bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of carbon sources on the rates of cyclic AMP synthesis, excretion, and degradation, and the ability to produce β-galactosidase in Escherichia coli
Authors: Fraser Ann D. E. , Yamazaki Hiroshi
Published: December 2009

We have determined the rates of adenosine 3′,5′-cyclic monophosphate (cAMP) synthesis, excretion, and degradation, and the cAMP pool size in Escherichia coli grown on various carbon sources. We have found that the cAMP pool size increases in approximate proportion to increases in the cAMP synthetic rate. Although the combined rate of excretion and degradation of cAMP is in approximate proportion to the cAMP pool size, no such regular relationship is seen between the cAMP pool size and either the excretion rate or the degradation rate. Using a method which we have developed for determining the cellular efficiency of enzyme production (termed 'cellular' rate), we have reexamined the relationship between cAMP pool size and the rate of β-galactosidase production. Although there exists an overall trend of increasing rate of β-galactosidase production with increasing cAMP pool size, large variations in the rates of β-galactosidase production are seen even under culture conditions which yield similar cAMP pool sizes. This suggests that the intracellular level of cAMP cannot be the unique regulator of β-galactosidase production.
From β-lipotropin to β-endorphin and 'pro-opio-melanocortin'
Authors: Chrétien M. , Benjannet S. , Gossard F. , Gianoulakis C. , Crine P. , Lis M. , Seidah N. G.
Published: December 2009

Studies on the biosynthesis of β-LPH on the one hand, and of ACTH on the other, have produced a new concept, that of a single precursor form which contains three active molecules. Thus, it is proper to name such a precursor 'pro-opio-melanocortin.' The concept that β-LPH was a precursor molecule was first put forward in 1967 and was based on both structural forms and biological activities. The discovery that morphine-like substances are part of the C-terminal fragment of β-LFH brought an additional important biological side product. That, together with the recent demonstration of ACTH as part of a still larger precursor, constitutes an exciting model for the study of peptide hormone biosynthesis. We have shown unambiguously that β-endorphin is the result of a maturation process from the large precursor, while β-LPH is an important and transient intermediary. Since it is also present in the brain, our recent results using pars intermedia cells can be applied to study the fabrication and degradation of these molecules in the brain. We expect to see it established that all other neuropeptides are also biosynthesized as larger precursor molecules whose structure at the site of cleavage could well be constituted of two basic amino acids like in the pro-opio-melanocortin.
Action of galactose oxidase on galactolipids
Authors: Lingwood C. A.
Published: December 2009

Exposure of galactose-containing glycosphingolipids and certain glycoproteins to galactose oxidase followed by treatment with sodium borotritide has been used to label specifically the terminal galactose and N-acetylgalactosamine moieties of these glycoconjugates. This labelling procedure was shown to be ineffective with a lipid isolated from rat testis, 1-O-palmityl-2-O-palmitoyl-3β-galactosyl-glycerol, under conditions which resulted in excellent labelling of galactosyl-ceramide. Monogalactosyldiacylglycerol was also poorly labelled by this procedure. It was shown that neither 1-O-palmityl-2-O-palmitoyl-3β-(3′-sulfogalactosyl)-glycerol nor sulfogalactosyl-ceramide ("classical sulfatide") was labelled by this procedure indicating that sulfate substitution of the galactose inhibits the action of galactose oxidase. The labelling of galactosyl-ceramide was not inhibited in the presence of the galactoglycerolipid, sulfogalactoglycerolipid, or sulfatide. The property of the lipid aglycone responsible for inhibiting galactose oxidase action remains to be determined.
The effect of membrane cholesterol depletion upon erythrocyte membrane-bound enzymes
Authors: Vickers J. D. , Rathbone M. P.
Published: December 2009

The cholesterol content of normal human erythrocyte membranes (ghosts) was reduced by incubation of the ghosts with phosphatidylcholine (PC) liposomes for 4 h at 37 °C. As controls, ghosts were stored at 4 °C, incubated alone or incubated with PC liposomes saturated with cholesterol; none of these treatments altered membrane cholesterol content. The activities of three membrane-bound enzymes, spectrin kinase, band-3 protein kinase, and phospholipid kinase were reduced in cholesterol-depleted ghosts compared with control ghosts (p < 0.02, p < 0.05, and p < 0.01, respectively). Reductions of the protein kinase activities ranged from 5 to 30% and were apparently related to the extent of the reduction of ghost cholesterol content. The reduction of phospholipid kinase activity was greater (30–60%) and showed no correlation with the extent of reduction of ghost cholesterol content. In contrast to the kinase activities, the activity of a fourth enzyme, NADH: cytochrome c oxidoreductase, increased in response to membrane cholesterol depletion. The relationship between the increase in oxidoreductase activity and cholesterol depletion was best described as linear (r = 0.76).The data demonstrate that these enzyme activities are affected by membrane cholesterol content. Since the alterations in the enzyme activities caused by the artificial alterations of membrane cholesterol content were comparable with those observed in vivo in various pathological conditions, we suggest that the observed changes may be due to mechanisms whereby membrane lipids influence membrane enzyme activities in vivo.
Hepatic 3-hydroxy-3-methyl-glutaryl-CoA reductase response to litter size in suckling rats
Authors: Hahn Peter , Walker Brian L.
Published: December 2009

Rats were raised in litters of 3 or 14 from the 3rd postnatal day. Already, 2 days later on day 5, those raised in groups of three had elevated blood cholesterol and insulin levels and a very much lower hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG CoA) reductase activity, than the other group. When aged 40 days, hepatic HMG CoA reductase activity was higher in the group raised three rats per litter than in the large litter group.
Evolution et controle de l'activité de l'amylo-1,6-glucosidase du foie foetal de rat
Authors: Vaillant R. , Bourgeois-Victor P. , Fairand A.
Published: December 2009

The development of amylo-1,6-glucosidase activity is studied in fetal rat liver. The activity of control fetuses is high on day 17.5, decreases from day 17.5 to day 19.5, and then rises during the next days. In hypophysectomised fetuses, the increase of the activity is suppressed but not the decrease. Moreover, if the mother is adrenalectomized the decrease and the increase are abolished in hypophysectomised fetuses. Growth hormone administration is quite effective in preventing the decrease in enzyme activity but cortisol treatment does not prevent it. In contrast, cortisol produces a precocious decrease of the activity in intact fetuses. These findings suggest that during fetal life, two hormonal regulation mechanisms are involved in the regulation of amylo-1,6-glucosidase activity: cortisol has a repressive effect on the enzymic activity while growth hormone acts as an inducer.
The synthesis of soluble and Insoluble elastin in chicken aorta as a function of development and age. Effect of a high cholesterol diet
Authors: Keeley F. W.
Published: December 2009

The synthesis of soluble elastin, newly synthesized insoluble elastin and total accumulated insoluble elastin was measured in aortic tissue of chickens ranging in age from the 11-day embryo to the adult chicken. Synthesis of soluble elastin reached a maximum in the 1st week after hatching, then decreased rapidly with a second transient increase between 4 and 6 weeks and thereafter decreased continuously until synthesis could no longer be detected in the 35-week-old adult. A portion of this newly synthesized soluble elastin was insolubilized even during 1 h of incubation by a β-aminopropionitrile-inhibited mechanism. Total insoluble elastin accumulated rapidly in aortic tissue in the late embryonic stages and reached a plateau about 1 week after hatching, after which time the proportion of the protein in the tissue remained constant. Synthesis of soluble elastin and total insoluble elastin was also determined in aortic tissue of chickens raised on a diet containing 2% cholesterol for 14 weeks after hatching. This cholesterol-rich diet had an early, but transient effect on the synthesis of soluble elastin, shifting the age of maximal synthesis to 1 to 2 weeks after hatching. However, by 6 to 8 weeks on the diet there was no difference in soluble elastin synthesis between normal and cholesterol-fed groups. Although prolonged cholesterol feeding resulted in serum cholesterol levels 10 times normal, aortic tissue cholesterol levels 3 times normal and grossly visible atherosclerotic lesions, no reinitiation of soluble elastin synthesis or alteration in the quantity and character of insoluble aortic elastin could be detected.
Phosphorescence evidence for the role of solvent–protein interactions in the energetics of conformational flexibility of liver alcohol dehydrogenase
Authors: Kishner Stephen , Trepman Elly , Galley William C.
Published: December 2009

When tryptophyl side chains are hidden within relatively inflexible domains of globular proteins, the lifetime of the phosphorescence from these residues provides a measure of the local conformational flexibility. The phosphorescence decay from the tryptophan buried at the base of the nucleotide-binding domain in liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was monitored between 1 and 40 °C to determine the energetics associated with the rate of local unfolding. The slow rate at which this process takes place is found to result from a high entropic barrier rather than from the disruption of strong intramolecular interactions. This observation along with the response of the system to solvent perturbations points to the significance of solvent–protein interactions in determining conformational flexibility.
The properties of purified low molecular weight phosphoprotein phosphatase from rabbit heart
Authors: Khandelwal Ramji L.
Published: December 2009

A simplified procedure for the purification of low molecular weight phosphoprotein phosphatase acting on muscle phosphorylase a has been described from rabbit heart. The enzyme was purified to homogeneity by acid precipitation, ethanol treatment, and chromatography on Sephadex G-75 and Sepharose–histone. The purified enzyme showed a single band when examined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis; the molecular weight calculated by this method was 34 000. The S20,W value and Stokes radius for the enzyme was 3.35 and 24.0 Å (1 Å = 0.1 nm), respectively. Using these two values, a molecular weight of 35 000 was calculated. Purified enzyme showed a wide substrate specificity and catalyzed the dephosphorylation of phosphorylase a, glycogen synthase D, phosphorylated histone, and phosphorylated casein. Kinetic studies revealed the lowest Km with glycogen synthase D and maximum Vmax for the reaction with phosphorylase a.
Analysis of a 5S RNA–protein complex isolated from the ribosomes of rye embryos
Authors: Dyer Tristan A. , Zalik Saul
Published: December 2009

Rye embryo ribosomes were dissociated into subunits and the large subunit fraction was treated with formamide. A low molecular weight complex of RNA and protein (RNP) was released. Electrophoresis of the RNP in polyacrylamide gels containing sodium dodecyl sulphate yielded an RNA band and a single protein band. The protein had a molecular weight of approximately 41 000 and the RNA of the complex was shown to be 5S ribosomal RNA.Embryos were germinated in the presence of [32P]orthophosphate and the labelled RNP was isolated from their ribosomes. The RNA component was partially digested with pancreatic A ribonuclease and the parts protected from degradation by the protein were determined by sequence analysis. Although the whole 5S RNA molecule was shielded to some extent, the portion most protected was between nucleotides 68 and 108. This is, therefore, probably the part of plant cytosol 5S RNA which is primarily involved in the interaction with protein in the complex and possibly in the ribosome as well.
Selective spin-labeling of the ribosomal proteins of 70S ribosomes from Escherichia coli
Authors: Dugas H. , Rodriguez A. , Brisson N.
Published: December 2009

We have used a series of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) maleimide spin labels of different length to label, covalently and selectively, the most reactive sulfhydryl groups of 70S ribosomal proteins of Escherichia coli. Under short periods of labeling (1–2 min), less than two spin labels per ribosome are incorporated and were shown to be distributed mainly on five ribosomal proteins in the following order: S18 > S21, L27 > S17, and S12. With a long period of labeling (3 h) up to 13 spin labels are attached to the ribosome, and protein S1 is the most labeled. The shape of the electron paramagnetic resonance (epr) signal shows two components with a predominance for the strongly immobilized orientation, and the percentage of these components in each spectra has been evaluated. When the distance between the nitroxide group and the maleimide-attaching group exceeds 6 Å (1 Å = 0.1 nm) the strongly immobilized orientation disappears. The effect of magnesium ions on these selectively spin-labeled ribosomes shows that the dissociation into subunits does not affect the epr signal, but more spin labels are incorporated into the subunits if labeling is performed under conditions of dissociation.
β-D-Xylosidase from Penicillium wortmanni: binding and hydrolysis of alkyl and aryl 1-oxygen and 1-thio-β-D-xylopyranosides
Authors: Deleyn F. , Claeyssens M. , Bruyne C. K. De
Published: December 2009

The influence of the chain length in n-alkyl β-D-xylopyranosides and of the para substituents in aryl β-D-xylopyranosides on the kinetic parameters (Ka, V) for hydrolysis of these substrates by a β-D-xylosidase from Penicillium wortmanni, has been investigated. Binding of the corresponding 1-thio derivatives as competitive inhibitors (Ki) was studied for comparative purposes. For the n-alkyl substrates, a slight dependency of V on the chain length is noted, whereas the aryl β-D-xylopyranosides show nearly constant V values. The influence of the aglycon on Ka and Ki values is complex; for the n-alkyl derivatives the contribution of the hydrophobicity of the aliphatic chain seems predominant, although steric factors cannot be neglected. These results, together with previous observations, can tentatively be interpreted in terms of a double-displacement mechanism.
Estrone β-glucosidase activity in human placenta
Authors: Labow Rosalind S. , Williamson Denis G. , Layne Donald S.
Published: December 2009

The 105 000g supernatant from human placental homogenates, prepared in the presence of sodium taurocholate and Cutscum, contained β-glucosidase activity towards estrone glucoside as well as towards 4-methylumbelliferyl glucoside (4-MU-glucoside) and glucocerebroside. After partial purification, the estrone glucosidase was found to be active only after the addition of negatively charged phospholipid, whereas the other β-glucosidases did not exhibit this requirement. The estrone glucosidase was separated from the 4-MU-glucosidase by chromatography on Sephadex G-200 with 0.1% sodium taurocholate in the eluting buffer. The estrone glucosidase was mainly contained in material with a pi of 4.7, while the 4-MU-glucosidase was distributed in fractions with pi values of 4.7 and 6.2 to 6.4. The partially purified estrone glucosidase had a pH optimum of 5.8, as distinct from that of 6.4 found for the 4-MU-glucosidase, and differed markedly from the 4-MU-glucosidase in its response to treatment with heat, sulfhydryl reagents, and detergents. Its sensitivity to changes in pH differed from those reported for glucocerebrosidase.
Effect of phosphatidylinositol and phosphatidylserine on membrane-bound galactosyltransferase
Authors: Ratnam Samuel , Fraser Ian H. , Mookerjea Sailen
Published: December 2009

Membrane-bound galactosyltransferase is solubilized and activated by exogenous lysolecithin or Triton X-100. A study on the effect of different phospholipids on the lysolecithin-solubilized enzyme showed that two charged phospholipids (i.e., phosphatidylinositol and phosphatidyl-serine) inhibited the membrane-bound enzyme in the presence of a wide range of lysolecithin concentration (up to 6 μmol/mg protein). In contrast, these phospholipids produced a biphasic effect on the enzyme solubilized with Triton X-100. In lower concentration of Triton (up to 2 μmol/mg protein), the charged phospholipids somewhat reduced the enzyme activity but a reversal of this effect was observed when Triton concentration was gradually raised (from 2 to 8 μmol/mg protein). This biphasic effect of the phospholipids was also demonstrated on purified membrane-bound galactosyltransferase in presence of low and high concentration of Triton. Electron microscopic evidence suggested that an increased concentration of phosphatidylinositol prevented membrane solubilization by lysolecithin or retained the membrane vesicular organization concurrent with a restraining effect on the enzyme. The results lend support to the hypothesis that the phospholipid microenvironment of the membrane may exert a control on the membrane-bound glycosyltransferases.
Rearrangements and reconstitution of mononucleosomes as monitored by thermal denaturation measurements
Authors: Chiu Susan S. , Lee Kay P. , Lewis Peter N.
Published: December 2009

Derivative melting profiles of calf thymus mononucleosomes have been examined for changes resulting from variations in solvent pH and ionic strength, histone H1 content, and DNA size. Samples of mononucleosomes were found to rearrange during freeze-drying to form an altered monomer and a series of noncovalent multimers. The derivative melting profiles of these particles differ significantly from those for the untreated monomer and dimer. The noncovalent dimer exhibited a new melting transition at 66 °C involving approximately 18 base pairs of DNA normally associated with the highest melting transition. Mononucleosomes were reconstituted from 6 M guanidine hydrochloride to give particles with physical properties including melting profile which were virtually indistinguishable from those of the starting material. This result confirms the notion that no structural domains exist in the histone core that can be irreversibly denatured by noncovalent perturbations.
Metabolism and positional distribution of isomers of 18-carbon dienoic fatty acids containing trans and cis double bonds by developing rat brain
Authors: Cook Harold W.
Published: December 2009

In addition to dietary linoleic acid (cis,cis-18:2), trans isomers of 18:2 may be ingested as minor components of partially hydrogenated oils. Developing rat brain has significant capacity for conversion of cis,cis-18:2 to more abundant polyenes by desaturation and elongation, but less is known about trans dienes. We have shown that incorporation of 1-14C-labeled cis,cis-, cis,trans-, and trans,trans-18:2 into brain lipids of 10-day-old rats is significant but that metabolism of the isomers between 0.2 and 72 h after intracerebral injection differs markedly. Incorporation of trans,trans-18:2 into phosphoglyceride and triacylglycerol was slower than that for cis,cis diene. Oxidation of all isomers occurred with formation of similar quantities of labeled cholesterol and palmitate. However, metabolism of trans dienes differed in two important respects: (i) desaturation and elongation of trans-containing diene was much less (< 5 versus > 30% for cis,cis-18:2 after 6 h); (ii) trans,trans-18:2 and palmitate appeared to be preferentially esterified to the 1 position, whereas cis,cis-18:2 and its products were mainly in the 2 position of choline and ethanolamine phosphoglycerides. Thus, developing brain does not appear to ensure exclusion of trans dienes from membrane lipids; however, conversion by desaturation and elongation and selectivity for position of acylation differ markedly for the cis- and trans-containing dienoic isomers.
The subcellular localization of testicular sulfogalactoglycerolipid
Authors: Klugerman Abbey , Kornblatt Mary Judith
Published: December 2009

The subcellular localization of sulfogalactoglycerolipid in rat testicular germinal cells was determined. The sulfolipid of young rats was labelled in vivo with Na235SO4. Rat testis cell suspensions were prepared, homogenized, and centrifuged on linear, continuous, sucrose gradients. The labelled lipid had the identical equilibrium density distribution pattern as alkaline phosphatase, an enzyme of the plasma membrane. The pattern of the sulfolipid was different from the patterns of enzyme markers for the Golgi apparatus, lysosomes, mitochondria, and endoplasmic reticulum. From these results, we conclude that sulfogalactoglycerolipid is located on the plasma membrane of rat testicular germinal cells.
Fractionation of large glycopeptides of human teratocarcinoma-derived cells by concanavalin A – Sepharose chromatography
Authors: Rasilo Maija-Liisa
Published: December 2009

Human teratocarcinoma derived cells, line PA 1, were labeled with radioactive monosaccharides and subsequently digested with pronase. Large sized glycopeptides (fraction A) were isolated by gel filtration on Bio-Gel P-10. Their chromatography on concanavalin A – Sepharose gave three subfractions, two of which were eluted with a sugar-free buffer and the third with 10 mM α-methyl mannoside. The first subfraction (fraction A – Con A Ia) incorporated label from [3H]galactose and [3H]glucosamine and contained the largest components of fraction A. The second and the third subfractions (fractions A – Con A Ib and A – Con A II) were glycopeptides which incorporated label from tritiated fucose, mannose, galactose, and glucosamine. Even these molecules were of large size eluting partially at the void volume from Bio-Gel P-60. The glycopeptides of fraction A – Con A Ib contained mannose, fucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Fucose and galactose residues occupied ultimate or penultimate positions at the nonreducing termini of the oligosaccharides. N-Acetyl-neuraminic acid, too, was present in the glycopeptides of fraction A.
Studies on the in vivo uptake and incorporation of 1-14C-labeled D-hexosamines in tissues, mucopolysaccharide–peptide complexes, and glycosaminoglycans of the domestic fowl
Authors: Kwai Hang Kwet Fane Ng , Anastassiadis Phoebus A.
Published: December 2009

The incorporation of radioactivity from D-[1-14C]glucosamine and D-[1-l4C]galactosamine into the tissues of skin, comb, wattle, liver, kidney, spleen, lung, heart, ovaries, egg yolk, infundibulum, magnum, isthmus, shell gland, and vagina of the White Leghorn laying hens was studied for different time intervals up to 120 h. A total of 28 laying hens were involved in this experimentation. The radioactivity was measured in the above whole tissue, the acetone extracts, the acetone-extracted tissues, the mucopolysaccharide–peptide complex (MPS-P), and in individual glycosaminoglycan (GAG) isolated from skin, comb, liver, kidney, and egg yolk. The radioactivities of the expired CO2 of the eggs and of the excreted urine and faeces were also measured in some cases. There was an increase in 14C associated with MPS-P with increasing time of experiment in all the tissues studied except in the case of the liver. The rate of increase of radioactivity was dependent on the tissue. A useful scheme for the study of the speed of the formation of GAG is to consider the acetone extract and the acetone-extracted tissue as macrocompartments from where the GAG draw hexosamines for their formation.
Reticulocyte membrane transferrin receptors
Authors: Ecarot-Charrier B. , Grey V. L. , Wilczynska A. , Schulman H. M.
Published: December 2009

A membrane protein with specific transferrin binding activity has been isolated from rabbit reticulocytes. The isolation procedure involved the immunoprecipitation by antibody to transferrin of transferring–receptor complexes from reticulocyte membrane proteins which had been solubilized with nonionic detergent. Receptor dissociated from the antibody–transferring–receptor complexes could bind transferrin saturably and reversibly. It migrated electrophoretically as a single band of glycoprotein with an estimated molecular weight of approximately 180 000 which was reduced to around 93 000 following complete dissociation with dithiothreitol.
Evidence for multiple forms of p-trifluoromethylbenzenesulfonyl-α-chymotrypsin
Authors: Ando M. E. , Gerig J. T. , Luk K. F. S. , Roe D. C.
Published: December 2009

p-Trifluoromethylbenzenesulfonyl-α-chymotrypsin, an analog of tosylchymotrypsin, has been prepared and shown to be stable enough to permit fluorine nuclear magnetic resonance experiments. Up to four distinct trifluoromethyl resonances can be observed for the modified protein at 94.1 MHz even when the enzyme derivative is prepared from protein which has been purified by several methods. The resonances observed appear to represent proteins which are grossly similar as regards molecular size and the ability to bind and hydrolyze substrates, but nonetheless distinctive enough in the active-site region to produce appreciable chemical-shift effects.
Jack bean urease (EC 3.5.1.5). II. The relationship between nickel, enzymatic activity, and the "abnormal" ultraviolet spectrum. The nickel content of jack beans
Authors: Dixon Nicholas E. , Gazzola Carlo , Asher Colin J. , Lee Dennis S. W. , Blakeley Robert L. , Zerner Burt
Published: December 2009

At low pH, EDTA promotes the loss of the tightly bound nickel ions from jack bean urease. The specific activity of soluble enzyme after partial EDTA-promoted inactivation is a linear function of the nickel content. The results are consistent with the presence of 2.0 nickel ions per 97 000-dalton subunit in pure urease. The time scale for loss of enzymatic activity and nickel under these conditions is similar to that for loss of the "abnormal" tail absorption in the ultraviolet and visible absorption spectrum of urease (including the shoulder at ~420 nm). This indicates that nickel in urease is essential for enzymatic activity and establishes that the metal ions are in part responsible for the tail absorption in the ultraviolet spectrum of urease. After partial inactivation in the presence of EDTA either at low pH or in 2.5 M guanidinium chloride at neutral pH, urease did not regain activity in the presence of Ni2+. As yet apourease has not been produced reversibly. Jack bean seeds grown hydroponically without added nickel were low in both urease activity and nickel (10 and 6%, respectively, of parent seeds). Several other metal ions were readily available. This result suggests that metal ions other than nickel cannot substitute for nickel in the formation of normally active urease.
Preparation of some long-chain N-acyl derivatives of essential amino acids for nutritional studies
Authors: Paquet Alenka
Published: December 2009

Long-chain N-acyl derivatives of methionine, tryptophan, threonine, and lysine (N6) have been obtained by the reaction of succinimidyl esters of fatty acids with the unprotected amino acids. Their physical properties have been characterized.
Determination of bioavailability of some long-chain N-substituted derivatives of L-methionine and L-lysine
Authors: Paquet Alenka , Sarwar Ghulam
Published: December 2009

The bioavailability of N-acyl-L-methionine derivatives has been determined using microbiological assay with Tetrahymena pyriformis. It was found that palmitoyl- and stearoyl-L-methionine, stearoyl-L-methionine ethyl ester, and stearoyl-L-methionine sodium salt were partially utilized (14–38%) for growth of the microorganism. These compounds partially inhibited utilization of free methionine added to the media. The shorter derivatives, acetyl-, hexanoyl-, lauroyl-, and myristoyl-L-methionine completely inhibited the growth of T. pyriformis. This effect was not reversed when DL-methionine was added to the media. N6-fatty acyl L-lysine derivatives gave low availability values (3–18%) in microbiological assessment with T. pyriformis. N2-Acetyl- and N6-acetyl-lysine did not inhibit the utilization of the added parent amino acid.Nutritional evaluation of L-methionine derivatives by the rat growth method using net protein ratio (NPR) as the performance index indicated complete availability of stearoyl-L-methionine, stearoyl-L-methionine sodium salt, and partial availability of stearoyl-L-methionine ethyl ester (52%), stearoyl-L-methionylglycine (32%), and lauroyl-L-methionine (75%).
Renal glutamine utilization: glycylglycine stimulation of γ-glutamyltransferase
Authors: Welbourne T. C. , Dass P. D. , Smith R. L.
Published: December 2009

Glycylglycine stimulation of renal glutamine utilization was studied on the homogenate, subcellular and purified enzyme level. The results clearly establish the existence of two glutamine utilizing pathways, the mitochondrial dependent L-glutamine amidohydrolase (PDG) and a second, extramitochondrial pathway. In contrast to the mitochondrial pathway which produces stoichiometric amounts of ammonia and glutamate, this second pathway hydrolyzes glutamine to produce ammonia and transfers the γ-glutamyl moiety, producing γ-glutamyl peptides. In the crude systems, containing cyclotransferase, the γ-glutamyl moiety appears mainly as 5-oxoproline; however, in the enzyme preparation, purified 112-fold, γ-glutamyl peptides (transpeptidation) and a small amount of glutamate (hydrolysis) appear. D-Glutamine was also hydrolyzed, in contrast to the stereospecific PDG, but at less than one-half the rate of the L-isomer. The molecular weight of this extramitochondrial D- and L-glutamine utilizing enzyme was estimated by gel filtration on a Sephadex G-200 column and found to be ≈70 000. Based on product formation, molecular weight estimation and copurification with the activity responsible for p-nitroanilide release from γ-glutamyl-p-nitroanilide, we conclude that this reaction is catalyzed by γ-glutamyltranspeptidase. Glycylglycine stimulated this enzyme to produce more ammonia while decreasing the appearance of glutamate; in contrast, the mitochondrial glutaminase was unaffected by glycylglycine. This extramitochondrial glutamine utilizing pathway can make a significant contribution to in vivo renal ammoniagenesis.
Amino group reactions of the sulfhydryl reagent methyl methanesulfonothioate. Inactivation of D-3-hydroxybutyrate dehydrogenase and reaction with amines in water
Authors: Kluger Ronald , Tsui Wing-Cheong
Published: December 2009

Methyl methanesulfonothioate (MMTS) has been used as a sulfhydryl reagent. However, it inactivates D-3-hydroxybutyrate dehydrogenase in a manner that suggests that an amino group may have reacted with the reagent. Nuclear magnetic resonance (NMR) analysis of solutions of glycylglycine, glycine ethyl ester, and imidazole with MMTS indicates that a reaction occurs which produces what is identified by its NMR signal as the methylsulfenamide of the amine. It is suggested that caution should be used when MMTS is employed where reaction with an amine could give an undesired product.
The effect of temperature on some calcium-binding properties of troponin C and calmodulin
Authors: McCubbin William D. , Hincke Maxwell T. , Kay Cyril M.
Published: December 2009

Some calcium-binding properties of skeletal and cardiac troponin C (TnC) have been measured as functions of temperature employing several physical and spectroscopic techniques. The degree of exposure of the tyrosine residues in brain calmodulin has also been determined by a new approach. Circular dichroism thermal unfolding profiles have been established for the three cases: metal-free protein, high-affinity sites filled, and fully saturated. In addition some thermodynamic parameters have been calculated for these reversible melting processes. It was found that the calcium-binding parameters n and K, where n is the fraction of the total conformational change and K is the apparent association constant, for both skeletal and cardiac TnC, did not vary significantly over the temperature range 10–38 °C, but at 50 °C differences became quite apparent, dramatically so in the case of the skeletal protein. The technique of thermal perturbation difference spectroscopy was applied to determine the degree of exposure of aromatic chromophores for the TnC(s) and calmodulin in the absence and presence of calcium. For skeletal TnC and calmodulin the results were in good agreement with previous observations, but the reduced degree of exposure of the tyrosine residues in cardiac TnC, in the absence of Ca2+, was contrary to the earlier work. Calcium-induced difference absorption spectra have been measured for the TnC(s) over the temperature range 10–70 °C. Cardiac TnC showed greater heat stability than its skeletal counterpart, in terms of the rate and the amount of change of the difference spectral maxima.
Modification of hemoglobin upon covalent coupling to dextran: enhanced stability against acid denaturation and reduced affinity for haptoglobin
Authors: Tam Siu-Cheung , Wong J. Tze-Fei
Published: December 2009

Alkylation of human hemoglobin by bromoacetylaminoethylamino-substituted dextran gave rise to a covalent dextran–hemoglobin complex with enhanced stability against acid denaturation, and reduced affinity for binding to haptoglobin, compared with free hemoglobin. These effects increased with increasing size of the dextran moiety and were not elicited by either free dextran or alkylation of hemoglobin by iodoacetamide. These observations are consistent with an inhibition by covalently attached dextran of the binding of β-subunit of hemoglobin to haptoglobin.
Enhanced sensitivity of tumorigenic cells to rapid rounding induced by phenylalaninol
Authors: Mak W. Wai-Nam , Pinteric Leslie , Wong J. Tze-Fei
Published: December 2009

This study describes a rounding reaction induced in mammalian cells by the addition of phenylalaninol. In the Chinese hamster ovary tsH1 line the rounding occurred rapidly with a half time of 1 min at 25 mM phenylalaninol. After the removal of phenylalaninol, the rounding was reversed, leading to the reflattening of the cells with a half-time of 3.5 min. Rounding was inhibited by dibutyryl-cAMP and testosterone, and reflattening by cytochalasin B. Either in the case of the tsH1 line and its growth-control revertant GRC+L-73, or in the case of SV40-transformed and untransformed human WI-38 cells, the transformed cells displayed a weakened resistance toward rounding. Likewise, rat cells transformed by the highly oncogenic adenovirus-12 were more sensitive to rounding than cells transformed by the poorly oncogenic adenovirus-5, which in turn were more sensitive than untransformed cells. However, drug-resistant cell-surface mutants of the Chinese hamster ovary GAT− line also exhibited an altered sensitivity to rounding. These findings suggest that more than one cellular component determines cellular sensitivity to phenylalaninol-induced rounding. One of these components is specifically altered, giving rise to an enhanced sensitivity, in the course of tumorigenic transformation.
Intermolecular hydrogen bonding between lipids: influence on organization and function of lipids in membranes
Authors: Boggs Joan M.
Published: December 2009

Biological membranes have unique lipid compositions suggesting a specific role for many lipids. Evidence is reviewed concerning the intermolecular forces between glycero- and sphingolipids and cholesterol, the dependence of many of these interactions on the state of ionization of lipids, pH, ionic strength, and divalent cation concentration. The effect of intermolecular interactions between certain lipids on lipid clustering, interaction with cholesterol, on the conformation of proteins, and on transitions to the hexagonal phase is considered. Other forces which cause lipid phase separation or clustering are discussed. It is concluded that lipids are in dynamic equilibrium with their environment and can act as receptors for certain intra- or extracellular stimuli, which they can translate into a response by undergoing changes in fluidity, phase transitions, or phase separation.
Membrane alterations in acute myocardial ischemia
Authors: Godin David V. , Tuchek John M. , Moore Margo
Published: December 2009

The molecular consequences of acute myocardial ischemia induced in rabbit hearts by ligation of the left circumflex branch of the coronary artery were assessed in terms of the biochemical properties of subcellular organelles. Mitochondrial alteration, as reflected in progressive decrease in the activity of azide-sensitive ATPase, was apparent as early as 5 min postligation, but the activity of another mitochondrial enzyme, cytochrome c oxidase, was unchanged, even following 60 min of coronary ligation. Sarcolemmal Na+,K+-ATPase exhibited a time course of inactivation similar to that of the mitochondrial ATPase, but differed from the latter in that the impairment was not reversed on reperfusion. Cellular levels of ATP, which decreased in parallel with the loss of ATPase activities, also remained depressed following reperfusion. Decreases in lysosomal enzyme latency were noted, but these occurred somewhat later than the sarcolemmal and mitochondrial alterations. Attempts to demonstrate the production of a population of labile lysosomal structures during ischemia were unsuccessful. Similarly, no alterations in the gel electrophoretic profiles of proteins or in the Pphosphatidylcholine/Pphosphatidylethanolamine ratio of isolated mitochondrial or sarcolemmal membranes from hearts subjected to ischemia and (or) subsequent reperfusion could be found. It is suggested that sarcolemmal Na+,K+-ATPase may serve as a sensitive and readily quantifiable index of irreversible cellular necrosis and, therefore, be of value in assessing the possible beneficial effects of pharmacological interventions.
pH-induced alterations in the structure and activity of cytochrome c oxidase
Authors: Kornblatt Jack A.
Published: December 2009

The effects of pH on the activity and structure of beef heart cytochrome c oxidase have been studied in the pH range 5.0–7.6. (i) A group with pK of approximately 5.45 has been readily detected in the pH vs. activity curve. This group must be deprotonated to achieve maximal activity. (ii) A group with a similar pK (5.45) has been detected and contributes to the spectral character of the reduced oxidase. Over the range pH 5.0–7.6 no other acid-sensitive group contributes to the spectrum of the reduced oxidase. (iii) The oxidized oxidase shows at least three acid-sensitive groups contributing to the spectrum. One occurs in the pH 7 range and another in the pH 5.6 range; below pH 5.2 additional pH-sensitive groups are apparent. Accurate estimation of the pK's of the groups responsible for the spectral changes in the oxidized oxidase has not been possible. (iv) The spectrum of the "oxygenated" (428 nm) conformer of the oxidized protein is invariant over the range pH 5.5–7. (v) The changes occurring in the spectrum of the purified oxidase also occur in the protein contained in phospholipid vesicles. (vi) The data are discussed in terms of the mechanism by which the oxidase, during its in situ catalytic cycle, may give rise to the primary events in energy coupling.
Differential scanning calorimetric studies of the thermotropic phase behaviour of membranes composed of dipalmitoyllecithin and mixed-acid unsaturated lecithins
Authors: Davis P. J. , Coolbear K. P. , Keough K. M. W.
Published: December 2009

The lecithins 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) have been synthesized by reacylation of the appropriate lysolecithins with fatty acid anhydrides. These lecithins have been used to make model membranes in mixtures with dipalmitoyllecithin (DPPC), and phase diagrams of the two bilayer systems have been constructed. These diagrams show that there is essentially no gel-state miscibility in the POPC–DPPC bilayers at any composition, and that SOPC–DPPC bilayers show gel-state immiscibility at DPPC concentrations of less than 50 mol%, and partial miscibility above 50 mol% DPPC. Analysis of the POPC–DPPC phase diagram on the assumption of athermal solution in the liquid-crystalline phase shows that the two lipids mix nearly randomly above the phase transition. The liquidus curve of SOPC–DPPC bilayers showed deviations from calculated ideal behaviour, which indicated that there is a small excess tendency for the formation of pairs of like molecules in SOPC–DPPC bilayers in the liquid-crystalline phase. Thus, in the liquid-crystalline phase, SOPC and DPPC do not pack quite as well as do POPC and DPPC.
Purification and stabilization of galactosyl transferase from serum and lysolecithin extracted microsomes
Authors: Fraser Ian H. , Wadden Patricia , Mookerjea Sailen
Published: December 2009

Rat liver microsomes treated with increasing concentrations of lysolecithin released, after a brief lag, progressively increasing amounts of UDPgalactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. A second extraction of the microsomes with lysolecithin (8 mM) resulted in a total release of about 45% enzyme. The specific activity of the enzyme in the second extract was 12 times higher than that of the first extract. The galactosyl-transferase present in the extract was purified 417-fold by an affinity column chromatographic technique using a column of activated Sepharose 4B coupled with α-lactalbumin. During purification, the column and elution buffers required 0.1% lysolecithin to keep the enzyme in active form. For purposes of comparison the soluble serum galactosyltransferase was also purified by identical techniques, which also required 0.1% lysolecithin in column and elution buffers to prevent the loss of enzyme activity. The pure serum and membrane galactosyltransferase contained no sialyltransferase and ran as a double band on polyacrylamide gels (molecular weight 63 000–64 000). The pure enzyme had an absolute requirement for Mn2+, not replaceable by Cu2+, Mg2+, Zn2+, and Co2+. The enzymes were active over a wide pH range, with optimum pH of 6.5. The apparent Km's for UDPgalactose for the serum and membrane enzymes were 12.05 and 11.8 μM, respectively. The specific activities of these two purified enzymes were also remarkably similar, 3.99 × 106 for serum and 3.84 × 106 for membrane enzyme. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose (DSG-fetuin). The enzyme activity with DSG-fetuin acceptor was inhibited to a lesser extent by α-lactalbumin.Effect of various additives on the stability of purified membrane and serum galactosyltransferase was studied at 0–5 °C and at −20 °C up to 60 days. At both temperatures, albumin was found to be the best stabilizer. Ammonium sulfate was a good stabilizer for the serum but not for the membrane enzyme. Glycerol showed some stabilizing effect for both enzymes. EDTA, p-methylbenzenesulfonyl fluoride, N-acetylglucosamine-Mn2+, and water did not offer any stabilization of the pure enzyme.
The PO glycoprotein of peripheral nerve myelin
Authors: Ishaque A. , Roomi M. W. , Szymanska I. , Kowalski S. , Eylar E. H.
Published: December 2009

The PO glycoprotein, the major protein of peripheral nerve myelin, is a hydrophobic glycoprotein which can be isolated in soluble and insoluble forms from rabbit sciatic nerve myelin following extensive defatting and mild acidic extraction. The PO glycoprotein was localized exclusively in peripheral nervous system (PNS) myelin of sciatic nerve and rootlets by the immunofluorescent technique using goat anti-PO serum which showed a single precipitin band in double diffusion and did not cross-react with the myelin basic protein or P2 protein. Central nervous system (CNS) myelin from brain and spinal cord was negative by the immunofluorescent procedure. The major glycoprotein bands in PNS myelin, in addition to the PO glycoprotein at 28K, exist at 23K and 19K, as shown by gel electrophoresis in dodecyl sulfate. These glycoproteins, isolated by gel filtration in 2% dodecyl sulfate, show identity to the PO glycoprotein in their monosaccharide profile and overlapping tryptic peptides on peptide mapping. We conclude that both the 23K and 19K glycoproteins are derived from the PO glycoprotein by in situ proteolysis; the 23K glycoprotein has the identical amino terminal sequence. The 19K glycoprotein, beginning with amino-terminal methionine, is identical with the TPO glycoprotein, shown previously to originate from tryptic hydrolysis of the PO glycoprotein in isolated myelin. A tryptic glycopeptide containing 27 amino acids was isolated from the PO glycoprotein and sequenced. It contained a relatively high proportion of aspartic acid (four residues) and glutamic acid (two residues), thus exhibiting a high negative charge. We conclude that the total carbohydrate of the PO, 23K, and 19K glycoproteins does indeed exist as a single nonasaccharide moiety linked through N-acetylglucosamine to Asp-14 of the glycopeptide in a N-glycosidic linkage. These results further support the role of the PO glycoprotein as a typical amphipathic membrane protein.
Regulation by calmodulin of the calcium affinity of the calcium-transport ATPase in human erythrocytes
Authors: Roufogalis B. D. , Mauldin D.
Published: December 2009

The Ca2+ affinity of (Mg2+ + Ca2+)-ATPase in human red blood cells is regulated by a number of intracellular factors, including the association of the enzyme with the cytosolic Ca2+ binding protein, calmodulin. Ghosts prepared by hypotonic lysis in the presence of 0.1 mM CaCl2, or by a gradual stepwise hemolysis procedure, contain an EDTA-extractable protein whose effects are mimicked by calmodulin, whereas ghosts prepared by extensive washes in the absence of added CaCl2 lack calmodulin and contain only a high molecular weight heat stable activator. Purified calmodulin from human red cells or bovine brain shifts the apparent Ca2+ affinity of (Mg2+ + Ca2+)-ATPase activity in extensively washed ghosts to a high Ca2+ affinity state. The shift was most apparent in ghosts in which the Ca2+ affinity was decreased by EDTA treatment. Calmodulin increased the velocity of (Mg2+ + Ca2+)-ATPase in the EDTA-treated ghosts about 36-fold at a low (1.4 μM) Ca2+ concentration, compared with 6-fold before EDTA treatment. The maximum shift in apparent Ca2+ affinity occurred only in the presence of saturating concentrations of calmodulin. It is concluded that red cell calmodulin confers to the Ca2+ transport ATPase the ability to increase its apparent Ca2+ affinity, as well as its maximum velocity, in response to increases in intracellular Ca2+.