bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the Structure and Formation during Spermatogenesis of the Sulfoglycerogalactolipid of Rat Testis
Authors: Kornblatt M. J. , Knapp A. , Levine M. , Schachter H. , Murray R. K.
Published: December 2009

Structural studies on the major glycolipid of rat testis have confirmed our earlier identification of this compound as a 1-O-alkyl-2-O-acyl-glyceryl-monogalactoside sulfate. The acyl and alkyl groups are predominantly palmitoyl and palmityl groups, respectively. The minor glycolipid found in the same tissue has been identified as the non-sulfated form of this glycolipid. Examination of the levels of the sulfoglycerogalactolipid in the testes of immature rats, hypophysectomized rats, and normal and sterile mice indicates that the majority of the compound is located in the germinal cells of the testis and that the primary spermatocytes are the earliest spermatogenic cells to contain high levels of the lipid. Galactolipid sulfotransferase also shows peak activity in the primary spermatocytes of rat testis.
Amino Acid Metabolism in Plants. V. Changes in Basic Indole Compounds and the Development of Tryptophan Decarboxylase Activity in Barley (Hordeum vulgare) during Germination and Seedling Growth
Authors: Schneider Elnora A. , Wightman F.
Published: December 2009

In barley seedlings, tryptophan is the precursor of the simple indole alkaloid gramine, and also of tryptamine, which is important as a potential precursor of the plant growth hormone 3-indoleacetic acid. The present investigation was designed to study the distribution of free tryptophan and its derivatives within the seedlings, and to follow the changes in these compounds with time. Development of the enzyme tryptophan decarboxylase, which catalyzes the conversion of tryptophan to tryptamine, was also studied. An increase in free tryptophan was detected within 2 h of soaking the seed; this compound reached high values in very young tissues, and then declined. Gramine and its precursors, 3-aminomethylindole and N-methyl-3-aminomethylindole, were confined to the shoots; all three compounds appeared together at the inception of shoot growth. Quantitatively, gramine was the most important compound present and reached a concentration of 623 μg/g fresh weight (25 times that of free tryptophan) on the 9th day, and then declined. Isolated embryos were capable of synthesizing gramine at about one quarter the normal rate, indicating that these embryos have a considerable inherent capacity for tryptophan synthesis and are not wholly dependent on tryptophan released by the endosperm. Tryptophan decarboxylase and tryptamine were found only in the shoot, and both enzyme and product appeared after the 1st week of growth, when the rate of gramine synthesis was beginning to decline. 5-Hydroxytryptamine began to accumulate in both shoot and root after about 2 weeks of growth, and N-methyl-5-hydroxytryptamine was also present in the roots. The close parallel between the gramine pathway of the barley shoot and the analagous hordenine pathway of the root, in which tyrosine is the precursor amino acid, is discussed.
Succinate Dehydrogenases of Mucor rouxii and Penicillium duponti
Authors: Boonsaeng V. , Sullivan P. A. , Shepherd M. G.
Published: December 2009

Succinate dehydrogenase (SDH) was isolated from several fungi and the properties of the Mucor rouxii and Penicillium duponti SDH's were examined in some detail. The level of SDH was found to be dependent on the carbon source used for growth. The SDH of M. rouxii was found in the soluble fraction whereas the SDH of P. duponti was particulate. There is no evidence of more than one form of SDH in either M. rouxii or P. duponti. The SDH's from M. rouxii and P. duponti have different migration patterns after electrophoresis on polyacrylamide gels and have molecular weights of 110 000 ± 10 000 and 160 000 ± 10 000, respectively. M. rouxii SDH had a Km succinate of 3.7 × 10−4 M, a Ki malonate of 27.0 μM, and a Ki fumarate of 1.5 mM. P. duponti SDH had a Km succinate of 4.6 × 10−4 M, a Ki malonate of 17.5 μM, and a Ki fumarate of 2.6 mM.
Localization of Phosphate-Independent Glutaminase in the Brush Border of Rat Kidney Cortex
Authors: Kalra J. , Brosnan John T.
Published: December 2009

A microsomal fraction that contains the highly enriched activities of NADPH – cytochrome c reductase, 5′-nucleotidase, phosphate-independent glutaminase, and alkaline phosphatase was isolated by differential centrifugation from rat kidney cortex. Continuous sucrose density gradient studies on this fraction have shown that the distribution pattern of phosphate-independent glutaminase is identical with that of alkaline phosphatase and the specific activity of these enzymes in peak fractions were 13- to 17-fold higher than in the whole homogenate. These results indicate that the phosphate-independent glutaminase is localized in the brush border of rat kidney cortex. The enzyme is truly membranous as it could not be removed by sonication, salt treatment, or pH alterations.
In Vitro Synthesis of Propionyl-CoA Holocarboxylase by a Partially Purified Mitochondrial Preparation from Biotin-Deficient Chicken Liver
Authors: Murthy P. N. Achuta , Mistry S. P.
Published: December 2009

A 15-fold purified preparation from biotin-deficient chicken liver mitochondria catalyzed propionyl-CoA holocarboxylase (PCHC) synthesis in the presence of ATP and d-biotin. UTP effectively replaced ATP in the reaction mixture; with CTP, GTP, or ITP, less than 50% of control activities was observed. Both AMP and ADP inhibited PCHC synthesis. A cytosolic preparation obtained from the same tissue stimulated PCHC synthesis.
A Radiolabeled Assay for Aldose Reductase
Authors: Maddox I. S.
Published: December 2009

A radiolabeled assay of aldose reductase has been developed based on the use of 14C-glyceraldehyde as substrate. The reaction is measured by the formation of 14C-glycerol, separation being effected by extraction with organic solvent. The Km value for glyceraldehyde has been estimated as 5.7 × 10−6 M. Inhibition by 2-oxoglutarate has been shown to be noncompetitive with a Ki value of 12.5 mM.
Control of Fatty Acid Synthesis in White Adipose Tissue by Insulin: Coordination Between the Mitochondrial Citrate Transporter and Pyruvate Dehydrogenase
Authors: Schiller Carol M. , Taylor Wayne M. , Halperin Mitchell L.
Published: December 2009

The transport of citrate out of adipose tissue mitochondria is inhibited by palmitoyl-CoA. This inhibition varied inversely with the concentration of extramitochondrial exchanging anion.When adipose tissue is incubated in vitro, the rate of citrate output into the medium was increased by the addition of insulin. The tissue citrate content did not change significantly. Norepinephrine caused an initial decrease in the rate of citrate output (2.5 min). The tissue citrate content was approximately twofold higher at this time.When rats were fasted for 36 h, less than 40% of adipose tissue pyruvate dehydrogenase was in the active form. Optimal interconversion to the active form was achieved by preincubation with 4 mM Mg2+ in the absence of added Ca2+ (endogenous Ca2+ was approximately 25 μM). Citrate addition to the preincubation medium decreased this activation of pyruvate dehydrogenase. The inhibition induced by citrate correlated best with the concentration of 'free' citrate when the 'free' Mg2+ concentration was sufficient to cause near-maximal activation of pyruvate dehydrogenase.A hypothesis regarding the coordination of regulation of pyruvate conversion to fatty acids is formulated based on these findings.
Repression of the Branched-Chain Amino Acid Biosynthetic Enzymes in Developing Conidia and Mycelia of Neurospora
Authors: Jobbágy Andrés J. , Wagner Robert P.
Published: December 2009

During germination of the conidia of wild type Neurospora crassa, the enzymes of the isoleucine–valine pathway increase in activity. This increase is partially repressed in the presence of a combination of exogenous isoleucine, leucine, and valine. Repressed levels of enzymic activity are also observed in mycelium during the logarithmic phase of growth. In both cases, isoleucine and leucine together are almost as effective without valine as when the three are present together. The synthesis of these enzymes does appear to be affected by their end products but not to the extent exhibited in bacteria.
Studies of the Metabolism of Asialotransferrins: Relationship Between the Carbohydrate Composition of Bovine, Canine, and Porcine Asialotransferrins and their Metabolic Behavior in the Rabbit
Authors: Hatton M. W. C. , Regoeczi E. , Wong K.-L.
Published: December 2009

The catabolism and distribution of bovine, canine, and porcine asialotransferrins relative to the corresponding parent proteins were studied in rabbits using a dual-isotope tracer technique. The capillary transfer rates and the partitions between intra- and extravascular spaces of these asialotransferrins did not differ significantly from the corresponding values for the control proteins but the asialotransferrins were catabolized faster. However, the difference in catabolic rates only amounted to 13–20%, which was in the same order as established previously for rabbit asialotransferrin (15%) but was considerably less than for human asialotransferrin (350%). This behavior clearly indicates that, in contradistinction to human asialotransferrin, bovine, canine, and porcine asialotransferrins are not eliminated from the plasma of rabbits via the Ashwell–Morell pathway for asialoglycoproteins.To explain the discrepancy in the behavior in vivo of the heterologous asialotransferrins, the carbohydrate compositions of the native proteins were measured and compared. The data show that bovine, porcine, dog, and rabbit transferrins contain approximately half the amount of carbohydrate found for the human protein. Furthermore, the human asialotransferrin carbohydrate chains terminate with galactosyl residues, which can be cleaved by β-galactosidase, whereas bovine, porcine, dog, and rabbit asialotransferrins do not liberate galactose in the presence of β-galactosidase.These observations are consistent with the hypothesis that the rapid elimination of a heterologous asialotransferrin can only be expected if the terminal galactose residues are accessible and that the slightly accelerated catabolism of homologous and certain heterologous asialotransferrins is due to loss of negative charge from the protein.
Evidence for Nonproductive Binding Subsites within the Active Site of Papain
Authors: Glick Bernard R. , Brubacher Lewis J.
Published: December 2009

The reactions of several alkylating reagents with the sulfhydryl group in papain have been studied in the presence of varying concentrations of the competitive inhibitor α-N-benzoyl-D-arginine ethyl ester. The ratio of the alkylation rate constant of the papain – α-N-benzoyl-D-arginine ethyl ester complex to the rate constant with free papain is 4.3, 1.2, and 0.0 for the alkylating agents 1-chloro-3-tosylamido-4-phenyl-2-butanone, N-ethylmaleimide, and 1-chloro-3-tosylamido-7-amino-2-heptanone, respectively. These results are rationalized, along with data for the effect of α-N-benzoyl-L-arginine ethyl ester, in terms of nonproductive binding.
DNA Synthesis in Isolated Yeast Mitochondria
Authors: Zeman L. , Lusena C. V.
Published: December 2009

Isolated yeast (Saccharomyces cerevisiae) mitochondria incorporate radioactive precursors into mitochondrial DNA. This in vitro labelled DNA was characterized by isopycnic and sedimentation velocity centrifugation both in the native and denatured state. The profiles of isopycnic CsCl gradients obtained by centrifugation in a fixed-angle rotor are skewed toward high density. The skew is neither due to the presence of in vitro labelled nuclear DNA nor due to random breaks in mitochondrial DNA which would reveal, then, its heterogeneity in base composition. The in vitro labelled DNA is reproducibly recovered as a class of molecules sedimenting at about 5–8 S, indicating a molecular weight of 1 × 105 – 4 × 105 daltons, while the smallest in vivo labelled fragments sediment at about 13–14 S, corresponding to 1.6 × 106 – 2.0 × 106 daltons. After denaturation, the in vitro labelled DNA molecules sediment at about 2–5 S, corresponding to a single-strand molecular weight of 1 × 104 – 7 × 104 daltons, which is about one hundred times less than the observed size of the denatured in vivo labelled molecules.
Template Specificity and Subunits of RNA Polymerase from Asporogenous Mutants of Bacillus subtilis
Authors: Nishimoto H. , Takahashi I.
Published: December 2009

In order to investigate relations between template specificity of RNA polymerase and sporulation, RNA polymerase activities in partially purified preparations from various asporogenous mutants were measured with poly[d(A-T)] or DNA from phage PBS 15 as template. Results obtained suggest that morphological changes occurring during sporulation may not be tightly linked temporally to transcriptional events.Subunits of RNA polymerase from these mutants were analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis after purification by (NH4)2SO4 precipitation, DEAE-cellulose chromatography, phosphocellulose chromatography, and glycerol gradient centrifugation. Phenylmethylsulfonyl fluoride was present throughout the purification procedure to prevent proteolytic degradation. It was found that β and β′ subunits were present in 1:1 ratio in all preparations. In addition to β, β′, and α subunits, a protein having a molecular weight of 95 000 was found in enzyme preparations from a wild-type strain and stage II mutants harvested at t5–t9. This protein was absent in stage 0 mutants and in all strains harvested in log phase. The enzyme containing this protein was eluted from phosphocellulose column with 0.6 M KCl rather than 0.35 M KCl, which eluted the enzyme without the 95 000 dalton protein. Furthermore the enzyme with this protein showed a sedimentation coefficient higher than that of the enzyme without the 95 000 dalton protein.
The Inactivation of Penicillopepsin with l,2-Epoxy-3-(p-nitrophenoxy)propane, an Active-Site Directed Reagent
Authors: Mains G. , Hofmann T.
Published: December 2009

Penicillopepsin was fully inactivated by the pepsin inhibitor 1,2-epoxy-3-(p-nitrophenoxy) propane, and 1.3 ± 0.3 mol of reagent became associated with each mole of protein. Inactivation was more rapid at pH 3.0 than at pH 6.0. Approximately 1 equivalent of the bound reagent was esterified to an aspartic acid side chain. Enzyme previously inactivated with diazoacetylnorleucine methyl ester did not react with the epoxide; and enzyme that was first inactivated with the epoxide did not react with the diazo inhibitor. The results add further evidence for the enzymatic similarity of porcine pepsin and penicillopepsin.
Differing Populations of Mitochondria Isolated from the Skeletal Muscle of Normal and Dystrophic Hamsters
Authors: Mezon B. J. , Wrogemann K. , Blanchaer M. C.
Published: December 2009

Polarographic studies of a magnesium-responsive, calcium-associated defect of oxidative phosphorylation in dystrophic hamster skeletal muscle suggested that all dystrophic mitochondria are defective. This is at variance with the typical histological picture of dystrophic muscle, which shows, besides defective muscle fibers, many apparently normal cells, with presumably normal mitochondria. This discrepancy was resolved by linear sucrose density-gradient centrifugation (39–51%, w/v), which can separate normal mitochondria from calcium-loaded organelles. Upon density-gradient centrifugation of control and dystrophic muscle mitochondria, both preparations were found to yield suspended bands of similar density. However, the dystrophic preparations also produced a pellet at the bottom (most dense region) of the tube with calcium levels 10–40 times higher than those in the suspended bands. The data show the existence in dystrophic muscle of a distinct population of mitochondria with very high calcium levels that is absent from normal preparations. During the course of oxidative phosphorylation measurements, loss of calcium from these particular organelles and its uptake by others appear to render all the mitochondria defective. This exchange can be prevented by ruthenium red. Density-gradient centrifugation allows the identification of dystrophic muscle mitochondria in early and/or mild disease stages in which polarographic testing of mitochondria isolated by conventional centrifugation fails to detect an abnormality.
A Study on the Effect of Lysolecithin and Phospholipase A on Membrane-Bound Galactosyltransferase
Authors: Mookerjea Sailen , Yung James W. M.
Published: December 2009

Addition of lysolecithin caused very marked activation of UDP-galactose:glycoprotein galactosyltransferase in rat liver microsomes and in Golgi-rich membranes. Lysolecithin activated galactosyltransferase when the enzyme was assayed both with endogenous acceptor and with exogenous proteins or monosaccharides as acceptors. Lactose synthetase activity in presence of α-lactalbumin was also stimulated by lysolecithin. Lecithin, lysophosphatidylethanolamine, lysophosphatidic acid, and glycerophosphorylcholine did not activate the enzyme, suggesting that both fatty acyl and phosphorylcholine groups of the lysolecithin molecule are required for the observed activation. The degree of activation was about the same when myristoyl-, palmitoyl-, oleoyl-, or stearoyllysolecithin were tested. The activation by lysolecithin was observed well within the physiological concentration of the lipid in the liver cell. Saturating amounts of Triton masked the effect of lysolecithin.Brief preincubation with phospholipase A activated the enzyme and generated lysolecithin in the membranes. Triton and lysolecithin activated the enzyme without any lag time, whereas phospholipase A activation was dependent on preincubation and also on an alkaline pH favorable for the hydrolysis of phospholipid. EDTA blocked the activation effect of phospholipase A but had no effect on activation by lysolecithin. Albumin and cholesterol opposed the effects of lysolecithin and phospholipase A on the enzyme. Two successive incubations of the microsomes with lysolecithin caused considerable release of the enzyme into the soluble fraction. The role of lysolecithin in the activation of the enzyme is probably related to the solubilization of the membrane and consequent enhanced interaction of the enzyme with substrate. Lysolecithin also activated N-acetylglucosaminyl- and sialyltransferase activities in microsomes. A possible role of lysolecithin is indicated in the regulation of glycosylation reactions in mammalian system.
Characterization of the Esterases of Feline Serum
Authors: Ecobichon D. J.
Published: December 2009

Various techniques, including electrophoresis, gel filtration, titrimetric, and spectrophotometric analysis with specific and nonspecific substrates and inhibitors, were used to characterize the serum esterases of male and female cats. Starch-gel electrophoresis yielded five bands of activity: three butyrylcholinesterase bands and two bands of nonspecific carboxylesterase activity. Gel filtration on Sephadex G-200 yielded two distinct peaks of activity, one containing the cholinesterase bands and the other, the carboxylesterase activity. Kinetic characteristics determined for feline serum esterases included: (1) optimal pH; (2) optimal substrates for cholinesterase and carboxylesterase activities; (3) Km values for the choline, α-naphthyl, and p-nitrophenyl esters. With the organophosphate paraoxon as a specific substrate for arylesterase activity, low levels of this enzyme could be detected in close association with the carboxylesterase.
Effects of Ethylene, Carbon Dioxide, and Ethylene – Carbon Dioxide Mixtures on the Activities of "Membrane-Containing" and "Highly Purified" Preparations of Adenosine Triphosphatase from Pea-Cotyledon Mitochondria
Authors: Malhotra S. S. , Spencer Mary
Published: December 2009

From pea-cotyledon mitochondria two types of adenosine triphosphatase (ATP phosphohydrolase) were prepared, "membrane-bound" and "highly purified." Activities of both enzyme preparations were dependent on Mg2+ and were stimulated by Na+ and K+. Ethylene had no effect on the activity of either the "membrane-bound" or the "highly purified" enzyme. Ethylene – carbon dioxide – air mixtures stimulated the activity of the "membrane-bound" but not the "highly purified" ATPase, when Na+ and K+ were present. When these ions were absent, the gas mixture greatly increased the activities of both enzyme preparations. The work shows a direct action of ethylene on an enzyme (mitochondrial ATPase), its dependence on the presence of certain levels of CO2, and the augmentation of the ethylene–CO2 effects by the presence of phospholipids.
Evaluation of the Hill Coefficient for Yeast Aspartate Transcarbamylase by Nonlinear Regression Analysis
Authors: Hurst R. O.
Published: December 2009

Primary data for yeast aspartate transcarbamylase reported by Kaplan et al. ((1967) Arch. Biochem. Biophys. 119, 541–551) have been evaluated for the degree of fit to hyperbolic and sigmoidal functions. The restricted nature of their experimental design does not provide an adequate basis for discrimination between the types of mechanisms considered. Comparison of the parameter estimates obtained by the Hill plot and by nonlinear regression analysis demonstrated that the Hill plot may give rise to gross errors in the determination of the kinetic constants.
A Flow Method for Determination of the Kinetic Parameters for Immobilized Enzymes
Authors: Ngo T. T. , Bunting P. S. , Laidler K. J.
Published: December 2009

A flow method is described for determination of the kinetic parameters (Vm and Km) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate of formation of product. To illustrate the technique, it is applied to the determination of the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing.
Molecular and Biological Studies on Cardiac Muscle Calcium-Binding Protein (TN-C)
Authors: Burtnick L. D. , McCubbin W. D. , Kay C. M.
Published: December 2009

TN-C was purified from bovine cardiac muscle. In the absence of Ca2+, cardiac TN-C has an intrinsic sedimentation coefficient of 1.93 S and a molecular weight of 18 000 daltons. Cardiac TN-C reverses the inhibitory effect of skeletal TN-I on the Mg2+-activated ATPase of a skeletal synthetic actomyosin preparation in the presence of skeletal tropomyosin. Circular dichroism (CD) studies indicate that cardiac TN-C undergoes a major conformational Change upon binding Ca2+. A similar response is elicited by Sr2+, whereas Mg2+ has a much less pronounced effect. The presence of Mg2+ does not alter the net effects of either Ca2+ or Sr2+. Cardiac TN-C is rich in acidic amino acid residues. UV absorption, near UV CD, and fluorimetric studies show that the protein lacks tryptophan and has a relatively high phenylalanine to tyrosine ratio. The results of this study invite direct comparisons with results reported for the skeletal muscle analogue of cardiac TN-C.
Isolation and Tissue Specificity of Chromatin Associated Proteins in Vicia faba
Authors: Mischke Barbara Grahek , Ward Oscar G.
Published: December 2009

A method is described that permits extraction of one class of non-histones in 8 M urea −0.14 M mercaptoethanol prior to acid extraction of histones and a second class in 0.05 M Tris – 1% sodium dodecyl sulfate following acid extraction of histones. Comparisons of histones and non-histones extracted by this method with those obtained by other procedures demonstrate two important advantages of the method: (1) histones obtained by this method are not contaminated by acid-soluble non-histones, and (2) non-histones are not subjected to acid or phenol during extraction. Changes in the distributions of chromatin-associated proteins in different tissues suggest that some species represent regulators of gene action.
Tissue Specificity of Nuclear Acidic Proteins Isolated from Bovine Brain and Adrenal Medulla
Authors: Davis Robert H. , Wilson Richard B. , Ebadi Manuchair S.
Published: December 2009

Nuclear acidic proteins from bovine brain and adrenal medulla demonstrated dissimilar electrophoretic and chemical profiles. The amino acid analysis of the acidic nuclear protein fraction isolated from these tissues revealed some variation in the ratio of acidic to basic amino acids. The estimation of free carboxylic acid groups confirmed the more acidic nature of the brain proteins. In comparing the acrylamide gels either visually or optically, several electrophoretically specific bands were apparent. Although the total number of protein bands from each tissue was approximately the same, the adrenal medulla contained a larger proportion of the more positively charged proteins. These observations are interpreted to indicate that the nuclear acidic protein from brain and adrenal medulla may show functional variation.
A Comparison of the Peptide Composition of Human Serum Low and Very Low Density Lipoprotein
Authors: Rubenstein Bernard , Steiner George
Published: December 2009

We have examined the apoprotein structure of human very low density lipoprotein (VLDL) and two subfractions of human low density lipoproteins, LDL-2 (d 1.026–1.046) and LDL-3 (d 1.046–1.063). Both LDL-2 and LDL-3 had similar peptide patterns on polyacrylamide gel electrophoresis and this was the same as VLDL. Column chromatography showed that the 'B' component of the apoproteins of VLDL contained 42–45% of the total protein while the 'non-B' component contained 55–58%. In both LDL-2 and LDL-3, the 'B' component was 95% of the total protein while the 'non-B' was 5%.An examination of the peptides of the 'non-B' portion of VLDL and LDL was carried out using DEAE-cellulose chromatography. Although both VLDL and LDL contained the same major peptides, the proportion of the total 'non-B protein' of each of these peptides in VLDL differed from that seen in LDL. ApoLP-Glu and apoLP-Ala of VLDL contained 13 and 65%, respectively, of the total 'non-B protein', while in LDL these peptides were 26 and 46%. These results indicate that apoLDL contains the 'non-B proteins' of VLDL, but in different proportions and in a much reduced percentage.
Evidence for NAD Nucleosidase in Rabbit-Liver Lysosomes
Authors: Mellors A. , Lun A. K. L. , Peled O. N.
Published: December 2009

A method is described for the isolation of secondary lysosomes from homogenates of rabbit liver. The uptake of Triton WR-1339 by rabbit-liver lysosomes when administered by intraperitoneal injection was used to decrease the density of secondary lysosomes. Lysosomal fractions prepared by this method contain an NAD nucleosidase (NAD glycohydrolase, EC 3.2.2.5), an enzyme which has previously been considered to be associated with other subcellular fractions. The enzyme has maximum activity at pH 6 and cleaves both NAD and NADP. It is inhibited by nicotinamide (Ki = 4.5 mM) and by HgCl2. Both nucleosidase and 2′-nucleotidase show in-vitro latency typical of lysosomal acid hydrolases. Rabbit-liver plasma-membrane fractions were isolated which contained most 5′-nucleotidase but relatively little nucleosidase, whereas rabbit liver lysosomes contain both 5′-nucleotidase and nucleosidase enzymes but little adenyl cyclase.
The Effect of a New Antibiotic, Cryptosporiopsin, on RNA Synthesis in L-Cells
Authors: Dessureault J. , Krause M. O.
Published: December 2009

The effect of cryptosporiopsin on RNA synthesis in L-cells was studied as part of an investigation on the mechanism of action and potential toxicity of the antibiotic in mammalian cells.RNA synthesis in vitro was tested in intact isolated L-cell nuclei, in conjunction with selective inhibitors of nucleolar and nucleoplasmic RNA synthetic activities. It was found that only the nucleoplasmic activity (polymerase II), was inhibited by cryptosporiopsin and that the drug showed no effect on the activity of the nucleolar enzyme (polymerase I).RNA synthesis in vivo was tested using double labelling with [14C]guanine and [3H]-uridine in an attempt at discriminating between G + C nucleolar RNA and high A + U nucleoplasmic RNA synthesis. Results revealed that the uptake of these precursors into both types of RNA was inhibited by cryptosporiopsin in intact cells. Measurements of the nucleotide pools in these cells indicated that the antibiotic affects uptake and phosphorylation of nucleosides and nucleotides, especially the production of ATP. These results suggest that the uptake inhibition observed in vivo could be due, at least in part, to energy and/or precursor shortage.
Rabbit Cardiac Myosin. I. Physical and Chemical Characterization of the Native Molecule
Authors: Wolodko William T. , Kay Cyril M.
Published: December 2009

Rabbit cardiac myosin, isolated from frozen tissue, was effectively purified by batchwise treatment with DEAE-cellulose in addition to using dilution–precipitation techniques. An extensive experimental program was subsequently carried out with respect to the enzymic, amino acid, optical, and physicochemical properties of native cardiac myosin. This program has included the following: examination of the effects of pH and varying concentrations of ATP, CaCl2, MgCl2, and PCMB on its ATPase activity; measurement of its circular dichroic spectrum in solvent buffers, at different pH or containing ATP in the absence or presence of Ca2+ or Mg2+ ions; study of the concentration dependence of its viscosity and sedimentation velocity at low temperatures; and investigation of its molecular weight by the Archibald method and low- and high-speed sedimentation equilibrium. The results of these studies were consistent with the interpretation that cardiac myosin is comprised of highly asymmetric, semi-rigid molecules with a molecular weight in the order of 4.7 × 105, which display non-ideality even in solvent buffers of high ionic strength at neutral pH. In addition, computer analysis of the high-speed sedimentation equilibrium data has provided evidence for the presence of a self-association reaction at low protein concentration. Even though the specific ATPase activity of cardiac myosin was found to be approximately one-third that reported for skeletal myosin in all cases, it was concluded, on the basis of the essentially analogous physical and chemical properties of rabbit cardiac and skeletal myosin, that the two proteins are very similar in terms of molecular size, shape, and secondary structure.
Rabbit Cardiac Myosin. II. Proteolytic Fragmentation with Insolubilized Papain
Authors: Wolodko William T. , Kay Cyril M.
Published: December 2009

The substructure of the cardiac myosin molecule was examined by the limited proteolytic digestion of the parent molecule with (dialdehyde starch)-methylenedianiline–mercuripapain, S-MDA–mercuripapain, at low temperatures and neutral pH, using moderate enzyme to myosin ratios. Pertinent properties of the insoluble enzyme complex were also examined. Kinetic, ultracentrifugal, and chromatographic observations of the fragmentation process revealed that a single type of lytic reaction occurs during the early stages, predominantly releasing heavy meromyosin subfragment 1 (HMM-S1) and myosin rods. With further time of digestion, the rods are additionally cleaved yielding light meromyosin and HMM-S2, and HMM-S1 is found to be partially degraded. The major proteolytic subfragments were isolated, purified, and characterized with respect to their enzymatic, optical, amino acid, and physicochemical properties. Only HMM-S1 exhibited Ca2+-activated ATPase activity, and at a level three- to fourfold higher than that of native myosin. Moreover, its hydrodynamic properties suggest that it is globular in structure. On the other hand, light meromyosin-A (LMM-A) (which consists mainly of rods), and HMM-S2 appear to be highly asymmetric, rigid, α-helical molecules devoid of the amino acid proline. Strong similarities were evident in all aspects upon comparison of these results with documented information concerning the skeletal system. On the basis of the physical and chemical properties of the proteolytic subfragments relative to that of native myosin, it was further concluded that the cardiac myosin molecule is a double-stranded, α-helical rod ending in two subfragment 1 globules, of which only one may be enzymatically active at a time.
Lysolecithin and Cholesterol Interact Stoichiometrically Forming Bimolecular Lamellar Structures in the Presence of Excess Water, or Lysolecithin or Cholesterol
Authors: Rand R. P. , Pangborn W. A. , Purdon A. D. , Tinker D. O.
Published: December 2009

The structural interaction of egg lysolecithin, derived from egg lecithin, and cholesterol in aqueous solution has been investigated using X-ray diffraction. When mixed in any proportions, either suspended in excess buffer or up to 85% lipid by dry weight, a separate lamellar phase containing equimolar proportions of lysolecithin and cholesterol forms, separate from excess water, or lysolecithin or cholesterol. The cholesterol disorders the crystalline chains of the lysolecithin. The equimolar phase is stable up to 50 °C unlike lysolecithin alone, which forms micelles. These results show that lysolecithin and cholesterol combine stoichiometrically in a stable complex. We propose as a structural model, that cholesterol fills the space of the missing fatty acyl chain making the lysolecithin more cylindrical rather than wedge shaped. This interaction could reduce both the lytic action of lysolecithin on membranes and its induction of cell fusion. It suggests another role of cholesterol in cell membranes: namely, to act as a stabilizer of bilayer structure by being a mobile component that can fill free volume in the hydrocarbon interior. Lysolecithin–cholesterol interaction may also be important in the early events of atherosclerosis where lysolecithin levels in vessel walls increase fivefold.
Excretion of Radioactive Daidzein and Equol as Monosulfates and Disulfates in the Urine of the Laying Hen
Authors: Chang Herbert Hang-Shing , Robinson Arthur Robin , Common Robert Haddon
Published: December 2009

The phytoestrogen, daidzein, was injected intramuscularly as [4-14C]daidzein into two laying hens. The radioactive materials in the urine for the succeeding 23 (hen 1) or 14 (hen 2) days were fractionated on a DEAE-Sephadex column by a gradient of NaCl and the fractions thus separated were further analyzed by solvent partition, susceptability to enzymic cleavage and thin-layer chromatography. The six following components were identified and quantitated: [14C]daidzein, [14C]equol, [14C]daidzein monosulfate, [14C]equol monosulfate, [14C]daizein disulfate, and [14C]equol disulfate. The urine from hen 2 yielded also the sulfate of an unidentified conversion product of [14C]daidzein. Repeated tests for glucuronides of [14C]daidzein or its conversion products gave negative results, excluding the possibility that any appreciable proportion of the radioactivity in the urine was in the form of β-glucuronide. It is concluded that the daidzein and the equol excretion in the urine of the laying hen are present for the most part as monosulfates and disulfates.
Characterization and Composition of the Purple and Red Membrane from Halobacterium cutirubrum
Authors: Kushwaha S. C. , Kates M. , Martin W. G.
Published: December 2009

The purple membrane (bacteriorhodopsin) isolated from cells of Halobacterium cutirubrum grown anaerobically in the light was shown to contain 77% protein and 20% lipids by weight. The protein component consisted of a single protein moeity, having a molecular weight of (19.6 ± 0.8) × 103, complexed with retinal in mole ratio of 2:1, respectively. The protein moeity is not glycosylated but may be phosphorylated (ca. 2 mol of phosphate per mole of protein). The red membrane contains 56% protein and 38% lipids, including bacterioruberins. Several polypeptide components are present including some which may be glycosylated and/or phosphorylated. The lipids of both membranes contained phosphatidyl glycerophosphate (52%) and phosphatidyl glycerol (3–4%) but the sulfated lipid components, glycolipid sulfate and phosphatidyl glycerosulfate, were present exclusively in the purple membrane, the red membrane containing instead two unidentified glycolipids. Neutral lipids (squalenes, vitamin MK-8, etc.) were present in both membranes to the extent of 7–9%.
The Diglycoside of 17α-Estradiol from Rabbit Urine: Comparison with Material Prepared by Synthesis
Authors: Paquet Alenka , Layne Donald S.
Published: December 2009

Two synthetic routes to methyl estra-1,3,5(10)-trien-17α-yl-2′-trifluoroacetamido-3′,4′,6′-tri-O-acetyl-2′-deoxy-β-D-glucopyranosid-3-yl-2′,3′,4′-tri-O-acetyl-β-D-glucopyranosidyl uronate are described, with the 3-acetyl or the 17α-trifluoroacetyl derivatives of 17α-estradiol as starting materials. Physical characteristics of the fully acetylated double glycoside were determined. This compound was converted to sodium estra-1,3,5(10)-trien-17α-yl-2′-acetamido-2′-deoxy-β-D-glucopyranosid-3-yl-β-D-glucopyranosidyl uronate, which was found to be identical with the sodium salt of material isolated from rabbit urine.
Formation and Repair of Papain Sulfenic Acid
Authors: Lin W. S. , Armstrong D. A. , Gaucher G. M.
Published: December 2009

The inactivation of highly purified papain (2 × 10−5 M in distilled water) by hydrogen peroxide, generated by the γ-irradiation of water, was examined. The kinetics of activity loss at 23 °C was second order (k = 3.7 × 103 M−1 min−1) for papain:peroxide molar ratios of 1:1 or 2:1. Loss of activity is accompanied by a parallel loss of sulfhydryl; however, the sulfhydryl losses, as determined with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) or p-hydroxymercuribenzoate (pHMB), are anomalously either too large or too small, respectively. These discrepancies resulted from the reaction of inactive papain with either the thiol anion product of the DTNB reaction, or with the pHMB reagent itself. The addition of 1.2 M urea to the DTNB reaction mixture significantly decreased this error. Inactive papain reacted with high concentrations of cysteine or cyanide to yield completely repaired active papain, and with benzylamine to yield non-repairable, inactive papain. Sodium arsenite, which is capable of reducing sulfenic acids but not disulfide bonds, readily repaired peroxide-inactivated papain. A completely inactive but repairable papain fraction was isolated by virtue of its lessened ability to bind to a tetrapeptide inhibitor immobilized on Sepharose. The cumulative results indicate that the peroxide inactivation of papain is due almost exclusively to the formation of papain sulfenic acid (Cys25—SOH).
Apomorphine: Stereotyped Behavior and Regional Distribution in Rat Brain
Authors: Butterworth Roger F. , Barbeau André
Published: December 2009

The distribution of apomorphine following subcutaneous injection of 20 mg/kg (as the hydrochloride) was measured spectrofluorometrically in specific regions of rat brain. Measurable concentrations were found in the brain within a few minutes of injection, the drug was still detectable for at least 60 min in all regions, and maximum concentration was observed 20 min after injection. Stereotyped behavior, characteristic of apomorphine action, followed a time course parallel to accumulation of the drug in the brain.
Chemical Modification of Ribosomes with Dimethyl Sulfate: a Probe to the Structural Organization of Ribosomal Proteins and RNA
Authors: Moore Graham
Published: December 2009

Ribosomal proteins from [14C]dimethyl sulfate treated 30S and 50S subunits of Escherichia coli ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and the degree of methylation of each protein was determined. Comparison of the results from this relatively non-specific chemical modification procedure with results from the milder lysine-specific reductive alkylation procedure reported previously (Moore, G. &Crichton, R. R. (1974) Biochem. J. 143, 607–612) has permitted a topographical classification of ribosomal proteins in terms of 'degree of exposure' in the 30S and 50S subunits.The reaction of dimethyl sulfate with ribosomal RNA, both in intact subunits and after isolation from the subunits, has indicated that approximately half of the RNA in 30S and 50S subunits is exposed on the surface of the ribonucleoprotein complexes, and that no large sections (extended sequences) of 16S RNA are concealed in the 30S subunit. It is proposed that modification of ribosomes with dimethyl sulfate is a potentially useful technique for probing exposed and hidden regions and also exposed single-stranded regions of RNA in ribosomes.
Rat Liver Nuclear Protein Kinases
Authors: Desjardins P. R. , Liew C. C. , Gornall A. G.
Published: December 2009

We have shown that nuclei isolated by two methods contain grossly different amounts of cyclic AMP-dependent histone kinase activity. Repeated washing of the isolated nuclei with a low ionic strength buffer removed the majority of the cyclic AMP-dependent histone kinase and cyclic AMP binding activity. Nuclear cyclic AMP-dependent histone kinase activity accounted for only 0.42% of the total cytoplasmic enzyme activity. Similarly, the lactate dehydrogenase activity associated with liver nuclei represented only 0.07% of the total cytoplasmic activity. The isolated liver nuclei contained only 0.27% of the total homogenate glutamate dehydrogenase activity and 1.7% of the total homogenate glucose-6-phosphatase activity. The cyclic AMP-dependent histone kinase behaves as a cytoplasmic rather than a nuclear enzyme. We have also shown that using crude extracts, one can achieve separation of the two nuclear casein kinases, NI and NII, on sucrose density gradients in the presence of 0.5 M NaCl. Nuclear casein kinases NI and NII had sedimentation coefficients of 3.0 and 5.3 S, respectively, in the presence of 0.5 M NaCl. Under conditions of low ionic strength, all of the casein kinase activity in the crude nuclear extract sedimented as one peak with a sedimentation coefficient of 7.3 S. The aggregation–disaggregation which occurred in the crude extract was reversible and was mainly due to the aggregative and disaggregative properties of casein kinase NII The two nuclear casein kinases have different affinities for chromatin. When nuclei were disrupted in a hypotonic solution and extracted with a buffer containing 0.14 M NaCl, casein kinase NII could be completely extracted from the viscous nuclear material. Although a significant amount of casein kinase NI was extracted by the buffer containing 0.14 M NaCl, re-extraction of the nuclear material with a buffer containing 0.5 M NaCl yielded substantial amounts of casein kinase NI, and a final extraction with a buffer containing 1.0 M NaCl yielded measurable amounts of casein kinase NI. No casein kinase NII activity could be detected in the 0.5 M and 1.0 M NaCl extracts.
Transient Kinetics of the Acetylcholinesterase Catalyzed Hydrolysis of N-Methylindoxyl Acetate
Authors: Sadar M. H. , Laidler K. J.
Published: December 2009

An experimental study has been made of the kinetics of the hydrolysis of N-methylindoxyl acetate catalyzed by electric-eel acetylcholinesterase, both in the steady state and the pre-steady state. Stopped-flow and temperature-jump experiments revealed a fast transient and a slow one. The fast transient is correlated with the conventional mechanism [Formula: see text]. The slow transient is attributed to conformational changes involving E or EA. Analysis of it revealed two exponential terms of the form e−λt, and the two λ values were obtained over the temperature range 5.0 to 25.0 °C. The results are interpreted in terms of two alternative mechanisms; in one, the enzyme undergoes a conformational change before it adds on the substrate molecule; in the other, the conformational change occurs after the substrate addition. Both mechanisms may be involved, but the results exclude a concerted mechanism in which the conformational change occurs concurrently with the addition of substrate. Kinetic parameters (ΔS≠ and E) are obtained for this conformational change and for the conversion of EA into EA′ + X.
The Metabolism of 4′-Chloro-4-biphenylol in the Rat
Authors: Safe S. , Hutzinger O. , Ecobichon D. J. , Grey A. A.
Published: December 2009

4′-Chloro-4-biphenylol, the major metabolite of 4-chlorobiphenyl in the rat, was given intraperitoneally to rats, and the urine and feces were examined for possible metabolic degradation products. The structure of the major urinary metabolite was elucidated by mass and nuclear magnetic resonance spectroscopy and shown to be 4′-chloro-3,4-biphenyldiol. Two chloromethoxybiphenylols (M+ 234) were also identified in the urine extracts but they could not be separated by chromatographic procedures. Demethylation of the mixture gave 4′-chloro-3,4-biphenyldiol as the sole product, thus indicating that the two components of the mixture were 4′-chloro-3-methoxy-4-biphenylol and 4′-chloro-4-methoxy-3-biphenylol. A fourth urinary metabolite was identified as 4′-chloro-4-methoxy-3,5-biphenyldiol. No 4′-chloro-4-biphenylol metabolites were isolated in the fecal extracts, and mass spectrometric analysis of the crude urine and feces extracts did not reveal any chlorine-containing degradation products that could be derived by oxidative fission of the biphenyl nucleus.
The Development of Kinetic Parameters of Hepatic Drug-Metabolizing Enzymes in Perinatal Rats
Authors: Bell J. U. , Ecobichon D. J.
Published: December 2009

The development of the apparent kinetic parameters Km and Vmax was studied in perinatal Wistar rats for three functionally diverse, hepatic enzymes (p-nitroanisole O-demethylase, carboxylesterase and bromosulphophthalein–glutathione conjugating enzyme), the period studied being from 3 days prepartum to 35 days postpartum. The kinetic parameters underwent marked quantitative changes during development, which appeared to be independent of sex for the first 5 weeks postpartum.
The Incorporation of 14C from [l-14C]Palmitate into Glucose and Glycogen in Mice
Authors: Pushpendran C. K. , Eapen Jacob
Published: December 2009

The incorporation of 14C from [1-14C]palmitate into blood glucose and liver and kidney glycogen in postnatal mice has been studied. Incorporation of 14C from [1-14C]palmitate into blood glucose and hepatic glycogen is relatively high in suckling mice. In contrast, the incorporation into kidney glycogen is low in suckling mice and high in adults. The study indicates the possible utilization of palmitate for glucose synthesis.
A Kinetic Study of the Reaction of Horseradish Peroxidase with Hydrogen Peroxide
Authors: Dolman D. , Newell G. A. , Thurlow M. D. , Dunford H. B.
Published: December 2009

A kinetic study has been carried out over the pH range of 2.63–9.37 for the reaction of horseradish peroxidase with hydrogen peroxide to form compound 1 of the enzyme. Analysis of the results, indicates that there are two kinetic influencing, ionizable groups on the enzyme with pKa values of 3.2 and 3.9. Protonation of these groups results in a decrease in the rate of reaction of the enzyme with H2O2.A previous study of the kinetics of cyanide binding to horseradish peroxidase (Ellis, W. D. &Dunford, H. B.: Biochemistry 7, 2054–2062 (1968)) has been extended down to pH 2.55, and analysis of these results also indicates the presence of two kinetically important ionizable groups on the enzyme with pKa values of 2.9 and 3.9.