bluuebunny/crossref_metadata_2025 huggingface dataset:
Presence of fucosamine in teichuronic acid of the alkalophilic Bacillus strain C-125
Authors: Aono R , Uramoto M
Published: August 2015

Cell walls of the alkalophilic Bacillus strain C-125 are composed of gamma-peptidoglycan, teichuronic acid and a polymer of glucuronate and glutamate. An amino sugar that was a main component of the teichuronic acid did not correspond to any of the commercially available hexosamines. The amino sugar was purified into crystalline form from the hydrolysate of the teichuronic acid by ion-exchange chromatography and then partition chromatography on a cellulose column. The amino sugar was identified as D-fucosamine (2-amino-2,6-dideoxy-D-galactose) by 400 MHz n.m.r. spectrometric analysis, measurement of optical rotation and elemental analysis.
CNBr cleavage of the light chain of human complement factor I and alignment of the fragments
Authors: Yuan J M , Hsiung L M , Gagnon J
Published: August 2015

The light chain and heavy chain of reduced and alkylated human complement Factor I were purified by high-pressure gel-permeation chromatography. CNBr cleavage of Factor I light chain yielded four major fragments, which were purified by gel filtration. N-Terminal sequence analysis of the CNBr-cleavage fragments allowed identification of 200 of the approx. 240 amino acid residues of the light chain. An alignment is proposed, based on sequence analysis of peptides obtained after cleavage at arginine residues of the light chain and on homology of the sequence determined with that of other serine proteinases. The sequence around the active-site serine residue was established and three potential attachment sites for carbohydrate moieties were identified.
Cell-cell recognition of host surfaces by pathogens. The adsorption of maize (Zea mays) root mucilage by surfaces of pathogenic fungi
Authors: Gould J , Northcote D H
Published: August 2015

The adsorption of radioactive mucilage by pathogenic fungi was shown to be dependent upon time, the composition of mucilage, the type of fungal surface (conidia, hyphae, hyphal apices), fungal species, pH and bivalent cations. All fungal adhesins were inactivated by either proteinase or polysaccharase treatments. Adsorption was not inhibited by the numberous mono-, di- and oligo-saccharides that were tested individually, but it was inhibited absolutely by several polysaccharides. This suggested that adsorption of mucilage by pathogens involved conformational and ionic interactions between plant and fungal polymers but not fungal lectins bound to sugar residues of mucilage. Several fractionation schemes showed that pathogens bound only the most acidic of the variety of polymers that comprise mucilage. There was not any absolute distinction between ability to bind radioactive mucilage and type of pathogen or non-pathogen. However, there were notable differences in characteristics of adsorption between two types of pathogen. Differences were revealed by comparison of the adsorption capacities of conidia and germinant conidia and chromatography of radioactive mucilage on germinant conidia. An ectotrophic root-infecting fungus (a highly specialized pathogen) bound a greater proportion of mucilage than did a vascular-wilt fungus (of catholic host and tissue range) with more than one class of site for adsorption. In contrast with the vascular-wilt fungus, sites for adsorption on the specialized pathogen were present solely on surfaces formed by germination.
Sequences encoding two trypsin inhibitors occur in strikingly similar genomic environments
Authors: Kingston I B , Anderson S
Published: August 2015

The nucleotide sequences of two approx. 4 kilobase pair segments of the bovine genome are presented. One segment contains a coding region for bovine pancreatic trypsin inhibitor (BPTI) and the other segment contains a coding region for a BPTI homologue. The two 4 kilobase pair sequences are strikingly similar over approx. 3.4 kilobase pairs of their sequence, including putative intron sequences, suggesting that they have evolved from a gene duplication event.
Inactivation of 3α-hydroxysteroid dehydrogenase by superoxide radicals. Modification of histidine and cysteine residues causes the conformational change
Authors: Kim H S , Minard P , Legoy M D , Thomas D
Published: August 2015

3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50) from Pseudomonas testosterone was inactivated by superoxide radicals generated by the aerobic xanthine oxidase reaction. Superoxide dismutase, NAD+, bovine serum albumin and histidine and cysteine as free amino acids partially protected the enzyme from inactivation. NADH-binding properties were determined by fluorescence spectroscopy, and no variation was found between native enzyme and the unmodified fraction of the partly inactivated one. The fluorescence emission maximum for the completely inactivated enzyme was shifted 10 nm to a longer wavelength when compared with the native one, and it seems possible that the modification of histidine and cysteine residues by superoxide radicals causes the conformational change of the enzyme and the consequent loss of catalytic activity.
Is the availability of substrate for the tricarboxylic acid cycle a limiting factor for uncoupled respiration in sycamore (Acer pseudoplatanus) cells?
Authors: Journet E P , Bligny R , Douce R
Published: August 2015

Protoplasts obtained from sycamore (Acer pseudoplatanus) cell suspensions were found to be highly intact and to retain a high rate of O2 consumption. If the protoplasts were taken up and expelled through a fine nylon mesh, all the protoplasts were ruptured, leaving the fragile amyloplasts largely intact. Distribution of enzymes of glycolysis in plastids and soluble phase of sycamore protoplasts indicated that the absolute maximum activity for each glycolytic enzyme under optimum conditions exceeded the estimates of the maximal rate at which sycamore cells oxidize triose phosphate. Passage of protoplasts through the fine nylon mesh produced a 3-5-fold decrease in O2 consumption. However, addition of saturating amounts of respiratory substrates and ADP restored an O2 consumption equal to that observed with uncoupled intact protoplasts. Taken together, these results demonstrated that neither the overall capacity of the glycolytic enzymes in sycamore cells nor the availability of respiratory substrates for the mitochondria is ultimately responsible for determining the rate of uncoupled respiration in sycamore cells.
Binding of [14C]malonyl-CoA to rat liver mitochondria after blocking of the active site of carnitine palmitoyltransferase I. Displacement of low-affinity binding by palmitoyl-CoA
Authors: Grantham B D , Zammit V A
Published: August 2015

The active site of the overt activity of carnitine palmitoyltransferase (CPT I) in rat liver mitochondria was blocked by the self-catalysed formation of the S-carboxypalmitoyl-CoA ester of (-)-carnitine, followed by washing of the mitochondria. CPT I activity in treated mitochondria was inhibited by 90-95%. Binding of [14C]malonyl-CoA to these mitochondria was not inhibited as compared with that of control mitochondria. When CPT I activity was inhibited, palmitoyl-CoA could markedly displace [14C]malonyl-CoA binding from the low-affinity site for the inhibitor [Zammit, Corstorphine & Gray (1984) Biochem. J. 222, 335-342], but not from the high-affinity site for malonyl-CoA binding. The saturation characteristics of the malonyl-CoA-binding component lost in the presence of palmitoyl-CoA were sigmoidal, and thus suggestive of co-operative binding at this site. It is suggested that the site hitherto considered to be a low-affinity malonyl-CoA-binding site may be effectively a second, allosteric, acyl-CoA-binding site on CPT I under conditions that prevail in vivo, whereas the high-affinity site for malonyl-CoA may be exclusive to the inhibitor. The possibility that the competitive-type interactions of malonyl-CoA and acyl-CoA on CPT I activity could arise from the effects of separate malonyl-CoA and acyl-CoA allosteric sites is considered. The possible significance of the large difference in the capacity of the two sites and their different saturation kinetics is also discussed.
Protein conformation of potato (Solanum tuberosum) lectin determined by circular dichroism
Authors: van Holst G J , Martin S R , Allen A K , Ashford D , Desai N N , Neuberger A
Published: August 2015

The structure of potato (Solanum tuberosum) lectin, which is a hydroxyproline-rich glycoprotein, has been investigated by circular dichroism. The spectra of the native lectin, and of the oxidized, reduced and carboxymethylated and deglycosylated derivatives were examined, as was a hydroxyproline-rich glycopeptide and its deglycosylated derivative. It is concluded that the lectin contains about 35% polyproline II conformation, 34% type II beta-turn and 31% irregular conformation. No indications were found for the presence of alpha-helix or beta-sheet conformations. The polyproline II conformation is heat-stable, but is markedly destabilized by deglycosylation. The type II beta-turn is destabilized by cleavage of disulphide bonds.
Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes
Authors: Sommarin Y , Heinegård D
Published: August 2015

The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.
Immunochemical characterization of phosphatidylinositol 4-phosphate kinase from rat brain
Authors: van Dongen C J , Kok J W , Schrama L H , Oestreicher A B , Gispen W H
Published: August 2015

Affinity-purified antibodies were used to identify a protein of molecular mass 45 kDa (45 kDa protein) in rat brain cytosol as phosphatidylinositol 4-phosphate (PtdIns4P) kinase. Antibodies were raised in rabbits by immunization with the purified 45 kDa protein. Anti-(45 kDa protein) immunoglobulins were isolated by affinity chromatography of the antiserum on a solid immunosorbent, which was prepared by coupling a soluble rat brain fraction, the DEAE-cellulose pool containing 10-15% 45 kDa protein, to CNBr-activated Sepharose 4B. The purified IgGs were specific for the 45 kDa protein as judged by immunoblot and by immunoprecipitation. The purified anti-(45 kDa protein) IgGs inhibited the enzyme activity of partially purified PtdIns4P kinase, whereas preimmune IgGs were ineffective. Immunoprecipitation of the 45 kDa protein from the partially purified enzyme preparation with the purified IgGs resulted in a concomitant decrease in the amount of 45 kDa protein and in PtdIns4P kinase activity. The amount of 45 kDa protein remaining in the supernatant and the activity of PtdIns4P kinase correlated with a coefficient of r = 0.87. The evidence presented lends further support for the notion that the catalytic activity of PtdIns4P kinase in rat brain cytosol resides in a 45 kDa protein.
Phosphoinositide reorganization in human erythrocyte membrane upon cholesterol depletion
Authors: M'Zali H , Giraud F
Published: August 2015

The effect of cholesterol depletion on the activity of phosphatidylinositol/phosphatidylinositol 4-phosphate and diacylglycerol kinases and polyphosphoinositide phosphodiesterase has been studied in isolated membranes of human normal and cholesterol-depleted erythrocytes. Polyphosphoinositide synthesis (phosphatidylinositol/phosphatidylinositol 4-phosphate kinase activities) were found to depend on the permeability and sidedness characteristics of the membrane vesicles, which could limit the accessibility of ATP for the enzymes. When measured under proper conditions, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate synthesis were decreased in cholesterol-depleted membranes as compared with control membranes. The same level of synthesis could be obtained in both membranes by the addition of phosphatidylinositol (and Triton X-100) or of phosphatidylinositol 4-phosphate. Phosphatidic acid synthesis (diacylglycerol kinase activity) was also decreased in cholesterol-depleted membranes as compared with control membranes when measured in the presence of Ca2+. Addition of diolein (and Triton X-100) caused a large increase in phosphatidic acid synthesis which reached approximately the same level in both membranes. This showed that the apparent inhibition of polyphosphoinositide and phosphatidic acid synthesis was not due to a loss or to an inactivation of the kinases. Ca2+-activated polyphosphoinositide phosphodiesterase promoted the hydrolysis of 65-70% of the polyphosphoinositides in control and of only 45-55% in cholesterol-depleted membranes without changing the Ca2+ concentration for half-maximum hydrolysis (1 microM). Upon addition of sodium oleate, the extent of polyphosphoinositide hydrolysis became identical in both membranes, indicating again that there was no loss nor inactivation of the polyphosphoinositide phosphodiesterase in the cholesterol-depleted membranes. Since the concentration of the polyphosphoinositides was not changed by cholesterol depletion [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200], the reduction in both their synthesis and degradation observed here could be attributed to a reorganization of the phosphoinositides in membrane domains where they were not accessible to the kinases and phosphodiesterase. The reduction in phosphatidic acid synthesis was likely caused by a reduction in the total amount of the substrate diacylglycerol in cholesterol-depleted membranes as already shown [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200].
Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions
Authors: Bergey E J , Levine M J , Reddy M S , Bradway S D , Al-Hashimi I
Published: August 2015

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.
The serC-aro A operon of Escherichia coli. A mixed function operon encoding enzymes from two different amino acid biosynthetic pathways
Authors: Duncan K , Coggins J R
Published: August 2015

Sub-cloning experiments aimed at precisely locating the E. coli aroA gene, which encodes the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase, showed that in certain constructions, which remain capable of complementing an auxotrophic aroA mutation, expression of aroA is reduced. DNA sequence analysis revealed that a sequence approx. 1200 base pairs (bp) upstream of aroA is necessary for its expression. An open reading frame was identified in this region which encodes a protein of 362 amino acids with a calculated Mr of 39,834 and which ends 70 bp before the start of the aroA coding sequence. This gene has been identified as serC, the structural gene for 3-phosphoserine aminotransferase, an enzyme of the serine biosynthetic pathway. Both genes are expressed as a polycistronic message which is transcribed from a promotor located 58 bp upstream of serC. Evidence is presented which confirms that the aroA and serC genes constitute an operon which has the novel feature of encoding enzymes from two different amino acid biosynthetic pathways.
Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease
Authors: Spivak W , Yuey W
Published: August 2015

We have developed an extremely rapid and efficient reverse-phase h.p.l.c. method for the measurement of bilirubin and its conjugates in human bile and in model bile systems. Our method involves the use of a Perkin-Elmer 3 mu C18 column and a methanol/sodium acetate/aq. ammonium acetate buffer system. Three isomers of bilirubin diglucuronide (BDG), two isomers of bilirubin monoglucuronide (BMG), three isomers of unconjugated bilirubin (UCB) and minor conjugates containing glucose and xylose were separated in 12 min. Initial quantification of BDG and BMG was based on the use of the ethyl anthranilate azo derivative of bilirubin (AZO UCB); however, the standard curves for BDG, BMG and UCB were similar enough to permit quantification to be later based on the UCB standard curve only, thereby simplifying the quantification process. Routine direct injection of 6 or 10 microliter of crude undiluted or diluted (1:1) bile sample was sufficient for analysis. The method was helpful in diagnosing biliary-tract obstruction in a newborn and a partial deficiency state of bilirubin conjugation (Crigler-Najjar syndrome) in a 10-year-old male. When the method was applied to biles of patients both with and without gallstones, levels of UCB were less than 2% of total pigment, consistent with previous reports. Because of its speed and efficiency, this method has the potential for a broad range of applications including enzymic, kinetic and bile sample analyses.
A direct interaction between Photosystem I and the chloroplast coupling factor
Authors: Huchzermeyer B , Loehr A , Willms I
Published: August 2015

A regulation of the ATP-synthesizing complex by electron-transport rate has been found. The site of regulation could be localized within the Photosystem I region. The regulatory effect probably is produced by direct interactions between neighbouring charged protein complexes. The primary result is an increase in the percentage of those binding sites adopting a low-affinity state. This seems to lead to an enhanced leakage of protons out of the thylakoids, especially under those experimental conditions employing low nucleotide concentrations. Changes in the P/2e ratio can be observed, especially if the total ADP + ATP concentration used in the experiment is below 200 microM.
Modulation of pyruvate dehydrogenase kinase activity in cultured hepatocytes by glucagon and n-octanoate
Authors: Fatania H R , Vary T C , Randle P J
Published: August 2015

The activity of pyruvate dehydrogenase kinase in extracts of mitochondria from rat hepatocytes cultured for 21 h in medium 199 was increased 2.5-fold by the presence of 55 nM-glucagon and 1 mM-sodium n-octanoate in the culture medium. The change was comparable with that induced in vivo by 48 h starvation. The potential contribution of branched-chain complex to estimates of PDH-complex activity in rat liver mitochondria has been defined.
Compartmentation of guanine nucleotide precursors for DNA synthesis
Authors: Nguyen B T , Sadée W
Published: August 2015

We have studied the kinetics of guanine incorporation into DNA in mouse T-lymphoma (S-49) mutant cells [PNPase (purine-nucleoside phosphorylase)- and HGPRTase (hypoxanthine: guanine phosphoribosyltransferase)-deficient] that are incapable of converting dGuo (deoxyguanosine) to Gua (guanine) ribonucleotides. Of the two possible pathways for an exogenous guanine source to reach DNA, firstly: dGuo→dGMP→dGDP→dGTP and secondly: Gua→GMP→GDP→dGDP→dGTP only the second pathway was found to be functional in providing guanine for DNA replication, although deoxyguanosine readily produced toxic cellular dGTP levels via the first pathway. The functional guanine-nucleotide-precursor pools for DNA are rather small; further, the depletion of the small GMP pool, but not that of GDP, GTP and dGTP, correlated well with the inhibition of DNA synthesis by mycophenolic acid, an IMP dehydrogenase inhibitor. These results support the hypothesis that guanine-nucleotide incorporation into DNA is highly compartmentalized and that a small functional guanine-nucleotide pool, e.g., the GMP pool, may serve a crucial role in limiting the availability of DNA precursor substrate.
Experimental hyperthyroidism does not induce hepatic insulin resistance in the miniature pig
Authors: Müller M J , Möring J , Seitz H J
Published: August 2015

The effect of hypo- and hyper-thyroidism on insulin-mediated alterations in tracer-determined glucose kinetics and the arterial concentration of gluconeogenic precursors were investigated in 24 h-starved conscious unrestrained miniature pigs. Hyperinsulinaemia (about 40 microunits/ml) decreased blood glucose and, transiently, glucose output at unaltered glucose utilization in all thyroid states: this effect was pronounced in hyperthyroid (-50%) and less in hypothyroid pigs (-25%) compared with euthyroid controls (-35%). We conclude that moderate experimental hyperthyroidism does not induce hepatic insulin resistance, whereas hypothyroidism slightly impairs insulin action with respect to the regulation of glucose output.
Quantification of the importance of individual steps in the control of aromatic amino acid metabolism
Authors: Salter M , Knowles R G , Pogson C I
Published: August 2015

The quantitative importance of the individual steps of aromatic amino acid metabolism in rat liver was determined by calculation of the respective Control Coefficients (Strengths). The Control Coefficient of tryptophan 2,3-dioxygenase for tryptophan degradation was determined in a variety of physiological conditions and with a range of activities of tryptophan 2,3-dioxygenase. The Control Coefficient varied from 0.75 with basal enzyme activity to 0.25 after maximal induction of the enzyme by dexamethasone. The remainder of the control for tryptophan degradation was associated with the transport of the amino acid across the plasma membrane, with only very small contributions from kynureninase and kynurenine hydroxylase. The Control Coefficients of tyrosine aminotransferase for tyrosine degradation were approx. 0.70 and 0.20 with basal and dexamethasone-induced tyrosine aminotransferase activities respectively; the Control Coefficients of the transport of the amino acid into the cell were 0.22 and 0.58 respectively. Phenylalanine hydroxylase was found to have a Control Coefficient for the degradation of phenylalanine of approx. 0.50 under conditions of basal enzyme activity; after maximal activation by glucagon, the Control Coefficient decreased to 0.12. The transport of phenylalanine was responsible for the remaining control in the pathway. These results have important implications, directly for the regulation of aromatic amino acid metabolism in the liver, and indirectly for the regulation of neuroamine synthesis in the brain.
The regulatory properties of yeast pyruvate kinase. Effect of pH
Authors: Kinderlerer J , Ainsworth S , Morris C N , Rhodes N
Published: August 2015

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the effector H+ in the pH range 5-6.6. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that the data could be described by the exponential model for a regulatory enzyme. On that basis, it was concluded that the binding of H+ is positively interactive and that the protonated enzyme is catalytically inactive. It was also found that H+ interacts positively with phosphoenolpyruvate but negatively with both ADP and Mg2+.
A method for preparing radiolabelled rat pulmonary surfactant
Authors: Lewis R W , Harwood J L , Richards R J
Published: August 2015

A method is described for the preparation of rat pulmonary surfactant, radiolabelled specifically in the phosphatidylcholine species, which may be used for degradative studies of the lipoprotein complex. Intravenously administered [methyl-14C]choline chloride is maximally incorporated into alveolar surface surfactant 8 h after injection, and more than 97% of this radiolabel is present in the phosphatidylcholine fraction of the surfactant and, of this, 75% is associated with the dipalmitoyl phosphatidylcholine species. Electron microscopy indicates that the isolated surfactant has a similar physical form to that found at the alveolar surface. The mineral alpha-quartz can be used to increase the yield of surfactant lavaged from the lung surface, but the complex isolated from rats treated in this manner has a low specific radioactivity (less than 1000 d.p.m./mg) compared with that prepared from control animals (22860 d.p.m./mg).
Rooster comb hyaluronate—protein, a non-covalently linked complex
Authors: Tsiganos C P , Vynios D H , Kalpaxis D L
Published: August 2015

Hyaluronate from rooster comb was isolated by ion-exchange chromatography on DEAE-cellulose from tissue extracts and papain digests. The preparations were labelled with [14C]acetic anhydride and subjected to CsCl-density-gradient centrifugation in 4 M-guanidinium chloride in the presence and absence of 4% ZwittergentTM 3-12. A radioactive protein fraction was separated from the hyaluronate when the zwitterionic detergent was also present. The protein could also be separated from the glycosaminoglycan by chromatography on Sepharose CL-6B eluted with the same solvent mixture. The protein fraction contained three protein bands of Mr 15,000-17,000 as assessed by polyacrylamide-gel electrophoresis in 0.1% SDS, and seemed to lack lysozyme activity. No evidence of other protein or amino acid(s) covalently linked with the hyaluronate was obtained. The hyaluronate-protein complex may be re-formed upon mixing the components, the extent of its formation depending on the conditions used. The results show that, as in chondrosarcoma [Mason, d'Arville, Kimura & Hascall (1982) Biochem. J. 207, 445-457] and teratocarcinoma cells [Prehm (1983) Biochem. J. 211, 191-198] the rooster comb hyaluronate also is not linked covalently to a core protein.
Fractionation and quantification of calcium-dependent proteinase activity from small tissue samples
Authors: Clark A F , DeMartino G N , Croall D E
Published: August 2015

Almost all mammalian tissues contain a cytoplasmic Ca2+-dependent proteolytic system consisting of two different proteinases (CDP I and CDP II) and an endogenous inhibitor specific for these proteinases. It was difficult to determine the relative activities of CDP I and CDP II directly and accurately without extensive purification, requiring relatively large amounts of tissue. We developed a simple technique based on Reactive Red-agarose affinity chromatography for quantitatively measuring CDP II activities in small (less than 100 mg) tissue samples. This technique is rapid, sensitive and highly reproducible. CDP II activities can be quantified in numerous tissue samples in a single day. Using this method, we analysed the components of the CDP system in various rat tissues and demonstrated quantitative differences in CDP II activities as well as relative differences in the amounts of CDP-inhibitor activity among the various tissues. This technique should prove useful in the efforts to define the currently unknown physiological function(s) of the Ca2+-dependent proteolytic system by allowing comparison of CDP activities in tissues under diverse conditions of protein metabolism.
Purification and characterization of human α2-macroglobulin conformational variants by non-ideal high performance size-exclusion chromatography
Authors: Gonias S L , Roche P A , Pizzo S V
Published: August 2015

Human alpha 2-macroglobulin (alpha 2M) was eluted as a single nondispersed peak from a TSK-G4000SW size exclusion chromatography column equilibrated in 20 mM-sodium phosphate/100 mM-NaCl, pH 7.2 (PBS). The void volume and total accessible volume of the column were 6.08 ml and 14.42 ml. The elution volume (Ve) of native alpha 2M was 9.20 +/- 0.04 ml. The Ve was altered minimally by changing the ionic strength or adding ethanol to the equilibration buffer. Ternary alpha 2M-trypsin, containing 2 mol of proteinase/mol of inhibitor, and alpha 2M-methylamine failed to be eluted in well-defined peaks when the column was equilibrated in PBS. The majority of either preparation was recovered slowly at Ve values greater than 14.5 ml, reflecting significant nonideal interactions with the support structure. Addition of 10% ethanol or increased ionic strength in the equilibration buffer independently caused either form of reacted alpha 2M to be eluted in a distinct peak at decreased Ve, suggesting that the nonideal interactions included hydrophobic and electrostatic adsorption. When the equilibration buffer was 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2, partial resolution of ternary alpha 2M-trypsin and alpha 2M-methylamine was obtained with a single column run. The Ve values of ternary alpha 2M-trypsin and alpha 2M-methylamine in this buffer were 13.15 +/- 0.08 ml and 11.94 +/- 0.14 ml, respectively. The Ve of native alpha 2M was 8.84 +/- 0.03 ml. The resolving capacity of TSK-G4000SW was exploited to purify native alpha 2M rapidly and efficiently from solutions that contained significant amounts of either ternary alpha 2M-trypsin or binary alpha 2M-trypsin (1 mol of proteinase/mol of inhibitor). This purification was complete within the limits of sensitivity of denaturing and nondenaturing polyacrylamide-gel electrophoresis. alpha 2M-plasmin was well resolved from native alpha 2M. The Ve of alpha 2M-plasmin was 12.88 +/- 0.32 ml in 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2. A number of procedures were used to prepare solutions with up to 90% binary alpha 2M-trypsin. The Ve of binary alpha 2M-trypsin in these various solutions was intermediate between the values of native alpha 2M and ternary alpha 2M-trypsin. The conformations of binary and ternary complex, as reflected by mobility in nondenaturing electrophoresis, were identical, confirming previous results. Finally, in the binary alpha 2M-trypsin complex, the single trypsin cleaved more than two, and as many as all four alpha 2M subunits.
Characterization of three-fraction mycobacillin synthetase
Authors: Mukhopadhyay N K , Majumder S , Ghosh S K , Bose S K
Published: August 2015

Mycobacillin synthetase lacks aspartic acid racemase, alanine racemase and glutamic acid racemase activities. The enzyme also does not respond to ATP-[32P]Pi exchange, nor does it catalyse the antibiotic synthesis in presence of amino acids of configuration opposite to that present in the molecule. Preincubation with optical isomers of opposite configuration inhibited the ATP-[32P]Pi exchange reaction to the extent of 60-90%. None of the three fractions of mycobacillin synthetase contained a pantothenic acid arm. Two molecules of ATP are required to synthesize one peptide bond of mycobacillin. Intermediate peptides of mycobacillin are not covalently linked to the three-fraction mycobacillin synthetase.
Formation of hexose 6-phosphates from lactate + pyruvate + glutamate by a cell-free system from rat liver
Authors: Stoecklin F B , Mörikofer-Zwez S , Walter P
Published: August 2015

A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors and gluconeogenic intermediates at near-physiological concentrations was shown to form hexose 6-phosphates linearly from lactate + pyruvate + glutamate at a rate of 0.82 +/- 0.05 mumol/min per g of liver (mean +/- S.E.M., n = 8, 37 degrees C). The indicated rates were measured between 20 min and 60 min incubation time, when the system was near steady state. Experiments with either [1-14C]lactate or [U-14C]glutamate revealed that the incorporation of radioactive label into hexose 6-phosphates was proportional to the utilization of lactate + pyruvate and of glutamate during incubation and that both served as gluconeogenic substrates at a ratio of about 2:1. When the [ATP]/[ADP] ratio was lowered from 60 to 19 by addition of ATPase, the rate of hexose 6-phosphate formation fell to one-third. This decrease in gluconeogenic flux was mainly due to a decreased flow through the phosphoglycerate kinase step. Hexose 6-phosphate formation could also be decreased by increasing the ratio [NADH]/[NAD+], either by addition of ethanol or by increasing the initial concentration of lactate + pyruvate at a fixed ratio of 10:1. The observed inhibition was linked to a limitation in the availability of oxaloacetate for the phosphoenolpyruvate carboxykinase reaction and to an increased formation of sn-glycerol 3-phosphate. Finally, the rates of hexose 6-phosphate formation in incubations with cytosols from fed rats were only 50% of those observed with cytosols from animals starved for 48 h. One of the limiting steps was found to be the flow through the phosphoenolpyruvate carboxykinase step.
Inhibition of oxidative metabolism by propionic acid and its reversal by carnitine in isolated rat hepatocytes
Authors: Brass E P , Fennessey P V , Miller L V
Published: August 2015

The present study was designed to study the interaction of propionic acid and carnitine on oxidative metabolism by isolated rat hepatocytes. Propionic acid (10 mM) inhibited hepatocyte oxidation of [1-14C]-pyruvate (10 mM) by 60%. This inhibition was not the result of substrate competition, as butyric acid had minimal effects on pyruvate oxidation. Carnitine had a small inhibitory effect on pyruvate oxidation in the hepatocyte system (210 +/- 19 and 184 +/- 18 nmol of pyruvate/60 min per mg of protein in the absence and presence of 10 mM-carnitine respectively; means +/- S.E.M., n = 10). However, in the presence of propionic acid (10 mM), carnitine (10 mM) increased the rate of pyruvate oxidation by 19%. Under conditions where carnitine partially reversed the inhibitory effect of propionic acid on pyruvate oxidation, formation of propionylcarnitine was documented by using fast-atom-bombardment mass spectroscopy. Propionic acid also inhibited oxidation of [1-14C]palmitic acid (0.8 mM) by hepatocytes isolated from fed rats. The degree of inhibition caused by propionic acid was decreased in the presence of 10 mM-carnitine (41% inhibition in the absence of carnitine, 22% inhibition in the presence of carnitine). Propionic acid did not inhibit [1-14C]palmitic acid oxidation by hepatocytes isolated from 48 h-starved rats. These results demonstrate that propionic acid interferes with oxidative metabolism in intact hepatocytes. Carnitine partially reverses the inhibition of pyruvate and palmitic acid oxidation by propionic acid, and this reversal is associated with increased propionylcarnitine formation. The present study provides a metabolic basis for the efficacy of carnitine in patients with abnormal organic acid accumulation, and the observation that such patients appear to have increased carnitine requirements (‘carnitine insufficiency’).
Carnitine palmitoyltransferase in liver and five extrahepatic tissues in the rat. Inhibition by dl-2-bromopalmitoyl-CoA and effect of hypothyroidism
Authors: Saggerson E D , Carpenter C A
Published: August 2015

Mitochondria were isolated from rat adult liver, foetal liver, kidney cortex, heart, skeletal muscle and interscapular brown adipose tissue. DL-2-Bromopalmitoyl-CoA inhibited the overt form of carnitine palmitoyltransferase (CPT1) in heart, skeletal muscle and brown adipose tissue, with an IC50 value (concentration giving 50% inhibition) of 1.3-1.6 microM. By contrast, the IC50 value for inhibition of the kidney or adult liver enzyme was 0.08-0.1 microM. CPT1 in near-term foetal liver differed from that in adult liver in that the IC50 for inhibition by 2-bromopalmitoyl-CoA was 0.57 microM. It is suggested that there may be tissue-specific forms of the catalytic entity of CPT1 and that foetal liver may contain a mixture of adult liver- and muscle-type enzymes. In rats made hypothyroid by administration of propylthiouracil and an iodine-deficient diet, hepatic CPT1 activity was decreased by 83%. However, CPT1 activity in extrahepatic tissues showed no adaptive decrease in hypothyroidism.
Immunological analysis of plant mitochondrial NADH dehydrogenases
Authors: Cottingham I R , Cleeter M W J , Ragan C I , Moore A L
Published: August 2015

Plant mitochondrial NADH dehydrogenases were analysed by two immunological strategies. The first exploited an antiserum raised to a preparation of SDS-solubilized mitochondrial-inner-membrane particles. By using a combination of activity-immunoprecipitation and crossed immunoelectrophoresis, it was shown that Triton X-100-solubilized membranes contain at least three immunologically distinct NADH dehydrogenases. Two of these were subsequently isolated by line immunoelectrophoresis and analysed for polypeptide composition: one contained three polypeptides with molecular masses of 75, 62 and 41 kDa; the other was a single polypeptide with a molecular mass of 53 kDa. The other approach was to probe plant mitochondrial membranes with antibodies raised to a purified preparation of ox heart rotenone-sensitive NADH dehydrogenase and subunits thereof. Cross-reactions were observed with the subunit-specific antisera against the 30 and 49 kDa ox heart proteins. However, the molecular masses of the equivalent polypeptides in plant mitochondria are slightly lower, at 27 and 46 kDa respectively.
Potentiation of specific association of insulin with HepG2 cells by phorbol esters
Authors: Blake A D , Strader C D
Published: August 2015

The effects of tumour-promoting phorbol esters on the receptor-mediated endocytosis of insulin were investigated in the human hepatoma cell line HepG2. Treatment of these cells with the biologically active phorbol 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with the non-tumour-promoting analogue 4 alpha-phorbol 12,13-didecanoate, resulted in dramatic morphological changes, which were accompanied by a 1.5-2.5-fold increase in specific 125I-insulin association with the cells at 37 degrees C. This increase in insulin binding was not observed when the binding reaction was performed at 4 degrees C. The potentiation of 125I-insulin association with TPA-treated cells at 37 degrees C could be completely accounted for by an increase in the intracellular pool of internalized insulin; there was no concomitant increase in cell-surface insulin binding. Dissociation studies showed that the enhanced internalization of insulin by cells after treatment with TPA resulted from a decrease in the rate of intracellular processing of the insulin after receptor-mediated endocytosis. The phorbol-ester-induced enhancement of internalized insulin in HepG2 cells was additive with the potentiation of endocytosed insulin induced by both the lysosomotropic reagent chloroquine and the ionophore monensin; this indicates that TPA affects the intracellular processing of the insulin receptor at a point other than those disrupted by either of these two reagents. The potentiation of insulin receptor internalization by tumour-promoting phorbol esters could be completely mimicked by treatment with phospholipase C, but not with phospholipase A, and partially mimicked by treatment with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol. By these criteria, the effects of phorbol esters on the insulin receptor in HepG2 cells appear to be mediated through protein kinase C. These results support the concept that the activation of protein kinase C by treatment with phorbol esters causes a perturbation of the insulin-receptor-mediated endocytotic pathway in HepG2 cells, reflected in a long-term decreased rate of dissociation of internalized insulin by the phorbol-ester-treated cells.
The effect of desferrioxamine on transferrin receptors, the cell cycle and growth rates of human leukaemic cells
Authors: Bomford A , Isaac J , Roberts S , Edwards A , Young S , Williams R
Published: August 2015

The effect of the iron chelator, desferrioxamine, on transferrin binding, growth rates and the cell cycle was investigated in the human leukaemic cell line, K562. At all concentrations of the chelator (2-50 microM) binding of 125I-transferrin was increased by 24 h and reached a maximum at 72-96 h. Maximum binding (6-8-fold increased) occurred in cells treated with 20 microM-desferrioxamine, in contrast with control cells which, at 96 h, showed a 50% decrease over initial binding. Scatchard analysis at 4 degrees C showed that this increased binding was due to an increase in the number of receptors, as the Kd was similar in induced (1.8 nM) and control (1.5 nM) cells. After 96 h cells, cultured with 20 and 50 microM-desferrioxamine accumulated 59Fe from bovine transferrin at over twice the rate found with control cells, reflecting the increase in transferrin receptors. Although iron uptake was unimpaired by the chelator there was a dose-dependent inhibition of cell growth, with control cells completing three divisions in 96 h and those in 10 microM-desferrioxamine only two divisions. At the highest concentration (50 microM), cell division was abrogated although cell viability was maintained (85%). In contrast, DNA synthesis was not markedly affected, except at 50 microM-desferrioxamine when incorporation of [3H]thymidine was 52% of that in control cells. Flow cytometry revealed that there was a progressive accumulation of the cells in the active phases of their cycle (S, G2 + M). Desferrioxamine may increase transferrin receptors in two ways: by chelating a regulatory pool of iron within the cell, and by arresting cells in S phase when receptors are maximally expressed.
Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase
Authors: Varalakshmi K , Savithri H S , Rao N A
Published: August 2015

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.
NADP-specific isocitrate dehydrogenase from the citric acid-accumulating fungus Aspergillus niger
Authors: Meixner-Monori B , Kubicek C P , Harrer W , Schreferl G , Rohr M
Published: August 2015

NADP-specific isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] was purified 200-300-fold from the citric acid-accumulating fungus Aspergillus niger. The enzyme consists of a single polypeptide chain with a molecular mass of 60 +/- 4 kDa and has a pI of 5.9 +/- 0.2. Only a single enzyme protein was found, although the enzyme appears to occur both in the mitochondrion and in the cytoplasm. Growth on organic acids as carbon sources or on NO3- as nitrogen source led to increased activities, whereas the presence of amino acids led to lower activities. The enzyme exhibits hyperbolic kinetics with respect to its substrates isocitrate and NADP+. Mn2+ and Mg2+ are obligatory for enzyme activity. The enzyme is inhibited by its products alpha-oxoglutarate and NADPH. Among various metabolites, ATP and citrate appear to inhibit the enzyme at a concentration considered to occur intracellularly. In both cases, however, the mechanism is a removal of the metal ion cofactor from the substrates. It is concluded that under physiological conditions, where the Mg2+ content is around 10 mM, the observed inhibition by ATP or citrate is of poor regulatory significance.
Cystine transport in purified rat liver lysosomes
Authors: Jonas A J
Published: August 2015

Amino acid efflux from highly purified rat liver lysosomes exposed to the methyl ester derivatives of leucine, methionine, tyrosine and cystine was examined. The lysosomal efflux of leucine, methionine and tyrosine was unaffected by the presence of MgATP, whereas cystine efflux was stimulated by MgATP. Exposure of lysosomes to 2 mM-MgATP resulted in lysosomal acidification and a 0.5 pH unit increase in the lysosomal pH gradient through the action of a proton-pumping ATPase. Cystine efflux was also stimulated when the lysosomal proton gradient was increased through changes in buffer pH. Decreasing the lysosomal proton gradient with ionophores resulted in diminished cystine efflux. Bivalent cations had no effect on the lysosomal efflux of leucine, methionine and tyrosine. However, cystine efflux was stimulated by the presence of bivalent cations even when the lysosomal proton gradient was minimized. Cation-stimulated cystine efflux was inhibited by the presence of the calcium ionophore A23187, which altered the lysosomal membrane potential. Cystine efflux from lysosomes appears to be uniquely dependent on pH gradients and cation concentrations.
Vitamin K antagonism of coumarin anticoagulation. A dehydrogenase pathway in rat liver is responsible for the antagonistic effect
Authors: Wallin R
Published: August 2015

In the liver, it appears that there are two different pathways for vitamin K reduction. One pathway is irreversibly inhibited by coumarin anticoagulant drugs. The other pathway has been shown in the present study to be composed of enzymes that are not effected by physiological ‘in vivo’ concentrations of these drugs. This pathway appears to be responsible for the antidotal effect of vitamin K in overcoming coumarin poisoning. In rat liver the pathway has been shown to be composed of DT-diaphorase (EC.1.6.99.2) and a microsomal dehydrogenase(s). The activity of the microsomal dehydrogenase(s) was 3.6-fold higher with NADH than with NADPH present in the test system. It appears that this enzyme is the physiologically important enzyme in the pathway. In contrast with DT-diaphorase, this enzyme(s) is shown to be tightly associated with the mirosomal membrane. The enzyme(s) is not identical with either of the quinone-reducing enzymes cytochrome P-450 reductase or cytochrome-b5 reductase. Our data thus postulate the existence of an as-yet-unidentified microsomal dehydrogenase that appears to have an important function in the pathway.