sn-1,2-diacylglycerols could not be excluded. Endogenous dilution varied from a minimum of 5% during triacylglycerol biosynthesis from monoacylglycerols to 15% during their synthesis from free fatty acids, and was characterized by a preferential placement of the endogenous acids in the sn-3 and 2 positions of the triacylglycerol molecules. Exogenous myristic acid was preferentially associated with the sn-3 position, and stearic acid became preferentially bound to the sn-1 position. The complexity of the triacylglycerol end products prevented an exact estimate of the contribution of the phosphatidic acid pathway, but the acylglycerol structures were compatible with a minimum of 20% of total triacylglycerol yield at all times.
Mitochondria with impaired phosphate transport: 32P uptake studies
Authors: Williams G. R. , Orr J. L.
Published: December 2009

Rat liver mitochondria which have been exposed to 0.15 M NaCl at 35 °C for 15 min subsequently take up 32Pi from an external medium only to about 5% of the extent of uptake by control mitochondria. The volume into which 32Pi distributes in a pellet of such 'aged' mitochondria is less than that available to 3H2O but is greater than that available to [3H]sucrose. Mitochondria treated in this manner cannot therefore accumulate Pi although limited penetration of the inner membrane can occur. These results confirm earlier findings by indirect methods (Williams, G. R. &Orr, J. L.: Dynamics of energy-transducing membranes (Ernster, L., Estabrook, R. W. &Slater, E. C, eds), Elsevier Scientific Publishing Company, Amsterdam, Netherlands, pp. 497–508 (1974)).
Ribosomal RNA: the message or matrix for ribosomal proteins
Authors: Miller D. R. , Matheson A. T. , Visentin L. P.
Published: December 2009

The known nucleotide sequence of Escherichia coli 16S ribosomal RNA has been converted to amino acid sequences in all possible ways, and compared to known ribosomal protein sequences. The degree of similarity is precisely what one would expect by chance alone, providing additional evidence that ribosomal proteins cannot be coded for by ribosomal RNA.
The 'adenosine cycle' is not a significant route of purine metabolism in mammalian cells
Authors: Brox Larry W. , Henderson J. Frank
Published: December 2009

It has been proposed that the so-called 'adenosine cycle' of purine metabolism is quantitatively important in mammalian cells. We show here that this process is in fact a very minor route of purine metabolism in a number of mammalian cells.
Perturbation of lecithin bilayer structure by globoside
Authors: Tinker D. O. , Pinteric L. , Hsia J. C. , Rand R. P.
Published: December 2009

The ultrastructure of aggregates formed by mixtures of pig erythrocyte lecithin, cholesterol and globoside in aqueous systems was studied by electron microscopy and X-ray diffraction. Globoside and lecithin in up to equimolar amounts formed a lamellar mesophase, although the structure of the lamellae was perturbed. Mixtures containing excess globoside formed complex tubular or reticular aggregates. Cholesterol appeared to promote mixing of lecithin and globoside. The flexibility gradient of the hydrocarbon (hc) region of the lipid bilayers was studied using electron spin resonance (esr) spectroscopy of various nitroxide-labelled stearic acid probes. Globoside in equimolar amounts greatly perturbed the order parameters of lecithin bilayers, reducing the fluidity of the hc region and flattening the flexibility gradient near the polar (p) surface. The effect of globoside on lecithin–cholesterol bilayers was not so pronounced, since the latter was already more ordered than lecithin bilayers. A phase transition of pure globoside at 55 °C, involving 'melting' of the hc chains was also detected using X-ray and esr spectroscopic techniques. The interbilayer spacing, dw, of equimolar lecithin–globoside lamellar phase increased by 42% from that of lecithin bilayers, indicating that the glycolipid p group may increase the net repulsive force between bilayers, as was previously predicted theoretically.
Low-affinity interaction of ethanol with the fluorescent complex between horse liver alcohol dehydrogenase and auramine O
Authors: Bronskill Patricia M. , Wong J. Tze-Fei
Published: December 2009

Quenching of the fluorescence of the complex between horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase (EC 1.1.1.1)) and auramine O complex is inconsistent with a simple competitive displacement of auramine O by ethanol. Instead, the action of ethanol requires an explanation in terms of a solvent effect, or the formation of an enzyme – auramine O – ethanol ternary complex. The latter complex would have to be the low-affinity variety similar to the enzyme–NADH–ethanol ternary complex encountered in the kinetic system.
Spin labels as probes for tetraphenylboron ion interaction with liposomes
Authors: Seidah N. G. , Roy G. , Laprade R. , Cyr T. , Dugas H.
Published: December 2009

The effects of tetraphenylboron (TFB) on the molecular organization of lipids within phosphatidylcholine (PC) liposomes were investigated using the spin-labeling method. Perturbations at the surface of the lipid were probed using stearamide and cholestane spin labels; perturbations in the hydrophobic portion were probed with spin-labeled amphiphilic fatty esters.The spin-labeling results showed that in the concentration range 1.5 mM to 10 mM of the lipid-soluble anion TFB, detectable perturbations affecting both surface and interior hydrophobic portions of the lipids were observed. For all the spin labels used, the motional correlation time (τc) is independent of TFB concentration for [TFB] [Formula: see text] and the Arrhenius plots (In τcvs. 1/temperature) are parallel, with a slope independent of TFB concentration, but show a decrease of motional freedom of the labels as TFB is added (i.e., concentrations below that which causes saturation). The effect of pH on surface perturbations is small but observable and indicates that the surface of the lipid is more positively charged for [Formula: see text]. All the results suggest that TFB adsorbs on the surface portion of the lipids and causes the largest perturbation there. Furthermore, the sites occupied by TFB and those occupied by the labels are fairly remote from each other and saturation of the TFB sites occurs before the local environment of the spin labels is affected.Conductance measurements on black lipid membranes were done in parallel, and the results showed that conductance saturation occurs at [TFB] [Formula: see text] for PC membranes and at [TFB] [Formula: see text] in the presence of equimolar ratio of PC and cholesterol. These measurements also showed the presence of a surface charge. These experiments have shown that for the case of TFB, conductance and spin-labeling results agree quite well.
Acid phosphatases: androgen dependent markers of rat prostate
Authors: Tenniswood M. , Bird C. E. , Clark A. F.
Published: December 2009

Our investigations on acid phosphatase (AP) were aimed at finding a biochemical assay marker for androgen actions in the rat prostate. We quantitatively examined the effects of l-tartrate or formaldehyde on AP activity in tissue filtrates from nine adult male rat tissues, plasma and hemolysed red blood cells (HRBC). There was significant inhibition of AP activity in all instances with the exception of HRBC with tartrate. The prostate inhibition results were not different from those for seminal vesicles and adrenals but were different from the other tissues studied. Ten days following castration the inhibition by tartrate was less in all tissues studied except plasma and HRBC; the formaldehyde inhibition percentages were not altered. Intraperitoneal testosterone enanthate administration begun 2 days after castration maintained the tartrate inhibitions in the ranges found for noncastrated rats. Gel electrophoresis of the tissue filtrates and staining of the gels for AP indicated two bands of AP activity for the prostate from normal rats and one band of activity for all other tissues. This second band of prostate AP activity was completely eliminated by the addition of formaldehyde and was not found for prostate tissue filtrates from castrated animals. However, it was found for the animals which had received testosterone replacement for 14 days. It would appear that AP can be used as a marker of androgen responsiveness for rat prostate.
Biosynthesis of sphingomyelin in rat liver endoplasmic reticulum
Authors: Lyman E. M. , Knowles C. L. , Sribney M.
Published: December 2009

Rat liver microsomal sphingomyelin synthetase (CDPcholine: N-acylsphingosine cholinephosphotransferase (EC 2.7.8.3)) has been shown to be markedly stimulated by ATP and pantothenic acid derivatives such as CoA, pantethine, pantetheine and 4′-phosphopantetheine. Dephospho CoA, oxidized CoA and 4′,4″-diphosphopantethine likewise stimulate the enzyme in the presence of ATP, but to a much less significant extent, while pantothenic acid and pantothenyl alcohol were inactive. These pantothenic acid derivatives or ATP when added to the incubation system singly do not have any stimulatory activity. The stimulatory activity of ATP and CoA or ATP and pantethine was found to be approximately 20-fold. The activation of sphingomyelin synthetase is probably not through formation of an acyl-CoA or acyl thioester because palmityl-CoA or acetyl-CoA had very little stimulatory activity. Ceramides having the erythro configuration must have an acetyl group in N-acyl linkage; longer fatty acyl derivatives did not act as acceptors of the phosphorylcholine moiety of CDPcholine in the presence of ATP and CoA or ATP and pantethine. The incorporation of N-acetyl- and N-propionyl-threo-sphingosine compounds into sphingomyelin was also stimulated by ATP and the pantothenic acid derivatives mentioned above. The possible mechanism of action of ATP and CoA is discussed and other properties of the rat liver microsomal enzyme are also described.
Purine catabolism in man: characterization of placental microsomal 5′-nucleotidase
Authors: Fox Irving H. , Marchant Pamela J.
Published: December 2009

Human placental microsomal 5′-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of seven electrophoretic variants were isolated during the preparation of six placentas. Only three to six variants were found in a single placenta. The isoelectric pH's were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86 500 and 43 500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Michaelis constants for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence.There was a wide range of substrate specificity among nucleoside 5′-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 74; GMP, 68; IMP, 63; XMP, 28 and UDP–glucose, 68. The Michaelis constants for AMP, GMP and CMP ranged from 12–18 μM, from 33–67 μM and from 170–250 μM, respectively. Although 5′-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4–9.8.
Protein binding and subunit association activity in particles reconstituted from Escherichia coli MRE600 50S ribosomal components
Authors: Chu F. K. , Maeba P. Y.
Published: December 2009

Reconstituted 37S and 48S ribonucleoprotein particles were constructed by incubating Escherichia coli ribosomal RNA with total 50S ribosomal proteins by a sequential incubation method. By comparing the protein compositions of the two types of particles, the proteins that bind to 37S complexes to form 48S particles have been determined. Although only 48S particles could associate with 30S subunits, isolated 37S reconstituted particles could do so if incubated with exogenous 50S proteins. The proteins that bind under these conditions and confer upon particles the ability to associate are L2, L11, L15, L18 and L25. The involvement of these proteins in 5S RNA binding is discussed.
Synthesis and some pharmacological properties of [2-tryptophan]-oxytocin
Authors: Ferrier B. M. , Branda L. A.
Published: December 2009

Previous studies have suggested that an aromatic amino acid residue at position 2 in oxytocin facilitates the expression of the hormone's biological activities. [2-Tryptophan]-oxytocin, in which a residue of tryptophan has replaced that of tyrosine in oxytocin, has been synthesized by the method of azide coupling of the N-terminal dipeptide and C-terminal heptapeptide amide. It was found to have approximately 0.1% of the potency of oxytocin in milk ejection and uterotonic biological activities.
Ethanol metabolism by the rat heart and alcohol dehydrogenase activity
Authors: Forsyth G. W. , Nagasawa H. T. , Alexander C. S.
Published: December 2009

Rat hearts perfused with oxygenated buffer containing [1-14C]ethanol metabolized small amounts of the ethanol to carbon dioxide. Very sensitive techniques are required to separate the resulting 14CO2 from the ethanol. This metabolism is not inhibited by levels of pyrazole which markedly inhibit NAD dependent liver alcohol dehydrogenase (EC 1.1.1.1). In vitro studies suggest that NADP functions as a cofactor for the rat heart alcohol dehydrogenase activity of crude heart homogenates. The kinetic parameters, the specific activity, and the pH dependence of the enzyme activity measured in these experiments suggest that it may have a minor role in ethanol metabolism by the rat.
Lipid composition of developing Xenopus laevis embryos
Authors: Mes-Hartree Mary , Armstrong John B.
Published: December 2009

The total lipid content, amount of phospholipid, proportions of major polar and neutral lipid classes, and the overall fatty acid composition were examined in Xenopus laevis embryos. No obvious differences were observed in any of the parameters between fertilization and hatching, or between eggs produced by different females. The average lipid content per egg was 113 μg, 31.6 μg of which was phospholipid. The major phospholipids were phosphatidylcholine and sphingomyelin. The major fatty acids were palmitic and oleic acids, but polyunsaturated fatty acids were also present in substantial amounts. The results suggest that significant de novo synthesis of lipids does not occur until after hatching.
Binding of methylmercuric hydroxide by 4-thiouridine of N-formylmethionyl-tRNA from Escherichia coli
Authors: Maguire R. James
Published: December 2009

The binding of methylmercuric hydroxide to N-formylmethionyl-tRNA of Escherichia coli has been studied. In the absence of magnesium ions, methylmercury binds strongly to 4-thiouridine in the eighth position from the 5′ end; the dissociation constant is (1.0 ± 0.2) × 10−5 M, and the forward and reverse rate constants for binding are (5.3 ± 0.5) × 104 M−1 s−1 and 0.38 ± 0.02 s−1, respectively. The presence of Mg2+ prevents this binding, presumably through some conformational change induced in the structure of N-formylmethionyl-tRNA.
The endogenous biosynthesis of prostaglandins by brain tissue in vitro
Authors: Wolfe Leonhard S. , Rostworowski Klara , Pappius Hanna M.
Published: December 2009

The capacity for biosynthesis of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) from endogenous precursors by brain tissue slices and homogenates was measured by a gas chromatography – mass fragmentography method using deuterated prostaglandins as internal standards. Mean biosynthesis in rat cerebral cortex slices incubated for 60 min was 60.2 ng PGF2α and 17.4 ng PGE2 per 100 mg of tissue. The corresponding values for homogenates were 78.1 ng and 28.9 ng. Synthetic capacity of cat cerebral cortex was considerably greater but that of human tissue was smaller than that found in rat brain. Cat cerebellum in contrast to other regions synthesized more PGE2 than PGF2α. The time-course of prostaglandin formation in slices was linear for the initial 60 min. Catabolism in cerebral tissues was found to be very small. Prostaglandins formed or added to the incubation media distributed between tissue and medium in a manner indicating some specific binding as well as nonspecific solubilization in tissue lipids. Norepinephrine, 3,4-L-dihydroxyphenylalanine, dopamine, adrenochrome and apomorphine greatly stimulated PGF2α formation probably through a nonenzymatic reduction of endoperoxides. Norepinephrine added to homogenates appeared to stabilize the fatty acid cyclo-oxygenase preventing it from inactivation. Indomethacin and Ketoprofen were potent inhibitors of biosynthesis. Paracetamol was found to be a less potent synthetase inhibitor than aspirin. The biosynthetic capacity of brain tissue in vitro appears to be orders of magnitude more than that of normal brain in situ.
Metabolism of [4-14C]estrone by sheep erythrocytes around the time of parturition
Authors: Tsang Charles P. W.
Published: December 2009

The metabolism of [4-14C]estrone in vitro by red blood cells of sheep in late pregnancy and after parturition has been studied. [14C]estrone (600 ng) was incubated with 0.5 ml erythrocytes plus 0.5 ml of Krebs–Ringer phosphate buffer, pH 7.4, for 2 h at 37 °C in an atmosphere of air. After incubation, [3H]estrogens were added to the incubation medium as internal standards for identification and for correction for procedural losses. Metabolites were isolated and purified by chromatography, acetate derivative formation, and recrystallization to a constant 3H/14C ratio. Approximately 20% and 2% of added estrone were converted to 17β-estradiol and 17α-estradiol, respectively. The remainder was recovered unchanged. Daily measurements of 17β-hydroxysteroid dehydrogenase activity in erythrocytes of five ewes, over the period 8 days prepartum to 4 days postpartum, showed no significant change in activity.
Covalent assembly of mouse immunoglobulin G subclasses in vitro: application of a theoretical model for interchain disulfide bond formation
Authors: Percy M. E. , Baumal R. , Dorrington K. J. , Percy J. R.
Published: December 2009

The pathways and kinetics of interchain disulfide bond formation have been determined in vitro for purified myeloma proteins representing the three major subclasses of mouse immunoglobulin G (IgG)using the reoxidation system described previously (Petersen, J. G. L. &Dorrington, K. J. (1974) J. Biol. Chem. 249, 5633–5641). Mixtures of oxidized and reduced glutathione were added to act as a disulfide interchange catalyst. The pathways of covalent assembly observed in vitro were qualitatively and quantitatively similar to those followed by the various subclasses in vivo. HH and HHL were the principle covalent intermediates seen with IgG1 (MOPC 31C) and IgG2a (MOPC 173 and clone 19). With IgG2b (MPC 11C), HL, HH and HHL were all prominant intermediates.The time courses of reoxidation were simulated using a theoretical model based on second-order reaction kinetics (Percy, J. R., Percy, M. E. &Dorrington, K. J. (1974) J. Biol. Chem. 250, 2398–2400). Two distinct phases were apparent in the reoxidation sequence. The first, which lasted for the initial 5–15 min, did not conform to the theoretical model. The second phase could be accounted for by the model and represented the remainder of the covalent assembly process. The physico-chemical basis for this biphasic phenomenon was explored. Sedimentation velocity studies showed that noncovalent association was incomplete at the beginning of the reoxidation step for all proteins except IgG2b (MOPC 11C). No dissociation was apparent in the reduced and alkylated proteins at pH 5 in the absence of prior exposure to acid conditions. Thus, exposure to acid appears to affect the affinity between the subunits in the native proteins. Transfer of the proteins from pH 5 to pH 8.2 (the pH at which reoxidation proceeds) is accompanied by the generation of an absorption difference spectrum over an 8–10-min period. These data suggest that a pH-dependent conformational relaxation process may influence the early stages of reoxidation.
Versatility of oxazolidine spin labels—a model study with acyl-α-chymotrypsin
Authors: Hsia J. C. , Panthananickal A.
Published: December 2009

The p-nitrophenyl ester of N-oxyl-4′,4′-dimethyl-3-oxazolidinebutyric acid was synthesized. The resonance spectrum of the acyl-α-chymotrypsin intermediate of this substrate was found to have more motional freedom at the enzyme active site as compared to the acyl-enzyme prepared from the p-nitrophenyl esters of 1-oxyl-2,2,5,5-tetramethyl-3-carboxypyrrolidine and 1-oxyl-2,2,6,6-tetramethyl-4-carboxy-1,2,5,6-tetrahydropyridine. The flexibility and the versatility of Keana's oxazolidine spin labels as covalent conformational probes is discussed.
Diphosphoinositide metabolism in bovine adrenal medulla
Authors: Lefebvre Y. A. , White D. A. , Hawthorne J. N.
Published: December 2009

(1) A phosphatidylinositol kinase (EC 2.7.1.67) ofa chromaffin vesicle membrane preparation isolated from bovine adrenal medulla was characterized. Its activity towards endogenous and exogenous phosphatidylinositol was very similar to the kinase activity of the microsomal fraction prepared from the same tissue.(2) Phosphomonoesterase (EC 3.1.3.36) and diesterase activity hydrolysing membrane bound phosphatidylinositol 4-phosphate was located mainly in the microsomal fraction. No hydrolytic activity was present in the vesicle membrane.(3) Phosphorylation of chromaffin vesicle membrane phosphatidylinositol did not increase calcium-binding by the membranes.
Synthesis of diphosphoinositide by a supernatant fraction from Acanthamoeba castellanii
Authors: Buckley J. Thomas
Published: December 2009

Homogenates of the free-living amoeba Acanthamoeba castellanii incorporate phosphate from [γ-32P]ATP into a lipid which co-chromatographs with diphosphoinositide on one- and two-dimensional chromatography. Incorporation into lipids similar in mobility to triphosphoinositide is not detected. The product co-chromatographs with diphosphoinositide whether exogenous phosphatidylinositol or total amoeba lipid is the substrate. The inositide kinase is almost entirely located in the supernatant fraction after centrifugation at 100 000 g. Incorporation of phosphate from [γ-32P]ATP is linear for at least 15 min in the presence of 0.5 mM phosphatidylinositol. The enzyme requires Mg2+ or Mn2+ as well as ATP and it is not affected by low concentrations of Ca2+. The apparent Km for phosphatidylinositol is 2 mM. Both ADP and cAMP inhibit the reaction.
Purification and characterization of tonin
Authors: Demassieux S. , Boucher R. , Grisé C. , Genest J.
Published: December 2009

Tonin was purified from rat submaxillary glands by differential centrifugation, ammonium sulphate precipitation, gel filtration on Sephadex G150, and by ion-exchange chromatography on DEAE-cellulose, phospho-cellulose, SP-Sephadex C25, and SP-Sephadex C50. Purified tonin was shown to be homogeneous by analytical electrophoresis and by analytical ultracentrifugation analysis. Purified tonin was very stable when stored in buffers of low pH values or when incubated at high temperatures in neutral solutions. The molecular weight estimated by sedimentation equilibrium was 28 700. The pH optimum was near 6.8 in a 0.1 M potassium phosphate buffer. The Michaelis–Menten constant for tonin using angiotensin I as substrate was about 4 × 10−5 M. Tonin activity was strongly inhibited by plasma. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by plasma was of the non-competitive type.
Effects of indomethacin and aminoglutethimide phosphate in vivo on luteinizing-hormone-induced alterations of cyclic adenosine monophosphate, prostaglandin F, and steroid levels in preovulatory rat ovaries
Authors: Armstrong D. T. , Dorrington J. H. , Robinson J.
Published: December 2009

Changes in levels of cyclic adenosine monophosphate (cAMP), prostaglandin F (PGF), progesterone, testosterone, and estradiol-17β, in preovulatory rat ovaries induced by exogenous luteinizing hormone (LH) have been measured. Ovarian cAMP reached maximal levels 15 min and 1 h after LH administration by intravenous and intraperitoneal routes, respectively, and then declined to pre-LH levels by 8 h. Progesterone levels in ovaries and serum rose approximately in parallel with cAMP, but remained elevated throughout the 8-h sampling period. Ovarian testosterone increased to maximal levels 1 h after LH injection, followed by a rapid decline to below pre-LH levels. Ovarian estradiol-17β concentrations declined steadily throughout the sampling period, reaching almost undetectable levels 8 h after LH treatment. Elevated ovarian PGF levels were observed only at the 4- and 8-h sampling times. Indomethacin treatment, 1 h before LH, prevented the LH-induced increase in ovarian PGF levels, depressed PGF values considerably in saline-injected controls, but produced no significant inhibition of ovarian cAMP and progesterone levels. Aminoglutethimide phosphate depressed ovarian concentrations of all three steroids (progesterone, testosterone, and estradiol-17β) to essentially undetectable levels, both in control and LH-injected rats, but did not alter the LH-induced changes in ovarian cAMP and PGF levels. These observations support the concept of cAMP as a mediator of the LH-induced alterations of ovarian steroidogenesis in vivo during the preovulatory period, but argue against an obligatory role of PGF in this process.
Enzymatic synthesis of C40 carotenes by cell-free preparation from Halobacterium cutirubrum
Authors: Kushwaham Santosh C. , Kates M. , Porter John W.
Published: December 2009

[14C]Mevalonate or [14C]isopentenyl pyrophosphate was found to be converted to trans-phytoene, trans-phytofluene, lycopene, and β-carotene by a cell-free 270 000 × g supernatant fraction prepared from Halobacterium cutirubrum cells that were broken by manual grinding with glass beads. Incubations were done under N2 in the dark at 37 °C in 4 M NaCl in presence of FAD, NADP, and MgCl2; ATP was also added when mevalonate was the substrate. This system was also capable of converting trans-[14C]phytoene to β-carotene via the intermediates trans-phytofluene, ζ-carotene, neurosporene, lycopene, and γ-carotene. Each of these labelled intermediates on incubation separately with the same enzyme system was shown to be converted to the intermediates farther down the pathway. The results of this study show that the biosynthetic pathway for the formation of C40 carotenes in H. cutirubrum proceeds as follows: isopentenyl pyrophosphate [Formula: see text] trans-phytoene → trans-phytofluene → ζ-carotene → neurosporene → lycopene → γ-carotene → β-carotene. This pathway differs from that in higher plants in that the cis isomers of phytoene and phytofluene are not on the main pathway of carotene biosynthesis, as they are in higher plants. Furthermore, trans-phytoene, which has not been reported to have any role in higher plants, appears to be the main intermediate in carotene biosynthesis in H. cutirubrum.