bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of cathepsin D by dithiophosgene and by 2,2-dichloro-1,3-dithiacyclobutanone
Authors: Rakitzis E T , Malliopoulou T B
Published: August 2015

Cathepsin D, purified from bovine spleen, is inactivated by the unstable inhibitors dithiophosgene and 2,2-dichloro-1,3-dithiacyclobutanone. Inhibition constants are identical for both of the compounds tested: Ki 96.1 μM;k/c0.406. It appears that the active species is 2,2-dichloro-1,3-dithiacyclobutanone, to which dithiophosgene is hydrolysed before cathepsin D inactivation.
Localization of inhibition by adenosine diphosphate of phosphoglycerate-dependent oxygen evolution in a reconstituted chloroplast system
Authors: Slabas A R , Walker D A
Published: August 2015

ADP was shown to inhibit phosphoglycerate-dependent O2 evolution in a simplified reconstituted chloroplast system containing 3-phosphoglycerate kinase and triose phosphate dehydrogenase. Rates of O2 evolution in the simplified system are comparable with those obtained by using stromal protein rather than purified enzymes. ADP does not inhibit O2 evolution with glycerate 1,3-biphosphate as substrate nor does it inhibit triose phosphate dehydrogenase. The inhibitory effect of ADP is attributed to an increase in the rate of conversion of glycerate biphosphate into phosphoglycerate. The results are discussed in terms of control by ADP of phosphoglycerate-dependent oxygen evolution.
Interactions of some acceptors with superoxide anion radicals formed by the NADPH-specific flavoprotein in rat liver microsomal fractions
Authors: Mishin V , Pokrovsky A , Lyakhovich V V
Published: August 2015

In rat liver microsomal fractions oxidation of adrenaline was effected by superoxide anion radicals (O2-), whereas cytochrome c, 2,6-dichlorophenol-indophenol and ferricyanide accepted electrons from NADPH-specific flavoprotein only directly. Nitro Blue Tetrazolium was reduced both by O2- and by the direct acceptance of electrons. Elevation of pH and addition of menadione shift the Nitro Blue Tetrazolium reduction towards the O2--dependent pathway. From the values of the kinetic constants for interaction of adrenaline and Nitro Blue Tetrazolium with NADPH-specific flavoprotein, the rates of generation of O2- in rat liver microsomal fraction were determined.
Protein kinase activity in rat testis interstitial tissue. Effect of luteinizing hormone and other factors
Authors: Cooke B A , van der Kemp A J. W. C. M
Published: August 2015

Protein kinase activity was determined in subcellular fractions of rat testis interstitial tissue after incubation of the intact tissue with LH (luteinizing hormone) in vitro. Various factors that might have changed the activity of this enzyme during preparation of the fractions before assay were also investigated. The following results were obtained. 1. LH and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) added together during incubation of the interstitial tissue caused a twofold increase in the protein kinase activity in the total tissue homogenate and subcellular fractions (12000g X 5 min pellet and 105000g X 60 min supernatant and pellet). 2. A decrease of approx. 40% in the total amount of protein kinase recovered in the soluble fraction (105000g supernatant) occurred in tissue incubated with LH and 3-isobutyl-1-methylxanthine when compared with the controls. No change in total activity was found in the other fractions. 3. LH and 3-isobutyl-1-methylxanthine caused an increase in cyclic AMP concentration in the soluble fraction (from 30 +/- 6 to 450 +/- 40 pmol/mg of protein, means +/- S.E.M., n = 4), but there was little or no increase in the particulate fractions [from 9 +/- 1 to 13 +/- 3 pmol/mg of protein (n = 3) and from 6 +/- 2 to 23 +/- 11 pmol/mg of protein (n = 3) in the 12000g and 105000g pellets respectively]. 4 Addition of 3-isobutyl-1-methylxanthine alone had little effect on protein kinase activity or cyclic AMP concentrations. 5. Little or no protein kinase activity could be demonstrated in subcellular particulate fractions unless Triton X-100 was added; the effect of this detergent was shown to be at least partly due to the inhibition of adenosine triphosphatase activity. 6. In the presence of Triton X-100 approx. 57% of the total protein kinase activity in the homogenate was found in the 105000g supernatant compared with 11% in the 105000g pellet and 32% in the 12000g pellet. 7. In contrast with adipose-tissue protein kinase [Corbin et al. (1973) J. Biol. Chem. 248, 1813-1821] the relative amounts of cyclic AMP-dependent and -dependent enzyme were not affected by dilution of the interstitial-tissue fractions. NaCl (0.5 M) decreased the estimated total amount of protein kinase activity.
Respiration-dependent efflux of magnesium ions from heart mitochondria
Authors: Crompton M , Capano M , Carafoli E
Published: August 2015

Energy-linked respiration causes a net movement of Mg2+ between rat heart mitochondria and the ambient medium. When the extramitochondrial concontration of Mg2+ is less that about 2.5 mM the net movement of Mg2+ constitutes an efflux, whereas a net influx of Mg2+ occurs when the external concentration of Mg2+ is greater than this. Both the efflux and the influx are induced to only a very small degree by externally added ATP. Evidence suggests that Pi may be required for the respiration-induced efflux of Mg2+.
The effects of benzopyrene and safrole on biphenyl 2-hydroxylase and other drug-metabolizing enzymes
Authors: McPherson F J. , Bridges J W. , Parke D V.
Published: August 2015

A study was made of the nature and specificity of the increase in biphenyl 2-hydroxylase activity after preincubation of liver microsomal preparations with various carcinogens in vitro. This enhancement of enzyme activity in vitro was investigated in mouse, hamster and rat, and although the rat appears to be atypical in the variation of the pattern of 2- and 4-hydroxylation with age, similar enhancements were detectable in each species examined. An increase in biphenyl 2-hydroxylase activity was apparent 2h after intraperitoneal administration of safrole or benzopyrene to mature Wistar albino rats and appeared to be similar in nature to that observed after preincubation of liver microsomal preparations with the same chemical in vitro. Investigation of other drug-metabolizing enzyme systems suggests that the enhancing effects of carcinogens in vitro are specific for biphenyl 2-hydroxylase. No correlation between the enhancement of biphenyl 2-hydroxylase and inhibtion of biphenyl 4-hydroxylase was apparent.
Comparison between the ribosomal ribonucleic acids from free and membrane-bound ribosomal fractions of HeLa cells
Authors: Khan M S N , Maden B E H
Published: August 2015

The rRNA species from the total cytoplasmic, free and membrane-bound fractions of HeLa cells were compared. With the use of T1 ribonuclease and combined T1 ribonuclease plus pancreatic ribonuclease ‘fingerprinting’ procedures, no significant differences were found between the rRNA species from the different subcellular fractions.
The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies
Authors: Robson B , Pain R H.
Published: August 2015

1. A homogeneous preparation of penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.
Ligand-apomyoglobin interactions. Configurational adaptability of the haem-binding site
Authors: Lind K E , Møller J V
Published: August 2015

1. The interaction of the haem-binding region of apomyoglobin with different ligands was examined by ultrafiltration, equilibrium dialysis and spectrophotometry, to study unspecific features of protein-ligand interactions such as they occur in, for example, serum albumin binding. 2. Apomyoglobin, in contrast with metmyoglobin, binds at pH 7, with a high affinity, one molecule of Bromophenol Blue, bilirubin and protoporphyrin IX, two molecules of n-dodecanoate and n-decyl sulphate and four molecules of n-dodecyl sulphate and n-tetradecyl sulphate. 3. The number of high-affinity sites and/or association constants for the alkyl sulphates are enhanced by an increase of hydrocarbon length, indicating hydrophobic interactions with the protein. 4. Measurements of the temperature-dependence of the association constants of the high-affinity sites imply that the binding processes are largely entropy-driven. 5. Binding studies in the presence of two ligands show that bilirubin plus Bromophenol Blue and dodecanoate plus Bromophenol Blue can be simultaneously bound by apomyoglobin, but with decreased affinities. By contrast, the apomyoglobin-protoporphyrin IX complex does not react with Bromophenol Blue. 6. Optical-rotatory-dispersion measurements show that the laevorotation of apomyoglobin is increased towards that of metmyglobin in the presence of haemin and protoporphyrin IX. Small changes in the optical-rotatory-dispersion spectrum of apomyoglobin are observed in the presence of the other ligands. 7. It is concluded that the binding sites on apomyoglobin probably do not pre-exist but appear to be moulded from predominantly non-polar amino acid residues by reaction with hydrophobic ligands. 8. Comparison with data in the literature indicates that apomyoglobin on a weight basis has a larger hydrophobic area avaialble for binding of ligands than has human serum albumin. On the other hand, the association constants of serum for the ligands used in this study are generally somewhat larger than those of apomyoglobin.
Inhibition of acetylcholinesterase by derivatives of 1,3,2-dioxaphosphorinane 2-oxide
Authors: Coult D B
Published: August 2015

Cholinesterases are inhibited by 2-fluoro-1,3,2-dioxaphosphorinane 2-oxide by a mechanism that involves a slow association step followed by a very slow phosphorylation step. No phosphorylation step was observed for the interaction between acetylcholinesterase and 2-S-[2′-(NN-diethylamino)ethyl]thio-1,3,2-dioxaphosphorinane 2-oxide.
The dissociation of exocytosis and respiratory stimulation in leucocytes by ionophores
Authors: Zabucchi G , Romeo D
Published: August 2015

By exploiting the unique characteristics of three ionophores, experimental conditions were found which permit the dissociation of respiratory stimulation from secretion in polymorphonuclear leucocytes. A marked stimulation of respiration was produced by ionophore X537A, which binds and transports both alkali-earth and alkali cations. The stimulatory activity of this ionophore was the same at either high or low Na+/K+ ratios in the medium and was virtually unaffected by extracellular Ca2+. A slight stimulation of oxygen consumption was also caused by the K+-selective ionophore valinomycin and by ionophore A23187, which complexes and transfers bivalent cations. Ionophore X537A and valinomycin were unable to stimulate selective release of granuleassociated β-glucuronidase and gradually increased cell fragility, as monitored by increased leakage of lactate dehydrogenase. Ionophore A23187 slightly increased exocytosis of β-glucuronidase. In a Mg2+-free medium, Ca2+, added simultaneously with ionophore A23187, greatly enhanced respiration and secretion of the granule enzyme. If Ca2+ was added a few minutes after the ionophore, exocytosis occurred, but no respiratory burst was observed. If the latter experiment was repeated in the presence of extracellular Mg2+, both secretion and respiration were stimulated. This effect was not produced by Mn2+ or Ba2+. It is proposed that Ca2+ is required for triggering selective secretion of granule enzymes from leucocytes is caused by an intracellular redistribution of cations, which may invovle Mg2+-dependent mechanisms.
The androgenic regulation of prostate proteins with a high affinity for deoxyribonucleic acid. Evidence for a prostate deoxyribonucleic acid-unwinding protein
Authors: Mainwaring W I P , Rennie P S , Keen J
Published: August 2015

1. When testosterone is injected into castrated rats in vivo, a significant increase in the incorporation of [35S]methionine into prostate proteins may be detected under conditions in vitro. 2. Studies based on DNA-cellulose chromatography show that the synthesis of prostate proteins with a high affinity for DNA is particularly enhanced by androgenic stimulation. 3. These changes in protein synthesis are negated when the anti-androgen, cyproterone acetate, is administered concomitantly with testosterone in vivo. 4. Two assays were developed for measuring the strand separation of prostate DNA; first, the retention of 3H-labelled native DNA on nitrocellulose membranes, and second, the activation of native DNA as a template for 9S prostate DNA polymerase. On the basis of these criteria, DNA-unwinding activity is present in the prostate gland and it is regulated by androgens in a steroid-and tissue-specific manner. 5. The results are discussed in the context of the mechanism of action of androgens, particularly since the changes provoked in DNA-unwinding activity by androgens precede the onset of DNA replication and mitosis.
Nuclear binding of progesterone in hen oviduct. Binding to multiple sites in vitro
Authors: Pikler G M , Webster R A , Spelsberg T C
Published: August 2015

Steroid hormones, including progesterone, are known to bind with high affinity (Kd approximately 1 × 10(-10)M) to receptor proteins once they enter target cells. This complex (the progesterone-receptor) then undergoes a temperature-and/or salt-dependent activation which allows it to migrate to the cell nucleus and to bind to the deoxyribonucleoproteins. The present studies demonstrate that binding the hormone-receptor complex in vitro to isolated nuclei from the oviducts of laying hens required the same conditions as do other studies of bbinding in vitro reported previously, e.g. the hormone must be complexed to intact and activated receptor. The assay of the nuclear binding by using multiple concentrations of progesterone receptor reveals the presence of more than one class of binding site in the oviduct nuclei. The affinity of each of these classes of binding sites range from Kd approximately 1×10(-9)-1×10(-8)M. Assays using free steroid (not complexed with receptor) show no binding to these sites. The binding to each of the classes of sites, displays a differential stability to increasing ionic concentrations, suggesting primarily an ionic-type interaction for all classes. Only the highest-affinity class of binding site is capable of binding progesterone receptor under physioligical-saline conditions. This class represent 6000-10000 sites per cell nucleus and resembles the sites detected in vivo (Spelsberg, 1976, Biochem. J. 156, 391-398) which cause maximal transcriptional response when saturated with the progesterone receptor. The multiple binding sites for the progesterone receptor either are not present or are found in limited numbers in the nuclei of non-target organs. Differences in extent of binding to the nuclear material between a target tissue (oviduct) and other tissues (spleen or erythrocyte) are markedly dependent on the ionic conditions, and are probably due to binding to different classes of sites in the nuclei.
The development of the intense circular-dichroic signal during granum formation in greening etiolated maize
Authors: Demeter S , Mustardy L , Machowicz E
Published: August 2015

In greening maize mesophyll, circular dichroism (c.d.) revealed the early formation of protein-chlorophyll complexes, followed by unorganized chlorophyll. The intense c.d. [Gregory & Raps (1974) Biochem. J. 142, 193-201] appeared later still, whereas membrane-membrane contact (stacking), measured from electron micrographs, appeared much earlier. Isolated grana, which still showed stacking, lost 92% of their original intense c.d.; intense c.d. is not therefore simply dependent on stacking.
The calcium conductance of the inner membrane of rat liver mitochondria and the determination of the calcium electrochemical gradient
Authors: Heaton G M , Nicholls D G
Published: August 2015

1. A method is described for establishing steady-state conditions of calcium transport across the inner membrane of rat liver mitochondria and for determining the current of Ca2+ flowing across the membrane, together with the Ca2+ electrochemical gradient across the native Ca2+ carrier. These parameters were used to quantify the apparent Ca2+ conductance of the native carrier. 2. At 23 degrees C and pH7.0, the apparent Ca2+ conductance of the carrier is close to 1 nmol of Ca2+-min-1-mg of protein-1 mV-1. Proton extrusion by the respiratory chain, rather than the Ca2+ carrier itself, may often be rate-limiting in studies of initial rates of Ca2+ uptake. 3. Under parallel conditions, the endogenous H+ conductance of the membrane is 0.3 nmol of H+-min-1-mg of protein-1-mV-1. 4. Ruthenium Red and La3+ both strongly inhibit the Ca2+ conductance of the carrier, but are without effect on the H+ conductance of the membrane. 5. The apparent Ca2+ conductance of the carrier shows a sigmoidal dependence on the activity of Ca2+ in the medium. At 23 degrees C and pH7.2, half-maximum conductance is obtained at a Ca2+ activity of 4.7 muM. 6. The apparent Ca2+ conductance and the H+ conductance of the inner membrane increase fourfold from 23 degrees to 38 degrees C. The apparent Arrhenius activation energy for Ca2+ transport is 69kJ/mol. The H+ electrochemical gradient maintained in the absence of Ca2+ transport does not vary significantly with temperature. 7. The apparent Ca2+ conductance increases fivefold on increasing the pH of the medium from 6.8 to 8.0. The H+ conductance of the membrane does not vary significantly with pH over this range. 8. Mg2+ has no effect on the apparent Ca2+ conductance when added at concentration up to 1 mM. 9. Results are compared with classical methods of studying Ca2+ transport across the mitochondrial inner membrane.
Effect of phenobarbitone on the nucleocytoplasmic transport of ribonucleic acid in vitro
Authors: Hazan N , McCauley R
Published: August 2015

The transport of nucleic acids from the nucleus to the cytoplasm is a potential site for modification of normal cellular processes by drugs and hormones. In this study the effect of phenobarbitone on nucleocytoplasmic transport of ribosomes was measured in an assay system in vitro. The transport of radioactive ribosomes from isolated rat hepatic nuclei to unlabelled post-microsomal supernatant was measured in rats treated with 80 mg of phenobarbitone/kg body wt. or saline 3h before death. With either treatment, transport was linear with time, and dependent on temperature and the presence of ATP. However, phenobarbitone treatment increased transport of ribonucleoproteins over saline-treated animals nearly twofold. The effect of phenobarbitone was mediated through the cytosol, but was not the result of altered stability of the RNA transported to the cytosol. Cycloheximide (5 mg/kg body wt.) given 3.5 h before death inhibited the stimulation of transport by phenobarbitone. The data indicate that phenobarbitone increased the transport of RNA by stimulating the synthesis of cytosol factors that regulate transport of RNA from the nucleus.
The purification and properties of ferritin from human serum
Authors: Worwood M , Dawkins S , Wagstaff M , Jacobs A
Published: August 2015

1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.
Autoxidation of soluble trypsin-cleaved microsomal ferrocytochrome b5 and formation of superoxide radicals
Authors: Berman M C , Adnams C M , Ivanetich K M , Kench J E
Published: August 2015

The rate and mechanism of autoxidation of soluble ferrocytochrome b5, prepared from liver microsomal suspensions, appear to reflect an intrinsic property of membrane-bound cytochrome b5. The first-order rate constant for autoxidation of trypsin-cleaved ferrocytochrome b5, prepared by reduction with dithionite, was 2.00 × 10(−3) +/− 0.19 × 10(−3) S-1 (mean +/− S.E.M., n =8) when measured at 30 degrees C in 10 mM-phosphate buffer, pH 7.4. At 37 degrees C in aerated 10 mM-phosphate buffer (pH 7.4)/0.15 M-KCl, the rate constant was 5.6 × 10(-3) S-1. The autoxidation reaction was faster at lower pH values and at high ionic strengths. Unlike ferromyoglobin, the autoxidation reaction of which is maximal at low O2 concentrations, autoxidation of ferrocytochrome b5 showed a simple O2-dependence with an apparent Km for O2 of 2.28 × 10(-4) M (approx. 20kPa or 150mmHg)9 During autoxidation, 0.25 mol of O2 was consumed per mol of cytochrome oxidized. Cyanide, nucleophilic anions, EDTA and catalase each had little or no effect on autoxidation rates. Adrenaline significantly enhanced autoxidation rates, causing a tenfold increase at 0.6 mM. Ferrocytochrome b5 reduced an excess of cytochrome c in a biphasic manner. An initial rapid phase, independent of O2 concentration, was unaffected by superoxide dismutase. A subsequent slower phase, which continued for up to 60 min, was retarded at low O2 concentrations and inhibited by 65% by superoxide dismutase at a concentration of 3 mug/ml. It is concluded that autoxidation is responsible for a significant proportion of electron flow between cytochrome b5 and O2 in liver endoplasmic membranes, this reaction being capable of generating superoxide anions. A biological role for the reaction is discussed.
Kinetic analysis of experiments involving the single turnover of an enzyme
Authors: Holbrook J J , Gutfreund H , Südi J
Published: August 2015

The complete solution to the kinetic equation for nucleotide fluorescence quenching on addition of pyruvate to the late dehydrogenase-NADH complex modifies previous interpretations of such experiments.
Instability of rat liver chromatin and other nuclear non-histone proteins in alkaline solution
Authors: Gronow M , Thackrah T M , Lewis F A
Published: August 2015

Non-histone proteins from rat liver nuclei and chromatin were shown to be hydrolysed in 0.1M or-1M-NaOH solutions both at 4 degrees and 18 degrees C; 24h in 1M-NaOH at 18 degrees C is sufficient to break down approx. 77% of these proteins to low-molecular-weight peptides. Loss of protein material banding in the region of pH5.5-8.0 has been demonstrated by isoelectric focusing in polyacrylamide gels, and fine high-molecular-weight bands are no longer visible on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results indicate that care must be taken when analysing non-histone-protein fractions to avoid exposure to alkaline pH conditions.
The subunit structure of thyroglobulin
Authors: Pitt-Rivers R
Published: August 2015

Human and rat thyroglobulin were reduced and alkylated in aqueous alkaline conditions in the absence of denaturants; the product of reduction in both cases has been found to have mol.wt. about 165000, or one-quarter that of the native molecule.
Serum glycoprotein synthesis after partial hepatectomy in the rat
Authors: Serafini-Cessi F
Published: August 2015

The incorporation in vivo of D-[1-14C]glucosamine into serum glycoproteins and proteins of liver microsomal fractions shows a decrease in the early stages (24h) after partial hepatectomy compared with sham-operated animals; 72h after partial hepatectomy the specific radioactivity of hexosamines bound to liver microsomal fractions reaches the same value as for sham-operated animals.
Polyamine and ornithine metabolism during the germination of conidia of Aspergillus nidulans
Authors: Stevens L , McKinnon I M , Winther M
Published: August 2015

1. The activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase and ornithine-2-oxoglutarate aminotransferase were studied during the first 24 h of conidial germination in Aspergillus nidulans. 2. Increases (over 100-fold) in the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase occurred during the emergence of the germ-tube and before the doubling of DNA and this was followed by a sharp fall in the activities of both enzymes by 16h. 3. The increase in ornithine decarboxylase could be largely suppressed if 0.6 mM-putrescine was added to the growth medium. 4. Low concentrations of cycloheximide, which delayed germination by 2h, caused a corresponding delay in the changes in ornithine decarboxylase activity. 5. Ornithine-2-oxoglutarate aminotransferase activity increased steadily during the first 24h of germination. 6. Ornithine or arginine in the growth medium induced higher activity of ornithine-2-oxoglutarate aminotransferase, but did not affect ornithine decarboxylase activity. 7. The significance of these enzyme changes during germination is discussed.
The effects of added purines on urate and purine synthesis de novo by isolated chick liver, kidney and lymphoid cells
Authors: Badenoch-Jones P , Buttery P J
Published: August 2015

1. Isolated chick lymphoid cells, together with isolated chick liver and kidney cells, incorporate [1-14C]glycine or [14C]formate into urate. 2. Of the cell types used, bursal cells incorporate 14C into urate at the fastest rate, although the output of total urate by bursal cells is only 10% that of liver cells. 3. When suspended in Eagle's medium the incorporation of 14C into urate is inhibited by adenine and guanine up to 1 mM. In contrast, the addition of 1 mM-AMP or -GMP results in a relatively large stimulation of this incorporation. 4. Added adenine is rapidly taken up by liver cells and then released in an unmetabolized form; AMP is taken up more slowly and is rapidly metabolized. The metabolites (possibly including adenine) are then released. 5. Intracellular liver 5-phosphoribosyl 1-pyrophosphate is approx. 0.7mM and remains constant or falls slightly during a 3 h incubation of the cells. 6. The addition of adenine or guanine, AMP or GMP, does not alter liver intracellular 5-phosphoribosyl 1-pyrophosphate concentrations. Added 5-phosphoribosyl 1-pyrophosphate is not taken up by liver cells. 7. The results are discussed in the context of the control of urate and purine synthesis de novo in the chick.
The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells
Authors: Yue B Y J T , Baum J L , Silbert J E
Published: August 2015

Confluent monolayer cultures of rabbit corneal endothelial and stromal cells were incubated independently with [35S]sulphate and [3H]glucosamine for 3 days. AFter incubation, labelled glycosaminoglycans were isolated from the growth medium and from a cellular fraction. These glycosaminoglycans were further characterized by DEAE-cellulose column chromatography and by sequential treatment with various glycosamino-glycan-degrading enzymes. Both endothelial and stromal cultures synthesized hyaluronic acid as the principal product. The cell fraction from the stromal cultures, however, had significantly less hyaluronic acid than that from the endothelial cultures. In addition, both types of cells synthesized a variety of sulphated glycosaminoglycans. The relative amounts of each sulphated glycosaminoglycan in the two cell lines were similar, with chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate as the major components. Heparan sulphate was present in smaller amounts. Keratan sulphate was also identified, but only in very small amounts (1-3%). The presence of dermatan sulphate and the high content of hyaluronic acid are similar to the pattern of glycosaminoglycans seen in regenerating or developing tissues, including cornea.
Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Location of specifically modified methionine residues
Authors: Cheng K W
Published: August 2015

Bovine lutropin (luteinizing hormone) was carboxymethylated at pH3.0 for 12 h at 37 degrees C with iodoacetic acid for specific modification of methionine residues. To facilitate the location of preferentially modified methionine residues, iodoE114C]acetic acid was added as tracer. The alpha and beta subunits of bovine lutropin were carboxymethylated with a 2- or 5-fold molar excess of iodoacetic acid either in the presence or absence of their counterpart subunits. The modified subunits were separated and isolated by counter-current distribution followed by gel filtration on Sephadex G-100. To locate the modified methiones, the isolated alpha or beta chain was reduced. S-carboxymethylated and subjected to tryptic hydrolysis. The tryptic peptides were fractionated by gel filtration on Bio-Gel P-10. From analyses of the purified 14C-labelled tryptic peptides, it was observed that methionine-8 and -33 in bovine lutropin alpha chain and methionine-52 in the beta chain were preferentially modified. Similar results were obtained when isolated alpha and beta subunits were individually carboxymethylated in the absence of their counterpart subunit under identical conditions. The fact that a recombinant of native human lutropin alpha chain, in which a valine residue is present in the position corresponding to methionine-8 of bovine lutropin alpha chain, and carboxymethylated bovine lutropin beta chain regenerated a substantial amount of receptor-site-binding activity indicated that methionine-8 in bovine alpha chain was biologically not essential. These studies showed clearly that both methionine-33 in the alpha chain and methionine-52 in the beta subunit were involved for optimum binding between bovine lutropin and its receptors for expression of hormonal activity.
Purification and properties of arylsulphatase A from rabbit testis
Authors: Yang C H , Srivastava P N
Published: August 2015

Rabbit testis arylsulphatase A was purified 140-fold with a recovery of 20% from detergent extracts of an acetone-dried powder by using DE-52 cellulose column chromatography, gel filtration on Sephadex G-200 and preparative isoelectric focusing. The purified enzyme showed one major band with one minor contaminant on electrophoresis in a 7.5% (w/v) polyacrylamide gel at pH8.3. On sodiumdodecyl sulphate/polyacrylamidegel electrophoresis, a single major band was observed with minor contaminants. The final preparation of enzyme was free from general proteolytic, esterase, hyaluronidase, beta-glucuronidase and beta-galactosidase activities. Rabbit testicular arylsulphatase A exists as a dimer of mol.wt. 110000 at pH7.1. At pH5.0 the enzyme is a tetramer of mol.wt. 220000. Arylsulphatase A appears to consist of two identical subunits of mol.wt. 55000 each. The highly purified enzyme has pI4.6. The enzyme hydrolyses p-nitrocatechol sulphate with Km and Vmax, of 4.1 mM and 80nmol/min respectively, but has no activity toward p-nitrophenyl sulphate. The pH optimum of the enzyme varies with the incubation time. By applying Sephacex G-200 chromatography and preparative isoelectric focusing, one form of enzyme was obtained. The enzyme has properites common to arylsulphatase A of other sources with respect to the anomalous time-activity relationship, pI, inhibition by PO42-, SO32- and Ag+ ions and substrate affinity to p-nitrocatechol sulphate. However, the enzyme shows the temperature optimum of arylsulphatase B of other species.
Arylsulphatase and glycosulphatase of Charonia lampas. Substrate specificity towards sugar sulphate derivatives
Authors: Hatanaka H , Ogawa Y , Egami F
Published: August 2015

The specificity of the arylsulphatase and glycosulphatase enzymes of the marine mollusc Charonia lampas towards a number of carbohydrate sulphate esters was examined. The results confirm that the relationship between the established nomenclature of the two enzymes and their respective specificities is inconsistent.
Inactivation of human thrombin in the presence of human α1-proteinase inhibitor
Authors: Matheson N R , Travis J
Published: August 2015

Both the clotting and esterase activities of thrombin are inhibited by α1-proteinase inhibitor (α1-antitrypsin). The inhibition is a time-and temperature-dependent reaction which is proportional to the molar ratio of thrombin to inhibitor. Both the active-site serine residue of thrombin and the reactive-site lysine residue of α1-proteinase inhibitor are involved. α1-Proteinase inhibitor forms a 1:1 complex with thrombin that is comparable with the complex formed with trypsin and other proteinases. Incubation of the inhibitor with excess of thrombin, however, results in inactivation of nearly all the enzyme, even though only as much complex is formed as α1-proteinase inhibitor present. A portion of the remaining thrombin apparently aggregates. These results suggest that the mechanism for inhibition of thrombin may not be exactly the same as for trypsin, which is inhibited only to the extent to which complex is formed.
Modification of glyceraldehyde 3-phosphate dehydrogenase activity by adsorption on phospholipid vesicles
Authors: Wooster M S , Wrigglesworth J M
Published: August 2015

1. The adsorption of [14C]carboxymethylated glyceraldehyde 3-phosphate dehydrogenase to negatively charged liposomes of phsphatidic acid/phosphatidylcholine (3:7, w/w) was investigated. The apparent association constant at I/2 = 60, pH 7.6, was 0.4 × 10(6)M-1. Adsorption decreased as ionic strength and pH were increased. 2. In the presence of negatively charged liposomes, the Km value for glyceraldehyde 3-phosphate of glyceraldehyde 3-phosphate dehydrogenase was increased and Vmax. decreased. In the presence of positively charged liposomes, the Km value for glyceraldehyde 3-phosphate decreased and there was no significant change in Vmax. Addition of Triton X-100 abolished the effect of both positively and negatively charged liposomes on the kinetic properties of the enzyme.
Demonstration of a new mammalian isoleucine catabolic pathway yielding an R series of metabolites
Authors: Mamer O A , Tjoa S S , Scriver C R , Klassen G A
Published: August 2015

1. Normal human urine contains small amounts (< 4 mg/g of creatinine) of 2-ethylhydracrylic acid, formed, we believe, by a previously undisclosed endogenous catabolic pathway for the oxidation of a newly described series of R metabolites of isoleucine. 2. Urinary excretion of 2-ethylhydracrylic acid is variably increased in defects of isoleucine oxidation at distal steps in the catabolic pathway (3-oxoacyl-CoA thiolase deficiency and methylmalonyl-CoA mutase deficiency) and is diminished when proximal steps of the oxidative pathway are blocked as in branched-chain oxo acid decarboxylase deficiency (‘maple-syrup-urine’ disease). 3. Precursors of R-pathway metabolites [R(-)-2-methylbutyrate and 2-ethylacrylate] lead to increased 2-ethylhydracrylate excretion in the mammal(rat, rabbit and dog); the corresponding S metabolites [S(+)-2-methylbutyric acid and tiglic acid], when given in equimolar amounts, have little effect on its excretion, suggesting that little or no interconversion between S and R metabolites occurs in vivo. 4. Studies with 2H-labelled precursors indicate that conversion of R 2-methylbutyrate into 2-ethylhydracrylic acid occurs by a direct pathway (apparently via 2-ethylacrylic acid). 5. The further oxidation of 2-ethylhydracrylic acid to ethylmalonic acid was demonstrated, and may be analogous to S-metabolite oxidation via methyl malonate. 6. Valine metabolites do not interact with the R-isoleucine pathway under the conditions of these experiments in vivo.
The effect of calcium ions on testosterone production in Leydig cells from rat testis
Authors: Janszen F H A , Cooke B A , Van Driel M J A , Van Der Molen H J
Published: August 2015

Leydig-cell suspensions, prepared from rat testes, were incubated with different amounts of Ca2+ with and without added luteinizing hormone. The basal testosterone production in the absence of luteinizing hormone was unaffected by the Ca2+ concentration in the incubation medium. The luteinizing hormone-stimulated testosterone production, however, was progressively decreased in the absence of Ca2+ to one-third of that with 2.50 mM-Ca2+. This decrease in luteinizing hormone-stimulated testosterone production was independent of the different concentrations of luteinizing hormone (0-10μg/ml) used and could be restored by the addition of Ca2+ to the incubation medium. The restoration of the stimulation was achieved within 30 min after the addition of Ca2+ to the medium. Activation of cyclic AMP-dependent protein kinase by luteinizing hormone was not decreased by omission of Ca2+ from the incubation medium, suggesting that Ca2+ may be involved in steroidogenesis at a stage beyond the luteinizing hormone receptor-adenylate cyclase-protein kinase system.
Phosphate transport into brush-border membrane vesicles isolated from rat small intestine
Authors: Berner W , Kinne R , Murer H
Published: August 2015

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.
Phosphatidylcholine biosynthesis and choline transport in the anaerobic protozoon Entodinium caudatum
Authors: Bygrave F L , Dawson R M C
Published: August 2015

Choline accumulation and phosphatidylcholine biosynthesis were investigated in the choline-requiring anaerobic protozoon Entodinium caudatum by incubating whole cells or subcellular fractions with [14C] choline, phosphoryl [14C] choline and CDP-[14C] choline. 2. All membrane fractions contained choline kinase (EC 2.7.1.32) and CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), although the specific activities were less in the cell-envelope fraction. Choline phosphate cytidylyltransferase (EC 2.7.7.15) was limited to the supernatant, and this enzyme was rate-limiting for phosphatidylcholine synthesis in the whole cell. 3. Synthesis of phosphatidylcholine from free choline by membranes was only possible in the presence of supernatant. Such reconstituted systems required ATP (2.5 mM), CTP (1 mM) and Mg2+ (5 mM) for maximum synthesis of the phospholipid. CTP and Mg2+ were absolute requirements. 4. Hemicholinium-3 prevented choline uptake by the cells and was strongly inhibitory towards choline kinase; the other enzymes involved in phosphatidylcholine synthesis were minimally affected. 5. Ca2+ ions (0.5 mM) substantially inhibited CDP-choline-1,2-diacylglycerol cholinephosphotransferase in the presence of 15 mM-Mg2+, but choline phosphate cytidylyltransferase and choline kinase were less affected. 6. No free choline could be detected intact cells even after short (10-180s) incubations or at temperatures down to 10° C. The [14C] choline entering was mainly present as phosphorylcholine and to a lesser extent as phosphatidylcholine. 7. It is suggested that choline kinase effectively traps any choline within the cell, thus ensuring a supply of the base for future growth. At low choline concentrations the activity of choline kinase is rate-limiting for choline uptake, and the enzyme might possibly play an active role in the transport phenomenon. Thus the choline uptake by intact cells and choline kinase have similar Km values and show similar responses to temperature and hemicholinium-3.
Vitamin D in the avian egg. Its molecular identity and mechanism of incorporation into yolk
Authors: Fraser D R , Emtage J S
Published: August 2015

The chemical identity of vitamin D in the egg of the domestic fowl was studied by analysing radioactivity in eggs from hens injected with [3H]cholecalciferol. Labelled molecules were found throughout the egg, but the concentration of total radioactivity in albumin was only 5-7% of that in yolk. In lipid extracts of yolk, more than 90% of the radioactivity was as unchanged cholecalciferol and 5% as 25-hydroxycholecalciferol. Only about 3% of the radioactivity in albumin was chloroform-soluble, and of this 40% was 25-hydroxycholecalciferol and 15% was cholecalciferol. Evidence is presented to support the idea that the specific transport of cholecalciferol into yolk is mediated by a cholecalciferol-binding protein in blood. This protein forms a complex with yolk proteins in transit from liver to ovary via the blood. A cholecalciferol-binding protein, chromatographically similar to that from blood, was found in egg yolk. It is postulated that cholecalciferol forms part of a complex with its specific binding protein, Ca2+ and the yolk phosphoprotein, phosvitin. This complex is then incorporated into yolk by the thecal cells of the ovarian follicle.
Peptidyltransferase activity of ribosomes and a ribosome precursor from a mutant of Escherichia coli
Authors: Sims P F , Wild D G
Published: August 2015

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that caused the accumulation of ribonucleoprotein (‘47S’) particles during exponential growth. These particles are precursors to 50S ribosomes that lack three ribosomal proteins. Peptidyltransferase activity and binding at the peptidyl site of the peptidyltransferase centre are greatly decreased in 47S particles. Both these activities are lower in the 50S and 70S ribosomes of strain 15-28 than in its parent. Unusual assembly of the larger ribosomal subunit in strain 15-28 may produce completed ribosomes with diminished biological activity.
The phosphorylation sites of troponin T from white skeletal muscle and the effects of interaction with troponin C on their phosphorylation by phosphorylase kinase
Authors: Moir A J G , Cole H A , Perry S V
Published: August 2015

1. The phosphorylation of troponin T from rabbit white sketetal muscle is catalysed by phosphorylase kinase, but not at a significant rate by bovine 3′:5′-cyclic AMP-dependent protein kinase. 2. The amino acid sequences adjacent to the three major phosphorylation sites of troponin T were determined. 3. The serine in the N-terminal peptide (Asx, SerP, Glx)Glu-Val-Glu, is that phosphorylated (SerP, phosphoserine) when the troponin complex is isolated. 4. The other two sites of phosphorylation are located in the sequence Ala-Leu-(Ser, SerP)-Met-Gly-Ala-Asn-Tyr(Ser, SerP)Tyr. 5. When troponin T is phosphorylated in the presence of troponin C, the extent of phosphorylation at each site is considerably decreased. 6. CNBr fragments of troponin T are also phosphorylated by phosphorylase kinase, but the rate of phosphorylation at each site in the CNBr fragments is considerably slower than in the native protein. 7. From these studies it is suggested that troponin C interacts with troponin T in the region containing the two closely situated phosphorylation sites.
Molar absorption coefficients of porphyrin esters in chloroform determined by copper titration
Authors: With T K , Pedersen J S
Published: August 2015

Chromatographically pure porphyrin esters free from metalloporphyrins were titrated with Cu2+ to determine the molar amount of porphyrin present. The end point of the titration was defined by a t.l.c. method detecting traces of metal-free porphyrin ester in admxture to its copper complex. From spectrometric measurement at the Soret maximum and the molar amounts found by the titration, epsilonM was calculated for proto-, copro-, penta-, hexa- and hepta-carboxylic and uro-porphyrin permethyl esters. The values for uroporphyrin showed perfect agreement with those of previous workers, whereas those for coproporphyrin were about 5% lower and those for protoporphyrin more than 20% lower. The implications of the findings are discussed. Determinations of epsilonM of some higher esters (ethyl to pentyl) and some partial methyl esters with one carboxyl group free are also presented.