bluuebunny/crossref_metadata_2025 huggingface dataset:
The circular dichroic spectra of cytochrome c oxidase in the visible-wavelength region
Authors: Yong Fook-Choy
Published: December 2009

Cytochrome oxidase purified from beef heart shows distinct visible circular dichroic spectra which are oxidation- and ligand-state dependent. The oxidized enzyme and its cyanide complex exhibit broad negative extrema at 570 nm ([θ] = −4 × 103 degrees-cm2∙dmol−1) and 620 nm. A single Cotton effect was resolved for each of these extrema, except in the case of the cyanide complex where two Cotton effects (558.9 and 572.4 nm) were located within the 570-nm extremum.The reduced enzyme displays a major positive α-elliptic band at 608 nm and minor ones at 570 and 518 nm in accordance with its absorption spectrum. Carbon monoxide and cyanide cause the α-elliptic band of the reduced enzyme to shift to 604 nm ([θ] = 26 × 103 degrees-cm2∙dmol−1) and 602 nm ([θ] = 33 × 103 degrees-cm2∙dmol−1) respectively. Computer-assisted resolution of these three spectra suggests a common profile whereby two major transitions (Cotton effects II and III) of about equal rotational strength constitute the main α-elliptic band observed between 602 and 608 nm. Carbon monoxide induces Cotton effect II of the reduced enzyme to shift from 612.6 to 601.6 nm but not Cotton effect III at 608.4 nm. In contrast, cyanide alters the band position, band width, amplitudes, and the rotational strength of these two. These results permit identifying Cotton effect II as arising from the heme chromophore of cytochrome a3, and Cotton effect III from cytochrome a. Carbon monoxide also induces a red shift to the β-elliptic band at 520 nm of the reduced enzyme. On the other hand, cyanide causes a blue shift to this same band. The dependency of this elliptic band upon the extrinsic ligand of cytochrome a3 is consistent with the charge transfer nature of this band.
A DNA endonuclease isolated from yeast nuclear extract
Authors: Bryant Douglas W. , Haynes Robert H.
Published: December 2009

We have isolated and partially purified a DNA endonuclease from nuclei of the yeast Saccharomyces cerevisiae. Although purified on the basis of its ability to degrade denatured DNA, the enzyme can also attack native DNA. Denatured oligonucleotide products of the enzyme are sensitive to venom phosphodiesterase (EC 3.1.4.1) but not to bovine spleen phosphodiesterase (EC 3.1.4.18). The enzyme has an estimated molecular weight of 6.6–7.5 × 104, more than twice as large as the endonucleases involved in DNA repair in Escherichia coli.When analyzed on glycerol gradients, the endonuclease sedimented as a single activity against both denatured DNA and closed circular DNA duplexes. The enzyme showed a 10-fold preference for denatured over native T7 DNA substrate, and appears to produce random nicks in a supercoiled replicative form of [Formula: see text] DNA (RFI) with no discernable preference for the unpaired bases in the supercoiled duplex. The endonuclease appears to be distinct from the yeast endonucleases previously described.
Wheat embryo ribonucleates. X. Metabolism of 3′-hydroxyl termini in the conserved mRNA and tRNA of imbibing wheat embryos
Authors: Kennedy T. D. , Lane B. G.
Published: December 2009

There are conserved complements of ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA) in dry wheat embryos. Although early labelling of RNA is largely directed toward the synthesis of complete molecules of nascent rRNA and mRNA, there is also selective labelling at 3′-hydroxyl termini in conserved polynucleotides when dry wheat embryos are subjected to short-term (0.5 h) imbibition in a medium that contains tritium-labelled adenosine, guanosine, cytidine, and uridine. Conserved tRNA is the principal mass component in NaCl-soluble RNA (sRNA) and most of the 'rapid labelling' of sRNA (rl-sRNA) is a result of labelling at 3′-hydroxyl termini in conserved tRNA. By contrast, although conserved rRNA is the principal mass component in NaCl-insoluble RNA (iRNA), most of the labelled 3′-hydroxyl termini in 'rapidly labelled' iRNA (rl-iRNA) do not appear to derive from rRNA. Although about 10% of the labelled 3′-hydroxyl termini in rl-iRNA originates in conserved poly(A)-rich mRNA, the available evidence leads to the conclusion that most of the labelled 3′-hydroxyl termini in rl-iRNA originates in an unusual NaCl-insoluble fraction of conserved tRNA. During the course of extended imbibition, between 0.5 and 5 h, there are characteristic changes in the chain lengths and labelling patterns for 3′-hydroxyl terminal poly(A) sequences in mRNA. Analytical and physiological implications of these data are subjects of discussion.
Possible regulation of nucleoside transport and RNA synthesis in Achlya
Authors: Stevenson Roselynn M. , LéJohn Herb B.
Published: December 2009

The transport systems for adenosine, guanosine, uridine, cytidine, and thymidine were analysed kinetically, physically, and for a possible system of regulation in the fungus Achlya. Transport of nucleosides was inhibited by metabolic energy uncouplers, 2,4-dinitrophenol, carbonylcyanide-p-chlorophenylhydrazone, citrate (which chelate cell wall membrane calcium ions), and N6-substituted adenines (cytokinins) which displace three highly polyphosphorylated dinucleosides (HS3, HS2, and HS1) from a phosphorylated proteoglycan (PPG) attached to the cell membrane. Neither citrate nor the substituted adenines (except the nucleoside forms) entered the cell within the short period of transport measurements. Transport of nucleic-acid bases, adenine, uracil, cytosine, and thymine was unaffected by these inhibitors of nucleoside transport. Nucleosides were probably transported without modification for they could be made to efflux unchanged after being concentrated inside the cell which was then poisoned with metabolic energy uncouplers. The optimum pH of transport for all nucleosides except guanosine was 6.5. Guanosine had two pH optima at 6.5 and 8.0. They (nucleosides) were transported optimally between 30 and 40 °C, except thymidine which used a much higher optimum-temperature transport system.Osmotically shocked Achlya cells do not transport nucleosides but they do transport nucleic-acid bases which are taken up by facilitated diffusion. Osmotic-shock treatment of cells released the membrane PPG with associated calcium ions and HS compounds. When the PPG and HS compounds were relaid on the membrane, nucleoside transport was resumed by the cells.These results suggest that nucleoside transport and the PPG–Ca(II)–HS complex may be linked and that concentrating the nucleosides in the cells is an energy-linked activity.
Glucose transport in Achlya: Characterization and possible regulatory aspects
Authors: Goh Swee Han , LéJohn Herb B.
Published: December 2009

The freshwater fungus Achlya transported D-(+)glucose (glucose) and 2-deoxy-D-glucose (deoxyglucose) by an energy-related system. Their transport4 was inhibited by uncouplers of metabolic energy such as 2,4-dinitrophenol, cyanide, azide, and carbonylcyanide-p-chlorophenylhydrazone. Besides inhibiting each other, glucose and deoxyglucose transport was inhibited by D-(+galactose, D-(+)mannose, and D-(+)xylose. Many other sugars tested failed to inhibit glucose transport implying a certain degree of specificity. Glucose transport was pH (optimum at 6.5) and temperature (optimum at 30–40 °C) dependent. Glucose transport was also inhibited by citrate, N6-substituted adenines (cytokinins), and iodine. None of these agents penetrated the cell membrane within the brief (1–3-min) period in which glucose transport was measured. In every case, transport was inhibited within 10 s (the shortest time in which measurements could be made).When cells were osmotically shocked to release a cell-wall membrane phosphorylated proteoglycan (PPG), they became incapable of transporting glucose for several hours until new PPG material was reisolable from the membrane by osmotic-shock treatment. The osmotically shocked cells could not transport glucose or deoxyglucose. No glucose-binding protein was detected in the shock fluid.Practically all of the glucose transported within 1–2 min was recovered as glucose-6-phosphate. No other phosphorylated sugar was detected suggesting that glucose may be phosphorylated in transport.Related studies have shown that citrate removed calcium bound by PPG; N6-substituted adenines were bound by PPG while three polyphosphorylated dinucleosides, HS3, HS2, and HS1, were displaced from it. Iodine formed stable complexes with the HS compounds. All of these agents inhibited glucose transport without entering the cell. It is therefore possible that HS compounds, calcium, and PPG may be involved in maintaining the cell membrane in proper form for glucose transport.
Some comparative properties and localization of porcine jejunal adenylate cyclase
Authors: Forsyth G. W. , Hamilton D. L. , Goertz K. E. , Oliphant L. W.
Published: December 2009

Cholera toxin is thought to cause intestinal secretion by activating adenylate cyclase and increasing intracellular 3′,5′-cyclic AMP concentrations in intestinal mucosa. Cholera toxin causes profuse secretion of fluid into ligated intestinal loops of both pigs and rabbits, but cholera toxin-induced increases in 3′,5′-cyclic AMP concentration are much lower in the pig than in the rabbit. Porcine jejunal adenylate cyclase was examined for unusual properties which might account for a lack of 3′-5′-cyclic AMP accumulation after treatment with cholera toxin. The divalent cation requirements, the pH optimum, and the stimulation by fluoride ion were unremarkable. The Km for ATP was 0.11 mM with negative cooperativity indicated by a Hill coefficient of 0.83. Triton X-100 was inhibitory and guanosine diphosphate methylene phosphate stimulated enzyme activity. Adenylate cyclase activity was highest in the basal and lateral membrane fractions of jejunal mucosa and relatively low in brush-border preparations. Pretreatment of pig jejunum with cholera toxin caused a 30–40% activation of the crude and of the partly purified enzyme. A relatively low activation of adenylase cyclase in pigjejunal mucosa, compared with rabbit, may account for the absence of 3′,5′-cyclic AMP accumulation after cholera-toxin treatment in the pig.
The lipase activity of a partially purified lipolytic enzyme of Escherichia coli
Authors: Nantel G. , Baraff Georgia , Proulx P.
Published: December 2009

Escherichia coli lipase was found to have a broad pH optimum between pH 8 and 10. Long-chain acyl triacylglycerols such as trioleoylglycerol were hydrolysed at a relatively slow rate, whereas, the shorter-chain acyl derivative tricapryloylglycerol was not. Triacylglycerols and diacylglycerols were broken down at a rate 10- to 15-fold greater than that for monoacylglycerol. Simple esters such as methyloieate and cetylpalmitate were hydrolysed at rates greater than that of triacylglycerol. Water-soluble esters such as p-nitrophenylacetate were not attacked. Hydrolysis of lipase substrates occurred more readily in the presence of an anionic detergent such as taurocholate. The enzyme had no marked preference for the 1- or 3-position of triacylglycerols but attacked these positions much more readily than position 2. The enzyme also catalyzed transacylation reactions with simple alcohols such as methanol or ethanol.
Regulation of human amidophosphoribosyltransferase: Interaction of orthophosphate, PP-ribose-P, and purine ribonucleotides
Authors: Kovarsky Joel , Evans Margaret C. , Holmes Edward W.
Published: December 2009

The present report describes the effects of orthophosphate on the kinetic and physical properties of human glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14). Enzyme activity was shown to be dependent on the concentration of Pi relative to that of PP-ribose-P and purine ribonucleotides. The Hill coefficient for PP-ribose-P decreased from 1.83 ± 0.08 to 1.11 ± 0.04 when Pi was increased from 0 to 50 mM. Twenty-five millimolar PPi, 50 mM SO4, and 100 mM KCl did not alter the Hill coefficient for PP-ribose-P. In 50 mM Pi, the purine ribonucleotides AMP and GMP increased the Hill coefficient for PP-ribose-P from 1.1 to 1.6 and 1.8 respectively. At Pi concentrations less than 6.25 mM, purine ribonucleotides did not enhance the PP-ribose-P cooperativity which was already demonstrable. Pi at a concentration of 50 mM did not alter the interaction coefficient for AMP or GMP. The S20,w) was 6.2 in both 6.25 mM Pi and 50 mM Pi in the absence of purine ribonucleotides. When both PP-ribose-P and purine ribonucleotides were included in the sucrose-density gradients, Pi did not alter the relative distribution between the small and large forms of the enzyme. It is concluded from these studies that Pi acts as a substrate analogue for PP-ribose-P, and this may explain the mechanism by which Pi modulates the response of amidophosphoribosyltransferase to alterations in the concentrations of PP-ribose-P and purine ribonucleotides.
Bovine transferrin glycopeptide: the relevance of its structure to interaction with the mammalian hepatic lectin that binds asialoglycoproteins
Authors: Hatton M. W. C. , Regoeczi E. , Kaur H.
Published: December 2009

After proteolytic digestion of bovine transferrin (phenotype AA), a glycopeptide fraction was isolated by gel filtration and high-voltage electrophoresis. Two glycopeptide bands were recovered, each of which contained one residue of aspartic acid, two of serine, two of galactose, three of mannose, and four of N-acetyl glucosamine together with some fucose. However, the bands differed with respect to sialic acid content (two and three residues respectively). Using the same isolation procedures, a commercial sample of human transferrin yielded four glycopeptide bands (two major, two minor) on high-voltage electrophoresis. On analysis, the two major bands differed in amino-acid residues but their carbohydrate compositions were very similar. The analyses largely resembled previously published analytical data for two human transferrin glycopeptides (Jamieson, G. A., Jett, M. &DeBernardo, S. L. (1971) J. Biol. Chem. 246, 3686–3693; Spik, G., Bayard, B., Fournet, B., Strecker. G., Bouquelet, S. &Montreuil, J. (1975) FEBS Lett. 50, 296–299) and the carbohydrate analyses showed many similarities to the bovine transferrin glycopeptide. From these data and earlier observations made on bovine and human transferrins (Hatton, M. W. C, Regoeczi, E. &Wong, K.-L. (1974) Can. J. Biochem. 52, 845–853), we have concluded that, in contrast with human transferrin, bovine transferrin contains only one heterosaccharide chain per molecule.Following tritiation, bovine transferrin asialoglycopeptide was compared with asialoglycopeptides from other serum proteins for their affinity to the rat liver in vivo. Considerable differences were observed and the following order of binding was established for the asialoglycopeptide preparations: bovine transferrin < human transferrin [Formula: see text] bovine fetuin [Formula: see text] human α1-acid glycoprotein.
Purification and some properties of NAD-degrading purine nucleosidase from Aspergillus niger
Authors: Kuwahara Masaaki , Fujii Tomoko
Published: December 2009

An enzyme which degrades NAD at the adenine–ribose linkage has been purified from the mycelial extract of Aspergillus niger. NADP, deamido-NAD, and purine nucleosides and nucleotides were also susceptible to the hydrolytic cleavage. Pyrimidine- and nicotinamide–ribose linkages were not attacked. The substrate specificity showed that the enzyme may be classified as a N-ribosyl-purine ribohydrolase (EC 3.2.2.1). The enzyme had a maximum activity in the pH range of 4.0–4.5 toward NAD. The Km values for NAD, 5′-AMP, and inosine were 3.0, 2.9, and 1.6 mM respectively.
Coisolation of glycophorin A and polyphosphoinositides from human erythrocyte membranes
Authors: Buckley J. Thomas
Published: December 2009

The lipid composition of purified erythrocyte membrane glycophorin was measured. Diphosphoinositide, triphosphoinositide, and phosphatidylserine are the major phospholipids in glycophorin preparations. Nearly all of the radioactive diphosphoinositide and triphosphoinositide extracted from erythrocyte membranes by lithium diiodosalicylate are recovered in purified glycophorin. There appeared to be no significant enrichment of other acidic membrane phospholipids in the protein. The results do not permit a firm conclusion as to whether the polyphosphoinositides are associated specifically with the membrane protein or whether fortuitous binding has occurred during purification.
Wheat embryo ribonucleates XI. Conserved mRNA in dry wheat embryos and its relation to protein synthesis during early imbibition
Authors: Cuming A. C. , Lane B. G.
Published: December 2009

It has been found that bulk poiy(A)-rich RNA from dry wheat embryos is broadly hetero-disperse when examined by polyacrylamide gel electrophoresis. The poly(A)-rich RNA from dry wheat embryos has been translated in a cell-free protein-synthesizing system from the same commerically supplied, roller-milled wheat embryos. Compatible with the electrophoretic heterodispersity observed for poly(A)-rich RNA, the radioactive products of its cell-free translation, when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, have mobilities that are broadly coincident with the many dye-stained (nonradioactive) proteins present in wheat extracts. With due allowance for the limitations of the cell-free system, which is known to translate, selectively, lower molecular-weight species of mRNA, it has been concluded that the conserved poly(A)-rich mRNA in dry wheat embryos probably has the translational capacity required to account for the highly eclectic protein synthesis that we have observed during early (40-min) imbibition of viable wheat embryos.
Neurospora endoexonuclease and its inactive (precursor?) form
Authors: Kwong S. , Fraser M. J.
Published: December 2009

Two nuclease activities which were shown previously to copurify from extracts of log-phase Neurospora mycelia, a single-strand specific endonuclease activity (with DNA and RNA), and a strand nonspecific exonuclease activity (with DNA only) have been found to be associated with a single polypeptide. The enzyme has therefore been classified as an endoexonuclease. In log-phase extracts, about 75% of this enzyme was found to exist in an inactive form which was activated in vitro either by endogenous phenylmethylsulfonyl fluoride sensitive proteinase(s) or by exogenous trypsin. The inactive form of endoexonuclease has been purified 45-fold in 15% yield free of the active enzyme. On electrophoresis in 6 M urea–polyacrylamide gels, it migrated at a much slower rate than the active enzyme, indicating that it is a less acidic and(or) larger protein than the active nuclease. The strong adsorption of this inactive enzyme on octyl-Sepharose suggests that the protein may have a relatively large hydrophobic domain. The protein may be a precursor of the active enzyme (a pronuclease) or a strong complex of enzyme with a proteinaceous inhibitor that is not dissociated in 6 M urea or during a variety of chromatographic procedures.
Increased purine nucleotide binding, altered polypeptide composition, and thermogenesis in brown adipose tissue mitochondria of cold-acclimated rats
Authors: Desautels M. , Zaror-Behrens G. , Himms-Hagen J.
Published: December 2009

Rapid increases in atractyloside-insensitive binding of purine nucleotides (ADP or GDP) and in a polypeptide of 32 000 occur in brown adipose tissue mitochondria of the rat during acclimation to cold. The increased binding is apparent within 1 h and reaches a maximum after 3–7 days of exposure to 4 °C. The increase in the 32 000 peptide occurs more slowly and reaches a maximum after 2–3 weeks. There is a simultaneous decrease in a polypeptide of 96 000, apparent after 1 day and reaching a maximum after 1–2 weeks. Results are interpreted in terms of the appearance of an increased amount of the purine nucleotide-sensitive proton conductance pathway in association with the development of an enhanced thermogenic capacity of brown adipose tissue mitochondria during acclimation of the rat to cold.
Amino acid sequence alignment of bacterial and mammalian pancreatic serine proteases based on topological equivalences
Authors: James Michael N. G. , Delbaere Louis T. J. , Brayer Gary D.
Published: December 2009

The three-dimensional structures of the bacterial serine proteases SGPA, SGPB, and α-lytic protease have been compared with those of the pancreatic enzymes α-chymotrypsin and elastase. This comparison shows that ~60% (55–64%) of the α-carbon atom positions of the bacterial serine proteases are topologically equivalent to the α-carbon atom positions of the pancreatic enzymes. The corresponding value for a comparison of the bacterial enzymes among themselves is ~84%. The results of these topological comparisons have been used to deduce an experimentally sound sequence alignment for these several enzymes. This alignment shows that there is extensive tertiary structural homology among the bacterial and pancreatic enzymes without significant primary sequence identity (<21%). The acquisition of a zymogen function by the pancreatic enzymes is accompanied by two major changes to the bacterial enzymes' architecture: an insertion of 9 residues to increase the length of the N-terminal loop, and one of 12 residues to a loop near the activation salt bridge. In addition, in these two enzyme families, the methionine loop (residues 164–182) adopts very different conformations which are associated with their altered substrate specificities.
Purification and properties of phosphatidylgkycerophosphate synthetase from mammalian liver mitochondria
Authors: McMurray W. C. , Jarvis E. C.
Published: December 2009

The enzyme which catalyzes the synthesis of phosphatidylglycerophosphate from sn-glycerol-3-phosphate and cytidine diphosphate diacylglycerol was released from rat or pig liver mitochondrial membranes by extraction with Triton X-100 or Nonidet P-40. The detergent-extracted enzyme, like the activity of intact mitochondria, did not require added cations or lipids.The Triton extracts were fractionated by column chromatography on Bio-Gel A-1.5. The fractions obtained from the columns exhibited little activity in the standard assay system unless divalent cations were included. Additional stimulation (about twofold) was observed in the presence of added phospholipids.The cation requirement of the purified enzyme was relatively nonspecific with Mg2+, Ba2+, or Ca2+ providing maximal activity in the 10 mM range. Either Mn2+ or Co2+ were stimulatory at somewhat lower concentrations but higher concentrations were inhibitory. Other cations such as Cd2+, Zn2+, Hg2+, or Cu2+ were ineffective as cofactors, and in the presence of Mg2+ inhibited the reaction at concentrations greater than 0.5 mM.The phospholipid stimulation was obtained specifically with phosphatidylethanolamines from natural or synthetic sources. Other diacylglycerophosphatides or lysophosphatides including lysophosphatidylethanolamine were ineffective.
The evolution of 5S RNA secondary structures
Authors: Sankoff David , Morin Anne-Marie , Cedergren R. J.
Published: December 2009

We have applied the Pipas–McMahon algorithm based on free energy calculations to the search for a 5S RNA base-pair structure common to all known sequences. We find that a 'Y'-shaped model is consistently among the structures having the lowest free energy using 5S RNA sequences from either eukaryotic or prokaryotic sources. Comparison of this 'Y' structure with models which have recently been proposed show these models to be remarkably similar, and the minor differences are explicable based on the technique used to obtain the model. That prokaryotic and eukaryotic 5S RNA can adopt a similar secondary structure is strong support for its resistance to change during evolution.
Spectroscopic studies on the binding of divalent cations to porcine intestinal calcium-binding protein
Authors: Dorrington K. J. , Kells D. I. C. , Hitchman A. J. W. , Harrison J. E. , Hofmann T.
Published: December 2009

Circular dichroism and ultraviolet absorption difference spectroscopy have been used to study the binding of a series of divalent and trivalent cations to porcine intestinal calcium-binding protein (CaBP). When calcium is bound to the single high-affinity site on CaBP, the aromatic optical activity is greatly increased. Analysis of the circular dichroic spectra, obtained in the presence and absence of calcium, suggested that although changes in the optical activity of the single tyrosyl residue accounted for much of the overall change observed upon binding calcium, one or more of the five phenylalanyl residues was also perturbed. All the cations tested, with the exception of lead which gave rise to unique spectral effects, caused the same changes in optical activity between 300 and 250 nm. In the peptide absorption region, CaBP exhibited optical activity typical of an α-helical protein and no significant changes were observed in the presence of any of the cations tested. Cation-binding curves obtained from the circular dichroic data for the cations bound with high affinity (i.e., calcium, strontium, and the trivalent lanthanide ions) showed that the apparent number of binding sites was inversely related to the protein concentration. This phenomenon was accounted for by the concentration-dependent aggregation of CaBP observed in earlier studies. The binding data, obtained using circular dichroism, clearly indicated that the affinity of CaBP for the various cations was related to their ionic radius. Absorption difference spectra were observed when calcium was bound to CaBP. The features of these spectra confirmed that phenylalanyl as well as tyrosyl transitions were perturbed upon calcium binding. The extent to which the tyrosyl side chain was exposed to solvent was determined by solvent perturbation difference spectroscopy using perturbing agents of differing molecular radius. The apparent degree of exposure increased as the perturbant size decreased suggesting that the side chain was located in a cleft. Bound calcium did not change the degree of exposure. These data, together with complementary data obtained with bovine CaBP, were discussed in terms of the geometry of the cation-binding site.
The structure and maturation of intermediates in bacteriophage T7 DNA replication
Authors: Langman Lyndall , Paetkau Verner , Scraba Douglas , Miller Jr. Robert C. , Roeder G. Shirleen , Sadowski Paul D.
Published: December 2009

Concatemeric DNA from T7-infected cells consists of phage genomes in a linear head-to-tail arrangement. Adjacent genomes within a concatemer overlap for the length of the terminal repetition. Fast-sedimenting T7 DNA contains single-stranded regions at roughly unit-length intervals but these interruptions are heterogeneously distributed and do not occur at the genetic termini. Mutations in either bacteriophage genes 9, 18, or 19 (required for DNA maturation and packaging) lead to the synthesis and persistence of DNA with fewer interruptions than normal.
A new double-labelling procedure for determination of amino acid composition: Application to bacteriorhodopsin
Authors: Kaplan H. , Cheng D. C. H. , Oda G. , Kates M.
Published: December 2009

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H: 14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions.When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149–152) for bacteriorhodopsin of H. halobium.
Troponin T fragments: Physical properties and binding to troponin C
Authors: Pearlstone Joyce R. , Smillie Lawrence B.
Published: December 2009

Fragments derived from rabbit skeletal troponin T (Tn-T) were tested for binding on a troponin C (Tn-C) – Sepharose affinity column in order to locate the binding site of Tn-C on Tn-T. The COOH-terminal fragments P2 (residues 159–209) and B2 (residues 206–258) were found to bind most strongly, confirming the earlier proposal (Pearlstone, J. R., Carpenter, M. R., Johnson, P. &Smillie, L. B. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1902–1906) that the highly basic COOH-terminal region of Tn-T may serve as a site of interaction for the acidic Tn-C protein. Results from circular dichroism experiments on the large fragments B1 (residues 1–205), B2, P1 (residues 91–154), and P2 indicated that most of the α-helical structure resides in CB2 (residues 71–151), the tropomyosin (Tm) binding site of Tn-T, while the remainder of the molecule including the Tn-C binding region (approximately residues 159–259) contains very little in the way of α and β structure. These results are in agreement with the secondary structural studies made previously (Pearlstone, J. R. &Smillie, L. B. (1977) Can. J. Biochem. 55, 1032–1038). The presence of calcium resulted in a stronger interaction between these fragments and Tn-C, illustrating the calcium-sensitive nature of this system. The addition of magnesium ions to the buffer system did not affect this binding.
The primary structure of staphylococcal protease
Authors: Drapeau Gabriel R.
Published: December 2009

The amino acid sequence of staphylococcal protease has been determined by analysis of tryptic peptides obtained from cyanogen bromide fragments. Selected peptides obtained from digests with staphylococcal protease, thermolysin, and chymotrypsin provided the information necessary to align the tryptic peptides and the cyanogen bromide fragments. The protease is a single polypeptide chain of some 250 amino acids and is devoid of sulfhydryl groups. The COOH-terminal tryptic peptide of the protease molecule contains some 43 residues, most of which are aspartic acids, asparagines, and prolines. The amino acid sequence of this peptide was not determined. The primary structure near the active serine residue indicates that staphylococcal protease is related to the pancreatic serine proteases. However, it has little or no additional sequence homologies with these enzymes except for the regions near histidine-50 and aspartic acid - 91. These regions have striking similarities with the corresponding regions of protease B and the trypsin-like enzyme of Streptomyces griseus.
Electrogenic sodium-dependent glycine transport in sheep reticulocytes
Authors: Benderoff Stephen , Johnstone Rose M. , Blostein Rhoda
Published: December 2009

Na+-dependent glycine transport has been studied in reticulocyte-enriched fractions of blood obtained after massive bleeding of sheep. The activity is dependent on the sodium electrochemical potential and the membrane potential. The sodium chemical gradient was varied by changing either external or internal Na+ and the membrane potential, by addition of valinomycin. Similar results were obtained with resealed reticulocyte ghosts. Under conditions optimal for sodium pumping (intracellular Na+ > 50 mM), ouabain inhibited glycine uptake prior to any measurable change in the cellular Na+ suggesting that in these cells an electrogenic sodium pump is sufficiently active to contribute to the membrane potential. Na+-dependent glycine transport undergoes a marked decrease during Song-term incubation at 37 °C. During this time, the cells maintain their integrity and ATP content but undergo maturation as evidenced in the decrease in cells with reticulocyte morphology.
Kinetics of hydrolysis of dispersions of saturated phosphatidylcholines by Crotalus atrox phospholipase A2
Authors: Tinker David O. , Purdon A. David , Wei Jane , Mason Eileen
Published: December 2009

Dispersions of lamellar phase dipalmitoyl phosphatidylcholine (DDPC) and dimyristoyl phosphatidylcholine (DMPC) in 0.01 M CaCl2 were subjected to hydrolysis by phospholipase A2 (EC 3.1.1.4) from Crotalus atrox venom. The reaction was followed continuously by titrating the released fatty acids. For hydrolysis of gel phase phosphatides, the steady-state initial velocities were hyperbolic functions of bulk lipid concentrations. At the 'pre-transition' temperature (34 °C for DPPC, 15 °C for DMPC), there was a large increase in the Michaelis parameter Vmax but no change in the parameter Km. A model was devised to account for these observations, in which the enzyme desorbs from the lipid surface after hydrolysis. The desorption rate constant is postulated to increase above the pretransition temperature.For hydrolysis of liquid crystalline phosphatides, the reaction consisted of a short initial burst of hydrolysis, a long 'lag' period of very slow reaction, followed by a dramatic increase in the reaction rate. Addition of 10 mol% lysolecithin or fatty acid abolished the 'lag' period. It was postulated that the enzyme adsorbs irreversibly to the surface of the liquid crystalline phase. Reaction products are postulated to stimulate desorption of enzyme from the surface. Thus, temperature-dependent changes in the rate of hydrolysis of dispersed phosphatidylcholines are attributed to changes in the rate of desorption of the enzyme from the lipid surface.
Some properties of a Ca2+- and (or) Mg2+-requiring nucleoside di- and tri-phosphatase(s) associated with the membranes of rat pancreatic zymogen granules
Authors: Harper Francis , Lamy François , Calvert Raymond
Published: December 2009

Membranes of rat pancreatic zymogen granules have been purified and found to be essentially free of contamination by mitochondria, microsomes, and plasma membranes. They possessed an acid phosphatase activity which derived probably from lysed lysosomes contaminating the purified zymogen granules from which the membranes were prepared. These membranes were found to contain a strong Ca2+- and (or) Mg2+-requiring activity toward all nucleoside di- and tri-phosphates. Various data support the tentative conclusion that a single protein catalyzes the hydrolysis of the nucleoside di- and tri-phosphates. This protein appears to be intrinsic with its active site localized on the internal face of the membranes.
Replacement of endogenous phospholipids in rat plasma lipoproteins during intravenous infusion of an artificial lipid emulsion
Authors: Kuksis A. , Breckenridge W. C. , Myher J. J. , Kakis G.
Published: December 2009

Male Wistar rats, weighing 250–300 g, were intravenously infused (0.33 ml/h per 100 g body weight) with an artificial emulsion of soybean triacylglycerols and egg yolk phospholipids (Intralipid) for various periods of time ranging from a few hours to a few days. About 0.5 h after the infusion, the animals were sacrificed, various classes of plasma lipoproteins were isolated by centrifugation, and the total lipid profiles of the fractions were determined by high temperature gas chromatography. The lipoproteins collected after 24 h of infusion were subjected to a complete analysis of the molecular species of the various lipid classes. It was shown that continuous intravenous infusion of Intralipid, despite rapid apparent clearance of the visible lipid particles, resulted in a gradual accumulation of exogenous phosphatidylcholine and sphingomyelin, free cholesterol, and plant sterols in the plasma, which displaced the endogenous phospholipids and sterols from the various lipoproteins. At 24 h, about 70% of the endogenous phospholipid of all the plasma lipoproteins was replaced by egg yolk phospholipids of the Intralipid. Under the same conditions, no displacement nor exchange was observed of endogenous cholesteryl esters for exogenous triacylglycerols in any of the lipoprotein classes. Intralipid infusion for periods up to 3 days results in a limited further displacement of endogenous phospholipids by exogenous lipids in the plasma lipoproteins, but a complete substitution was not achieved because of a continued secretion of lipoproteins containing endogenous phospholipids. The infusion of Intralipid led to a significant increase in the proportion of the phospholipid and free sterol in all lipoproteins, which decreased the calculated diameter of the particles by about one-half. Only minor changes were seen in the lipid to protein ratios of the lipoproteins and in their apoprotein composition.
A comparison of some immunological characteristics of very low density lipoproteins of normal and hypercholesterolemic rat sera
Authors: Dolphin Peter J. , Wong Laurence , Rubinstein David
Published: December 2009

The immunological characteristics of very low density lipoproteins (VLDL) from normal and hypercholesterolemic rat sera were compared using polyspecific antisera to VLDL and high density lipoproteins (HDL) and monospecific antisera to apo-B, apo-C, apo-A-I, and apo-E. Ultracentrifugally isolated VLDL from normal serum were studied by immunodiffusion and found to contain both discrete and associated (with apo-B) apo-C and apo-E, probably in the form of lipid-containing lipoproteins. However, immunoelectrophoresis of whole serum revealed only an associated form of the lipoprotein having pre-β mobility (i.e., VLDL), suggesting that the presence of discrete lipoproteins in isolated VLDL, each containing a single apoprotein family, may represent ultracentrifugal artifacts. Ultracentrifugally isolated VLDL from diet-induced hypercholesterolemic rat serum contained only trace amounts of apo-C and large quantities of apo-E, both of which were totally associated with apo-B. VLDL isolated by ultracentrifugation from perfusate of normal and hypercholesterolemic livers contained only associated lipoprotein complexes made up of apo-B, apo-C, and apo-E in the former but only apo-B and apo-E in the latter. These data suggest that normal VLDL are secreted as lipoprotein complexes containing apo-B, apo-C, and apo-E which may become destabilized in the circulation. However, VLDL from hypercholesterolemic serum show a marked diminution in the quantity of apo-C as indicated by the relative incorporation of [3H]leucine in vivo and by polyacrylamide gel electrophoresis of apo-VLDL.
A procedure for obtaining initial estimates of parameters appearing in steady-state rate or equilibrium binding equations
Authors: Darvey I. G. , Walker E. J.
Published: December 2009

A 'peeling' procedure is described for obtaining initial estimates of the parameters in the equation:[Formula: see text]where P(x) and Q(x) are polynomials in x. The method is illustrated, in the context of enzyme kinetics, using data which are fitted to the following equation:[Formula: see text]where v denotes the initial steady-state velocity at an initial substrate concentration S, and a1, a2, b1 and b2 are non-negative constants. The applicability and limitations of the method for data fitting in fields such as enzyme kinetics and ligand-binding studies are discussed.
Control of glucose transport in adipose tissue of the rat: Role of insulin, ATP, and intracellular metabolites
Authors: Halperin Mitchell L. , Mak Marina L. , Taylor Wayne M.
Published: December 2009

The purpose of this study was to elucidate some of the mechanisms of control of the glucose transport step in adipose tissue. Glucose transport was studied by monitoring the conversion of [1-14C]glucose to 14CO2 in a system where glucose transport was made rate limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent which results in rapid rates of reoxidation of NADPH. The maximum velocity for the apparent rate of glucose transport was increased significantly by insulin. There was no change in the glucose concentration required for half-maximal rates of 14CO2 production. Glucose transport was also monitored by directly measuring the rate of glucose uptake. Glucose uptake was increased by phenazine methosulphate. The intracellular glucose-6-phosphate concentration was decreased by phenazine methosulphate. These two agents, insulin and phenazine methosulphate, seemed to act by independent mechanisms as their optimal effects on glucose uptake were additive.The apparent rate of glucose transport was decreased by ATP which resulted in a decrease in maximal velocity but did not affect the affinity for glucose. This effect of ATP was seen in the presence or absence of insulin.
Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns
Authors: Yousef I. M. , Murray R. K.
Published: December 2009

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3–CaCl2, and K2HPO4–KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3–CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (> 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to actin were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris–HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3–CaCl2 appeared to be the best buffer and Tris–HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.
Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10–12 °C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid – microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10–12 °C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.
Mechanism of heparin and serum lipoprotein interaction: Effects of calcium, phosphorylcholine, and a serum fraction
Authors: Mookerjea Sailen
Published: December 2009

The mechanism of formation of an insoluble complex between heparin and rat serum lipoprotein has been studied. Optical density changes during the reaction, counting of the fatty acid labelled lipoproteins in the precipitates, and complexing of [14C]palmitate-labelled lipoprotein with heparin–CNBr–Sepharose were used to quantitatively determine the formation of insoluble complexes. The maximal heparin–lipoprotein complex formation requires 25–30 mM of Ca2+, but with micromolar amounts of phosphorylcholine, the reaction was saturated at only 10 mM of Ca2+. The effect of phosphorylcholine in promoting the reaction was lost when purified chylomicrons or very low density lipoproteins were used. The effect of phosphorylcholine in promoting the interaction between heparin and pure chylomicrons or very low density lipoproteins was regained when a crude serum protein factor of unwashed chylomicrons was added to the system, suggesting that rat serum contains a protein factor(s) which normally inhibits the heparin–lipoprotein interaction by raising the requirement for Ca2+. Phosphorylcholine counteracted the effect of this protein, thereby favouring the precipitation reaction in the presence of much lower concentration of Ca2+. The results have been discussed with special reference to the possibility of a relationship between mucopolysaccharides, Ca2+, lipoproteins, and arterial phospholipids in the pathogenesis of atherosclerosis.
Levels of coenzyme A – glutathione mixed disulfide in Escherichia coli
Authors: Loewen Peter C.
Published: December 2009

The pool of coenzyme A – glutathione mixed disulfide (CoASSG) rapidly increased 2.0 times in response to oxygen starvation and 1.5 times in response to glucose starvation but did not change following ammonia starvation. The increase in the CoASSG pool resulted from an increase in the CoASSG fraction of the CoA pool from 42 to 66–93%. Fluoride, cyanide, chloramphenicol, and rifampicin all caused similar increases. Aerobic growth on fermentable sugars resulted in CoASSG making up 40–55% of the CoA pool while growth on nonfermentable carbon sources or anaerobic fermentation resulted in CoASSG replacing acetyl CoA and free CoA to make up 85–95% of the CoA pool. The CoASSG:ATP ratio varied inversely with the growth rate in two groupings of carbon sources made up of either fermentable or nonfermentable molecules. Cultures grown aerobically on fermentable sugars exhibited a lower CoASSG:ATP ratio reflecting the lower proportion of CoASSG in the CoA pool.
A direct radioimmunoassay for tonin
Authors: Gutkowska J. , Boucher R. , Demassieux S. , Garcia R. , Genest J.
Published: December 2009

A sensitive radioimmunoassay for the measurement of tonin is described. It is based on the use of antibodies produced in rabbits against highly purified tonin obtained from rat submaxillary gland. A radioiodinated enzyme with high specific activity was obtained by the chloramine-T method. Optimal conditions for radioimmunoprecipitation were established and the double-antibody method was used to separate bound and free tonin. The radioimmunoassay may be used to measure tonin in amounts as low as 150 pg. This radioimmunoassay was applied to the measurement of tonin in rat saliva and in homogenates of submaxillary glands. Excellent correlation was found between tonin activity measured fiuorometrically and tonin concentration obtained by the radioimmunoassay.