bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of dermatan sulphate proteoglycans from bovine sclera
Authors: Cöster L , Fransson L A
Published: August 2015

1. Proteoglycans were extracted from sclera with 4 M-guanidine hydrochloride in the presence of proteinase inhibitors and purified by ion-exchange chromatography and density-gradient centrifugation. 2. The entire proteoglycan pool was characterized by compositional analyses and by specific chemical (periodate oxidation) and enzymic (chondroitinases) degradations. The glycan moieties of the molecules were exclusively galactosaminoglycans (dermatan sulphate-chondroitin sulphate co-polymers). In addition, the preparations contained small amounts of oligosaccharides. 3. The scleral proteodermatan sulphates were fractionated into one larger (I) and one smaller (II) component by gel chromatography. Proteoglycan I was eluted in a more excluded position on gel chromatography in 0.5 M-sodium acetate than in 4.0 M-guanidine hydrochloride. Reduced and alkylated proteoglycan I was eluted in the same position (in 0.5 M-sodium acetate) as was the starting material (in 4.0 M-guanidine hydrochloride). The elution position of proteoglycan II was the same in both solvents. Proteoglycans I and II had s0 20,w values of 2.8 × 10(-13) and 2.2 × 10(-13) s respectively in 6.0 M-guanidine hydrochloride. 4. The two proteoglycans differed with respect to the nature of the protein core and the co-polymeric structure of their side chains. Also proteoglycan I contained more side chains than did proteoglycan II. The dermatan sulphate side chains of proteoglycan I were D-glucuronic acid-rich (80%), whereas those of proteoglycan II contained equal amounts of D-glucuronic acid and L-iduronic acid. Furthermore, the co-polymeric features of the side chains of proteoglycans I and II were different. The protein core of proteoglycan I was of larger size than that of proteoglycan II. The latter had an apparent molecular weight of 46 000 (estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis), whereas the former was greater than 100 000. In addition, the amino-acid composition of the two core preparations was different. 5. As proteoglycan I altered its elution position on gel chromatography in 4 M-guanidine hydrochloride compared with 0.5 M-sodium acetate it is proposed that a change in conformation or a disaggregation took place. If the latter hypothesis is favoured, aggregation may be due to self-association or mediated by an extrinsic molecule, e.g. hyaluronic acid.
The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin
Authors: Holmer E , Kurachi K , Söderström G
Published: August 2015

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.
Age-related changes in the chemical composition of bovine articular cartilage. The structure of high-density proteoglycans
Authors: Garg Hari G. , Swann David A.
Published: August 2015

Proteoglycans were extracted from the articular cartilage of foetal, calf and adult bovine metacarpal–phalangeal joints with 4m-guanidinium chloride. After extraction, the high-density proteoglycans (PG-I fractions) were prepared by sedimentation in two sequential CsCl-density-gradient procedures [Swann, Powell & Sotman (1979) J. Biol. Chem.254, 945–954]. The PG-I fractions from foetal, calf and adult tissues accounted for 75%, 52% and 46% respectively of the extracted components. The glucosamine, galactose, N-acetylneuraminic acid and protein contents increased with age. The overall amino acid compositions of PG-I fractions were similar. Fractionation of PG-I-fraction samples on a Bio-Gel A-50m column indicated that the molecular weight decreased with age. The PG-I fractions were specifically 3H-labelled by treatment with galactose oxidase followed by reduction with NaB3H4. The 3H radioactivity was incorporated into both galactose and galactosamine residues of different carbohydrate side chains. The elution profiles of alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples on a Sepharose 6B column showed that the molecular weights of chondroitin sulphate chains were 13500, 12000 and 10500 in foetal, calf and adult tissues respectively. Fractionation of the alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples and 3H-labelled calf and adult PG-I-fraction samples on a Bio-Gel P-10 column showed that there was an inverse relationship between the low-molecular-weight O-linked oligosaccharides and the higher-molecular-weight sialic acid-containing constituents at different ages. The oligosaccharide components of foetal, calf and adult PG-I-fraction samples represented 79%, 69% and 36% respectively of the total sialic acid content of the proteoglycans. Similarly in the 3H-labelled calf and adult samples 75% and 30% of the total radioactivity were present in the oligosaccharide components respectively. Digestion with chondroitinase AC-II and infrared analyses showed that the PG-I-fraction F and C samples contained primarily chondroitin 4-sulphate chains whereas PG-I-fraction sample A was 6-sulphated. These studies show that the major proteoglycans (PG-I fractions) in the articular cartilage of foetal, calf and adult animals differ in the content, types and structure of the chondroitin sulphate, keratan sulphate and oligosaccharide constituents. These changes in proteoglycan structure reflect the gross age-related changes in the chemical composition of the tissue.
Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy
Authors: Johnson M K , Eglinton D G , Gooding P E , Greenwood C , Thomson A J
Published: August 2015

Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)–CN in the partially reduced state. Hence as haem a3+(3)–CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)–CN . Cu+a3.
Photolabelling of membrane proteins with photoactive phospholipids
Authors: Bisson R , Montecucco C
Published: August 2015

Photoactive probes have been introduced recently to study the hydrophobic sector of integral membrane proteins. A simple procedure to synthesize a new series of highly radioactive aryl azido-phospholipids is presented. They effectively exchange with the boundary lipids and, on illumination, the cross-link to several membrane proteins with high efficiency. The procedure and analysis of labelling of ATPase from sarcoplasmic reticulum is reported as an example. The advantages in using these photoactive phospholipids are discussed together with some information obtained on their use.
Cyclic AMP-independent casein/glycogen synthase kinases from pig polymorphonuclear leucocytes
Authors: Pena J. Manuel , Cussó Roser , Itarte Emilio
Published: August 2015

1. Two cyclic AMP-independent casein/glycogen synthase kinases were purified from pig polymorphonuclear leucocytes by chromatography on phosphocellulose followed by affinity chromatography on casein–Sepharose 4B or gel filtration on Bio-Gel A-1.5m. When the affinity step was used, the specific activities were 86 and 43units/mg of protein for casein kinase 1 and 2, respectively, whereas these values were 94 and 90units/mg of protein when the gel-filtration step was used. 2. These kinases differ as follows: (a) the molecular weight of casein kinase 1 (38000) is very much lower than that of casein kinase 2 (185000); (b) the Km for casein (0.46mg/ml) and Ka for Mg2+ (0.3mm) of casein kinase 1 are lower than those of casein kinase 2 (0.90mg/ml and 1.7mm respectively); (c) KCl stimulates the phosphorylation of casein by casein kinase 1, whereas it inhibits phosvitin phosphorylation by this enzyme; on the contrary, the effect of KCl on casein kinase 2 is very similar with either casein or phosvitin as substrate; (d) although both kinases phosphorylate rabbit muscle glycogen synthase I, the ratio of glycogen synthase to casein phosphorylation by casein kinase 1 is about 4-fold greater than that by casein kinase 2. Furthermore, 32P incorporation into glycogen synthase promoted by casein kinase 1 (3.6mol of 32P/mol of 85000-dalton subunit) is twice that observed with casein kinase 2 (1.8mol of 32P/mol of 85000-dalton subunit). Such a phosphorylation results in a decrease in the glucose 6-phosphate-independence ratio of glycogen synthase to 10–15 with casein kinase 1 and to 35–45 with casein kinase 2. 3. The activity of both kinases is neither stimulated by cyclic AMP, Ca2+ and calmodulin nor inhibited by cyclic AMP-dependent protein kinase inhibitor protein. 4. No phosphorylation kinase activity was observed with casein kinase 1 and 2 at either pH6.8 or 8.2 in the presence of Ca2+. 5. Activities of both kinases on casein and glycogen synthase decreased in parallel when incubated at 50°C.
The effects of bivalent cations on ribulose bisphosphate carboxylase/oxygenase
Authors: Christeller J T
Published: August 2015

The half-saturation constants for binding of the bivalent cations (Mg2+, Ni2+, Co2+, Fe2+ and Mn2+) to ribulose bisphosphate carboxylase/oxygenase from Glycine max and Rhodospirillum rubrum were measured. The values obtained were dependent on the enzyme and the cation present, but were the same for both oxygenase and carboxylase activities. Ribulose bisphosphate rather than its cation complex was the true substrate. The kinetic parameters Vmax.(CO2), Vmax.(O2), Km(CO2), Km(O2), and K1(O2) were determined for both enzymes and each cation activator. The evolutionary and mechanistic implications of these data are discussed.
Fatty-acid desaturation and microsomal lipid fatty-acid composition in experimental hyperthyroidism
Authors: Faas F H , Carter W J
Published: August 2015

We have studied the influence of experimental hyperthyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty-acid composition. Tri-iodothyronine treatment (25 micrograms/100 g body weight) daily for 3 weeks caused no significant changes in delta 9 (stearate) desaturation but a 24% decrease in delta 6 (linoleate) desaturation. Much larger doses of tri-iodothyronine increased delta 9 desaturation. Liver microsomal fatty-acid composition in hyperthyroidism is altered with significantly increased proportions of stearate and arachidonate and decreased proportions of palmitate, palmitoleate, linoleate (C18:2) and eicosa-8,11,14-trienoate (C20:3). These changes, other than the decreases proportion of C20:3 fatty acid, which may be due to the diminished delta 6 desaturase activity, cannot be attributed to changes in fatty-acid desaturation. Most of these changes were also found to be due not simply to the decreased weight gain or the increased food intake of the hyperthyroid animals. Only the decreased C18:2 fatty-acid proportions could be mimicked by restricting food intake of control animals and none of the changes were prevented by restricting food intake of hyperthyroid animals. Thus most of the changes in microsomal lipid fatty-acid composition are likely to be due to a thyroid hormone effect on peripheral lipid mobilization or lipid degradation.
Desmosines in human urine. Amounts in early development and in Marfan's syndrome
Authors: Gunja-Smith Z , Boucek R J
Published: August 2015

Desmosines from 24 h human urine samples were isolated, characterized and quantified. The desmosines are in peptidyl form (1000–1500 molecular weight), and their amount is decreased by two-thirds between 7 and 25 years of age. Patients with Marfan's syndrome have significantly lower urinary amounts of desmosines than do comparable controls during the early development period.
Neutral glycolipids and gangliosides of concanavalin A-selected SV3T3 revertant cells and of normal and SV40-transformed Balb/c 3T3 cells
Authors: Mugnai G , Coppini R , Tombaccini D , Fallani A , Ruggieri S
Published: August 2015

The structural analysis of neutral glycolipids and gangliosides of the SV40 transformed Balb/c3T3 cells (SV3T3 cells) and concanavalin A-selected SV3T3 revertant cells, both compared with untransformed Balb/c3T3 cells, has shown: (i) a content of neutral glycolipids in revertant cells near to that found in the untransformed parental cells; (ii) a similar decrease of the higher gangliosides in transformed and revertant cells; (iii) a content of ganglioside GM3 in revertant cells much higher than that found in both SV3T3 and untransformed Balb/3T3 cells. The possible role of ganglioside GM3 in growth control is discussed.
Dependence on androgens of the specific DNA-binding activity of rat ventral-prostate non-histone chromosomal proteins
Authors: Lesser B H , Elliot N L
Published: August 2015

Interactions between rat prostate non-histone chromosomal proteins and DNA were studied by using a nitrocellulose-filter-binding technique to monitor the formation of DNA–protein complexes. The total binding activity of the non-histones, as measured by binding of proteins to a trace quantity of labelled DNA, displays no preference for rat DNA relative to Escherichia coli DNA. Sequestration of non-specific binding proteins by preincubation with unlabelled bacterial DNA enables detection of a fraction of rat prostate non-histones that binds preferentially to labelled rat DNA relative to labelled E. coli DNA. After castration of adult male rats, both total and specific binding activities decrease. Administration of 5 alpha-dihydrotestosterone to castrated rats stimulates both total and specific DNA-binding activities of prostate non-histones; specific binding is stimulated to a greater extent than total DNA, indicating that the specific binding proteins constitute a larger fraction of the non-histone proteins in the presence of androgens. The specific DNa-binding activity is dependent on the dose of steroid administered.
Oxygen-dependent 1,25-dihydroxycholecalciferol-induced calcium ion transport in rat intestine
Authors: Kendrick N C , Kabakoff B , DeLuca H F
Published: August 2015

O2-dependent CA2+ uptake by rat duodenal discs has been characterized and used in a revised assay for 1,25-dihydroxycholecalciferol-induced intestinal Ca2+ transport. Although both muscle and mucosal surfaces are exposed in this free-floating-disc assay, the Ca2+ influx across the muscle surface is small, not O2- or vitamin D-dependent, and can be subtracted out. Depriving the animals of food for 9-14 h before assay increases the O2-dependent uptake by about 75%. Half-saturation values for O2-dependent Ca2+ uptake as determined with this assay are: 0.8mM-Ca2+ (fed) and 0.5mM-Ca2+ (food-deprived) for vitamin D-deficient rats, and 0.9mM-Ca2+ (fed) and 1.5mM-Ca2+ (food-deprived) for rats dosed with 1,25-dihydroxycholecalciferol. The maximum velocity of uptake varies from 6.7nmol of Ca2+ per cm2/min (fed) to 7.0nmol of Ca2+ per cm2/min (food-deprived) for vitamin D-deficient rats and 16.7nmol of Ca2+ per cm2/min (fed) to 29 nmol of Ca2+ per cm2/min (food-deprived) for 1,25-dihydroxycholecalciferol-treated rats. By using a 5 min preincubation and 15 min incubation with 1.0mM-Ca2+, duodenal tissue taken from vitamin D-treated rats shows about a 3-fold increase in O2-dependent Ca2+ uptake when compared with tissue taken from vitamin D-deficient animals. The calcium ionophore A23187, depending on concentration, either has no significant effect on or inhibits the O2-dependent uptake, rather than increasing it. Actinomycin D, at a dose of 2 micrograms/g, inhibits the O2-dependent uptake in intestinal discs from both vitamin D-deficient and vitamin D-treated rats by 58 and 80% respectively, when administered in vivo 3 1/2 h before assay.
Metabolic aspects of the secretion of stored compounds from blood platelets. The effect of NaF at different pH on nucleotide metabolism and function of washed platelets
Authors: Mürer E H , Davenport K , Siojo E , Day H J
Published: August 2015

The purpose of this study was to investigate the response of human blood platelets to fluoride at different pH. The results were as follows. (1) Fluoride induced secretion faster and at a lower concentration when pH was lowered. (2) Platelets exposed to 2 mM-fluoride at 0 degrees C at pH 5.3 underwent secretion when first pH and then temperature was raised, although no secretion was seen at 2 mM-fluoride concentration in the absence of the preincubation at low pH. (3) The concentration of [14C]ATP in platelets decreased steeply in response to fluoride before induction of secretion. Addition of antimycin blocked or partly inhibited secretion. Fluoride thus exerts an inhibitory effect on platelet glycolysis before induction of secretion. (4) Fluoride accumulated in the platelet pellet by a time course that preceded secretion. The accumulation was faster and greater at pH 6 than at 7.4. These four points are taken as indirect evidence that fluoride has to penetrate to the interior of the platelet to induce secretion. The activation takes place over a wide range of acid pH in contrast with induction of platelet function via the outside of the plasma membrane. In addition evidence is presented that the salvage pathway may under special circumstances play an important role in the re-synthesis of platelet adenine nucleotides.
Removal of glycosaminoglycans from cultures of human skin fibroblasts
Authors: Gill P J , Adler J , Silbert C K , Silbert J E
Published: August 2015

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.
The effect of all-trans-retinoic acid on the synthesis of epidermal cell-surface-associated carbohydrates
Authors: King Ian A. , Tabiowo Anne
Published: August 2015

1. all-trans-Retinoic acid at concentrations greater than 10−7m stimulated the incorporation of d-[3H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl2-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of 35SO42−, l-[14C]fucose and U-14C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[3H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10−5m-all-trans-retinoic acid. The labelling of other epidermal components with d-[3H]glucosamine, 35SO42−, l-[14C]fucose and U-14C-labelled l-amino acids was not significantly affected by 10−5m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[3H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10−5m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[3H]-glucosamine into extracellular epidermal glycosaminoglycans in 10−5m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10−5m) did not affect the rate of degradation of this material in cultures ‘chased’ with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10−5m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[3H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10−5m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.
Metabolism of pent-4-enoate in rat heart. Reduction of the double bond
Authors: Hiltunen J K , Davis E J
Published: August 2015

1. Soluble extracts from rat heart and liver mitochondria were used to evaluate the early steps in the conversion of pent-4-enoyl-CoA into tricarboxylic acid-cycle intermediates. Hitherto the unresolved problem was the reduction of the double bond of pent-4-enoate. 2. Soluble extracts from heart mitochondria reduced pent-4-enoyl-CoA and penta-2,4-dienoyl-CoA in the presence of NADPH at rates (nmol/min per mg of protein) of 0.9 +/- 0.1 and 132 +/- 8 and from the liver mitochondria at the rates of 1.9 +/- 0.2 and 52 +/- 6 respectively. No reduction of acryloyl-CoA was found. 3. We show that primarily the double bond in position 4, not in position 2, of penta-2,4-dienoyl-CoA is reduced. 4. It is concluded that the principal metabolic pathway of penta-4-enoate is reduction of the double bond in position 4 after an initial oxidation of penta-2,4-dienoyl-CoA. The pent-2-enoyl-CoA thus formed can be further metabolized by the usual enzymes of beta-oxidation, and by the further metabolism of propionyl-CoA to tricarboxylic acid-cycle intermediates.
The presence in serum of proteins which are immunologically cross-reactive with Tamm-Horsfall glycoprotein
Authors: Lynn K L , Marshall R D
Published: August 2015

Affinity chromatography, with rabbit anti-(human Tamm-Horsfall glycoprotein) IgG, was applied to the isolation from normal human serum of protein, which is immunologically cross-reactive with the urinary glycoprotein. The antigen-antibody complex was dissociated with the use of sodium thiocyanate solution, a medium which fails to dissociate urinary Tamm-Horsfall glycoprotein-antigen complex. The cross-reactive serum proteins were isolated in amounts of 19-24 mg/l of serum. They have apparent molecular weights, assessed by disc-gel electrophoresis in the presence of sodium dodecyl sulphate, of 125 000, 84 000 and 74 000 respectively, with mobilities differing from that of urinary Tamm-Horsfall glycoprotein. They have a much lower immunoreactivity towards the antibody than does the urinary glycoprotein. Tamm-Horsfall glycoprotein could not be demonstrated in normal serum by the techniques used. The implications of these findings are discussed in terms of pathology involving Tamm-Horsfall glycoprotein.
Inactivation of oestrogen receptor in vitro by nuclear dephosphorylation
Authors: Auricchio F , Migliaccio A , Rotondi A
Published: August 2015

1. Nuclei of the calf uterus are endowed with an activity inactivating crude oestrogen-receptor complex. This activity has been partially purified. It shows a very high affinity for the oestrogen-receptor complex (Km = 0.8 × 10(-9) mol of specific [3H]oestradiol-17 beta-binding sites/l) as well as for the oestrogen-free receptor (Km = 1.5 × 10(-9) mol of specific [3H]oestradiol-17 beta binding sites/l). 2. The nuclear receptor-inactivating activity is enhanced by dithiothreitol and inhibited by several phosphatase inhibitors as well as by 4-nitrophenyl phosphate, as well known phosphatase substrate. This inhibition shows that a dephosphorylation process is required for the receptor inactivation. 3. The purified nuclear activity also inactivates pure receptor and phosphatase inhibitors prevent this inactivation. From these observations it appears that receptor inactivation is due to a nuclear phosphatase directly acting on the oestrogen receptor. 4. The nuclear localization of the receptor-inactivating activity, its high affinity for specific oestrogen binding sites and, as previously reported, its presence only in oestrogen target tissues suggest that this activity is the same as that involved in the nuclear loss of the receptor observed in intact cells.
UDP-galactose:ceramide galactosyltransferase of rat central-nervous-system myelin
Authors: Koul O , Jungalwala F B
Published: August 2015

The properties of ceramide galactosyltransferase associated with myelin and microsomal fractions of rat brain were studied. The enzyme from both the fractions had similar properties during development and synthesized the same molecular species of the product cerebroside. The results suggested that during myelination the turnover rate of enzyme protein is altered instead of regulatory modulation of the enzyme protein.
Changes in mitochondrial components of hamster brown adipose tissue in response to cold acclimation
Authors: Yacoe M E
Published: August 2015

Cold acclimation of hamsters was found to result in an increase in cytochrome concentration relative to ATPase activity in brown adipose tissue mitochondria, but not in liver mitochondria. These data lend support to the hypothesis that the uncoupled respiration characteristic of non-shivering thermogenesis is the result of an adaptive change in the composition of brown adipose tissue mitochondria in response to cold acclimation.
Inhibition of H2O2 generation in rat liver mitochondria by radical quenchers and phenolic compounds
Authors: Swaroop A , Ramasarma T
Published: August 2015

Generation of H2O2 by rat liver mitochondria with choline, glycerol 1-phosphate and proline as substrates has been shown by using high-concentration phosphate buffer. Rates obtained under these conditions were higher and more consistent as compared with the earlier reports with high-concentration mannitol/sucrose/Tris buffer. Sulphate ions could replace phosphate indicating a requirement for a high concentration of oxygen-containing anions. H2O2 generation was dependent on the presence of native mitochondria and substrate. Maximal rates with various substrates were found to be the same as with succinate. Values of Km and Vmax for H2O2 generation were considerably less than those obtained for respective dehydrogenase activities, measured by dye reduction. Scavengers of O2-. and OH. inhibited generation of H2O2. ATP, ADP, thyronine derivatives and a number of phenolic compounds also showed very potent inhibitory effects of H2O2 generation, whereas phenyl compound had no effect. Phenolic compounds did not have any effect on mitochondrial superoxide dismutase and choline dehydrogenase activities as well as on O2-. generation by the xanthine-xanthine oxidase system. Inhibition by phenolic compounds may have potential for regulation of the intracellular concentration of H2O2, that is not considered to have a ‘second messenger’ function.
Separation of haemopoietic cells for biochemical investigation. Preparation of erythroid and myeloid cells from human and laboratory-animal bone marrow and the separation of erythroblasts according to their state of maturation
Authors: Harrison F L , Beswick T M , Chesterton C J
Published: August 2015

The separation of haemopoietic bone-marrow cells by centrifugation through discontinuous density gradients of Percoll is described. This method was used to prepare fractions enriched in erythroblasts, myeloid blast cells or reticulocytes from bone marrow of anaemic and non-anaemic rabbits, from the marrow of other anaemic laboratory animals and from human samples. It is a simple, rapid, reproducible and inexpensive technique that can be readily adapted to suit individual requirements. Secondly, a convenient method is presented for the separation of large quantities of bone-marrow cells into fractions enriched in erythroblasts at different stages of maturation, by velocity sedimentation through a linear gradient of 1-2% sucrose at unit gravity. In vitro, erythroblasts adhere together strongly via a mechanism almost certainly involving a beta-galactoside-specific surface lectin termed erythroid developmental agglutinin. Since the efficiency of cell-separation techniques depends heavily on the maintenance of a single cell suspension in which each unit can move independently, the presence of an adhesive molecule at the cell surface is of considerable significance. The effect of washing the marrow with a lactose-containing medium, which has been shown to remove the agglutinin, was therefore investigated in relation to both methods. The separation on Percoll gradients is considerably enhanced by this treatment. In addition, the unit-gravity sedimentation gradient can be loaded with 5-10 times more cells after lactose extraction in comparison with intact marrow. Although enrichment is less, a useful fractionation according to maturation is still obtained.
Dephosphorylation of pig heart pyruvate dehydrogenase phosphate complexes by pig heart pyruvate dehydrogenase phosphate phosphatase
Authors: Kerbey A L , Randle P J , Kearns A
Published: August 2015

1. Pig heart pyruvate dehydrogenase phosphate complex in which all three sites of phosphorylation were completely phosphorylated was re-activated at a slower rate by phosphatase than complex predominantly phosphorylated in site 1. The ratio of initial rates of re-activation was approx. 1:5 with a comparatively crude preparation of phosphatase and with phosphatase purified by gel filtration and ion-exchange chromatography. 2. The ratio of apparent first-order rate constants during dephosphorylation of fully phosphorylated complex averaged 1/3.8/1.3 for site 1/site 2/site 3. Only site-1 dephosphorylation was linearly correlated with re-activation of the complex throughout dephosphorylation. Dephosphorylation of site 3 was linearly correlated with re-activation after an initial burst of dephosphorylation. 3. Because dephosphorylation of site 1 was always associated with dephosphorylation of site 2, it is concluded that dephosphorylation cannot be purely random. 4. The ratio of apparent first-order rate constants for dephosphorylation of site 1 (partially/fully phosphorylated complexes) averaged 1.72. This ratio is smaller than the ratio of approx. 5 for the initial rates of re-activation. Possible mechanisms involved in the diminished rate of re-activation of fully phosphorylated complex are discussed.
Preparation and characterization of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis
Authors: Giancotti V , Cosimi S , Cary P D , Crane-Robinson C , Geraci G
Published: August 2015

The separation and purification of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis is described. Physical studies were used to compare this histone H1 molecule with H1 histones from other species. C.d. and 270 MHz n.m.r. spectroscopy indicate that, despite significant compositional differences from other sea-urchin sperm H1 histones, their secondary and tertiary structures are very similar. A large difference in helicity was, however, found between S. granularis histone H1 and calf thymus histone H1, and their n.m.r. and fluorescence spectra also differ considerably. It is concluded that secondary structure and tertiary structure have not been conserved in the evolution of the H1 histone family.
Mössbauer spectroscopic studies of the terminal dioxygenase protein of benzene dioxygenase from Pseudomonas putida
Authors: Geary P J , Dickson D P E
Published: August 2015

Mössbauer spectra obtained from the terminal dioxygenase protein of the benzene dioxygenase system from Pseudomonas putida show that it contains [2Fe--2S] centres similar to those of the two-iron plant-type ferredoxins. In the oxidized form the two iron atoms within the centre are high-spin ferric but with considerable inequivalence. In the reduced form the centre contains one extra electron, and this is localized on one of the iron atoms, which becomes high-spin ferrous.
Calmodulin-free skeletal-muscle troponin C prepared in the absence of urea
Authors: Cox J A , Comte M , Stein E A
Published: August 2015

A method is described for the rapid preparation of electrophoretically pure troponin C from rabbit skeletal-muscle myofibrils that avoids the use of urea. The three-step procedure includes extraction od the myofibrils with EDTA-containing buffers, one-step elution from DEAE-Sephadex and Sephadex G-100 chromatography in the presence of EDTA. The procedure gives yields comparable with those of currently used methods that involve dissociation of the troponin complex with urea. Except for the thiol-group reactivity, troponin C produced by our method is physicochemically and functionally indistinguishable from that obtained by the classical procedure. Purified troponin C always contains traces of calmodulin. However, this contamination can be decreased to less than 0.02% by means of a second Sephadex G-100 chromatography step in the presence of EDTA.
Absence of the iron-containing superoxide dismutase in mitochondria from mustard (Brassica campestris)
Authors: Salin M L , Bridges S M
Published: August 2015

Mitochondria were isolated from mature leaves as well as etiolated seedlings of Brassica campestris (mustard), a eukaryote previously shown to possess the iron-containing isoenzyme of superoxide dismutase. On the basis of KCN- and H2O2-sensitivity, and on polyacrylamide-gel analysis, only the cuprozinc and mangano superoxide dismutases were found in mitochondria. The iron-containing enzyme was absent. The mangano enzyme was found in the mitochondrial matrix, whereas the cuprozinc enzyme appeared to be localized in the intermembrane space.
Polymeric structure of pig small-intestinal mucus glycoprotein. Dissociation by proteolysis or by reduction of disulphide bridges
Authors: Mantle M , Mantle D , Allen A
Published: August 2015

Pig small-intestinal mucus glycoprotein, of molecular weight 1.72 X 10(6), is cleaved by Pronase digestion into glycoprotein subunits of molecular weight 4.5 X 10(5). Of the protein component of the native glycoprotein 29% by weight was lost on Pronase digestion, with no loss of carbohydrate. The non-glycosylated region of the protein that was lost with proteolytic digestion had a broad spectrum of amino acid residues, in contrast with the glycosylated region of the protein, which was resistant to proteolysis and was rich in serine, threonine and proline residues. Reduction with 0.2M-mercaptoethanol dissociated the Pronase-digested glycoprotein subunits into smaller glycoprotein subunits of molecular weight 2.7 X 10(5). On reduction, the native glycoprotein was dissociated into subunits of molecular weight 2.4 X 10(5), a similar size to those obtained from reduction of the Pronase-digested glycoprotein. On reductive dissociation of the native glycoprotein, in addition to glycoprotein subunits, protein was also released principally as a component of 90000 molecular weight. This protein was separated quantitatively from the reduced glycoprotein in amounts compatible with one 90000-mol.wt. protein molecule per 1.72 X 10(6)-mol.wt. native glycoprotein molecule. No 90000-mol.wt. protein was released on reduction of the isolated Pronase-digested glycoprotein. Pig small-intestinal mucus glycoprotein is therefore a covalent polymer of glycoprotein subunits joined by disulphide bridges. This polymeric structure differs in important respects from that previously shown for gastric mucus, in particular with respect to the size and number of component subunits per native molecule.
Apurinic endonuclease from Saccharomyces cerevisiae
Authors: Thielmann H W , Hess U
Published: August 2015

An endonuclease cleaving depurinated and alkylated double-stranded DNA has been purified 500-fold from Saccharomyces cerevisiae, strain MB 1052. The enzyme has an Mr of 31 000 +/- 2000, a sedimentation value of 3.2S and a diffusion coefficient of 9.5 X 10-7 cm2/s. The enzyme was active only at apurinic/apyridiminic sites, regardless of whether they were produced by heating the DNA at acidic pH or by alkylation with the ultimate carcinogen methyl methanesulphonate. Native DNA was not acted upon. U.v.-irradiated DNA and DNA treated with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene were cleaved to an extent related to the extent of apurinic/apyridiminic sites. Enzymic activity was not dependent upon Mg2+, but was stimulated approx. 3-fold by 4mM-Mg2+. The enzyme did not bind to DEAE-cellulose or CM-cellulose at KCl concentrations greater than 160 mM. The endonuclease was obtained free of exonuclease and 3-methyladenine-DNA glycosylase activity in five chromatographic steps.
Determination of ionization constants by paper electrophoresis
Authors: Tate M E
Published: August 2015

Dimensionless apparent ionization constants of charged low-molecular-weight species may be obtained from paper-electrophoretic data at 20-25 degrees C with buffers (I0.1-0.5) of measured pH (1.5-12.5) containing oxalate ions. Relative mobilities rather than absolute mobilities were measured by using glycerol and m-nitrobenzenesulphonate respectively as standards of zero and unit mobility. Application of the procedure to ionizations of adenine, adenosine, 2′-deoxyadenosine, 3′-deoxyadenosine, 3′:5′-cyclic AMP, ADP, ADP-glucose-agrocin 84 and ATP is described.
The effect of insulin on plasma-membrane and mitochondrial-membrane potentials in isolated fat-cells
Authors: Davis R J , Brand M D , Martin B R
Published: August 2015

1. A recently developed technique for the measurement of plasma-membrane and mitochondrial-membrane potentials in intact cells by using the distribution of 86Rb+ and [3H]methyltriphenylphosphonium+ has enabled us to characterize a novel insulin effect on fat-cell mitochondria. For control cells the plasma-membrane and mitochondrial-membrane potentials were 75 mV and 152 mV respectively. Insulin (10 mu units/ml) caused a 9 mV hyperpolarization of the plasma membrane and a 19 mV depolarization of the mitochondrial membrane. 2. The insulin-dependent mitochondrial depolarization was observed at physiological insulin concentrations (10 mu units/ml) and was apparent when the cells metabolized a wide variety of substrates. 3. Evidence from the uptake of the weak acid 5,5-dimethyloxazolidine-2,4-dione by fat-cells was interpreted as indicating that the mitochondrial pH gradient was increased by insulin. 4. Insulin alters the balance between the electrical and pH-gradient components that form the mitochondrial protonmotive force. A model is proposed.
Activity of phosphorylase in total global ischaemia in the rat heart A phosphorus-31 nuclear-magnetic-resonance study
Authors: Bailey I A , Williams S R , Radda G K , Gadian D G
Published: August 2015

1. The uptake and subsequent phosphorylation of deoxyglucose into perfused rat hearts was monitored by 31P n.m.r. 2. The accumulated deoxyglucose 6-phosphate provided (a) an independent method for measuring cytosolic pH in the normoxic and ischaemic heart tissue and (b) a way of studying the activity of phosphorylase during ischaemia. 3. The cytosolic pH measured from the 31P n.m.r. resonance position of deoxyglucose 6-phosphate is in good agreement under all conditions studied with that obtained previously from the Pi resonances. This eliminates any possible doubts about the use of Pi for measuring intracellular pH. 4. Deoxyglucose 6-phosphate in vitro inhibits phosphorylase b but not phosphorylase a. Its inhibitory effect on glycogenolysis during ischaemia is monitored by measuring tissue acidosis by n.m.r. In the initial stages of ischaemia phosphorylase activity is not inhibited, whereas after about 5 min approx. 50% of the activity is inhibited. These observations are interpreted in terms of the relative contributions of phosphorylase a and the AMP-dependent phosphorylase b activities during ischaemia.
Foetal and maternal rates of glucose production and utilization in chronically catheterized ditocous ewes
Authors: Hodgson J C , Mellor D J , Field A C
Published: August 2015

1. Five well-nourished Scottish Blackface ewes carrying twin lambs were used. An umbilical artery and vein of each foetus in two sheep and one foetus in three sheep were catheterized at 109-112 days of gestation and both maternal jugular veins were catheterized 2 days before experiment. Two-day experiments were conducted between days 124 and 141 of gestation when [2-3H,U-14C]glucose was infused intravenously over 9 h into either the mother or one foetus, separate days being used for each infusion. 2. Plasma glucose specific radioactivities of the mother and infused foetus at plateau were used to determine the glucose flux rates within and between mother and foetus. The corresponding values for the uninfused foetus were calculated by using the relative weights of each twin foetus and were used to correct the maternal flux rates for the presence of the second foetus. 3. The net rate of glucose utilization by the foetus was 11.2 +/- 1.3 mg/min per kg of foetus (mean +/- S.E.M; n = 11). The corresponding rate in the mother was 1.1 +/- 0.1 mg/min per kg of live weight (n = 11). 4. The net rate of foetal uptake of glucose from the mother accounted for only 56 +/- 4% of the glucose used by the foetus. The remainder was provided by an input of 4.9 +/- 0.7 mg/min per kg of foetus to the foetal glucose pool via foetal gluconeogenesis. 5. The rate of glucose futile cycling in the foetus was high, equal to 95 +/- 19% of the net foetal glucose-utilization rate. 6. The rate of glucose utilization (mg/min per kg of foetus) decreased progressively over the observational period of 124-141 days of gestation. There was no increase in the rates of net foetal glucose uptake or of foetal gluconeogenesis to counter this decline.
Turnover of the creatine kinase subunits in chicken myogenic cell cultures and in fibroblasts
Authors: Caravatti M , Perriard J C
Published: August 2015

The rates of degradation of creatine kinase subunits, M-CK and B-CK subunits, were measured in cultured myogenic cells and in subcultured fibroblasts. In differentiated myogenic cells, the myotubes, both M-CK and B-CK subunits are synthesized. Their rates of degradation were compared. The M-CK subunits is slightly more stable and is degraded with an average apparent half-life of 75 h, whereas that of the B-CK subunit was shorter with 63 h. The turnover properties of M-CK subunit from soluble and of myofibril-bound MM-CK homodimeric creatine kinase isoenzyme isolated from breast muscle of young chickens were identical. The apparent half-life of the B-CK subunit was also determined in subcultured fibroblasts and 5-bromo-2′-deoxyuridine-treated cells, and found to be shorter than in myotubes (46 h and 37 h respectively). Similar observations were made for myosin heavy chain, actin and total acid-precipitable material. It appears therefore that proteins are in general degraded more slowly in differentiated myogenic cells. The differences in the stability of M-CK and B-CK subunits in myotubes probably do not reflect a major regulatory mechanism of the creatine kinase isoenzyme transition.
Changes in membrane proteins associated with inhibition of the general amino acid permease of yeast (Saccharomyces cerevisiae)
Authors: Woodward J R , Kornberg H L
Published: August 2015

The general amino acid permease (‘Gap’) system of the wild-type yeast (Saccharomyces cerevisiae) strain Y185 is inhibited by the uptake and accumulation of its substrate amino acids. Surprisingly, this inhibition persists even after ‘pools’ of amino acids, accumulated initially, have returned to normal sizes. Recovery from this inhibition depends on a supply of energy and involves the synthesis of a membrane protein component of the Gap system.
The use of proteinases to determine the topological location of cytochrome P-450 in vesicles derived from smooth endoplasmic reticulum of rat liver
Authors: Cooper M B , Estall M R , Rabin B R
Published: August 2015

1. The phospholipid bilayer of intact vesicles from smooth endoplasmic reticulum is impermeable to macromolecules. Specific and non-specific proteinases were used to investigate the site of membrane proteins in the transverse plane of the bilayer. 2. When two proteinases were used in conjunction, denaturing effects additional to proteolysis were observed on cytochrome P-450 content and glucose 6-phosphatase activity which did not depend on the integrity of the phospholipid bilayer. 3. When lipid peroxidation was inhibited, these effects were not observed.
Identification of Limulus polyphemus haemocyanin messenger RNA
Authors: Wood E J , Bonaventura J
Published: August 2015

Total RNA was isolated from cyanoblast-containing tissue taken from behind the compound eye of the horseshoe crab, Limulus polyphemus. Poly(A)-containing RNA separated from this by affinity chromatography on oligo(dT)-cellulose was translated in the rabbit reticulocyte haemolysate system in the presence of L-[35S]methionine. By using an antiserum to Limulus haemocyanin, polypeptides were isolated from the translation products which had a similar mobility to the authentic Limulus haemocyanin polypeptides as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.