bluuebunny/crossref_metadata_2025 huggingface dataset:
Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay
Authors: Haystead T A , Hardie D G
Published: August 2015

Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect on acetyl-CoA carboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.
New pteridine substrates for dihydropteridine reductase and horseradish peroxidase
Authors: Armarego W L F , Ohnishi A , Taguchi H
Published: August 2015

The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported.
Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution
Authors: Pozzan T , Di Virgilio F , Vicentini L M , Meldolesi J
Published: August 2015

Ca2+ homoeostasis was investigated in pheochromocytoma neurosecretory (PC12) cells both before and after treatment with nerve growth factor, which induces a neuronal-like differentiation accompanied by a large increase in the number of muscarinic receptors. The resting concentration of free cytosolic Ca2+, [Ca2+]i, measured by the quin2 technique, was found to be higher and more variable in differentiated cells. Moreover, the [Ca2+]i rises induced by the Ca2+ ionophore ionomycin and by depolarizing concentrations of KC1 were greater and more transient. Exposure to carbachol induced modest, but long-lasting, [Ca2+]i rises, which were faster and greater in differentiated than in non-differentiated cells. These effects were due to the activation of the muscarinic receptor, because they were unaffected by nicotinic blockers (hexamethonium and D-tubocurarine) and completely eliminated by low concentrations of the muscarinic antagonists atropine and pirenzepine [IC50 (concn. causing 50% inhibition) = 2 and 60 nM respectively]. The muscarinic-receptor-dependent [Ca2+]i rises were the result of two concomitant processes: (1) redistribution of Ca2+ from cytoplasmic stores to the cytosol, possibly mediated by generation of inositol 1,4,5-trisphosphate as a consequence of the muscarinic-receptor-coupled hydrolysis of polyphosphoinositides, and (2) increased Ca2+ influx through a pathway of the plasmalemma insensitive to verapamil and thus different from the voltage-dependent Ca2+ channel. The existence of this second process was documented: (a) by the difference of the [Ca2+]i responses brought about by carbachol in Ca2+-containing and Ca2+-free media; (b) by the occurrence of [Ca2+]i rise and increased 45Ca accumulation in cells exposed to 1 mM-CaCl2 after having been treated for 2 min with carbachol in Ca2+-free medium; (c) by typical differences in the quin2 signal kinetics observed in parallel samples of PC12 cells loaded with different concentrations of the dye.
Translation of rat intestinal RNA yields two alkaline phosphatases
Authors: Sussman N L , Seetharam S , Blaufuss M C , Alpers D H
Published: August 2015

After translation of total rat intestinal RNA, immunoprecipitation using monospecific antiserum against rat intestinal alkaline phosphatase yielded two polypeptides in the adult duodenum and jejunum (molecular masses 62 and 65 kDa). Immunoprecipitation of both bands was blocked by a single purified alkaline phosphatase. In the adult ileum and in the entire small intestine of suckling pups, only the 62 kDa translation product was found. After fat feeding, translated alkaline phosphatase increased by an amount proportionate to the increase in enzyme activity previously seen in the serum. A small fraction of nascent alkaline phosphatase was translocated into microsomal vesicles, producing peptides of 65 and 69 kDa. Tunicamycin-treated membranes demonstrated a different signal peptide for each translation product. N-Terminal sequencing of the translation products showed leucine residues at similar positions, but overlap with the mature protein sequence was not demonstrated. On the basis of these data, we propose the presence of two mRNAs encoding alkaline phosphatase in the rat intestine.
Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the ‘16:3’ plant Arabidopsis thaliana
Authors: Browse J , Warwick N , Somerville C R , Slack C R
Published: August 2015

The kinetics of [1-14C]acetate incorporation in Arabidopsis thaliana L. (Heyn) showed almost equal labelling of phosphatidylcholine (PC) and diacylgalactosylglycerol (DGG) at early times and the transfer of radioactivity from PC to DGG and diacyldigalactosylglycerol (DDG) at longer times. These kinetics demonstrated the parallel operation of the prokaryotic and eukaryotic pathways of lipid synthesis [Roughan & Slack (1982) Annu. Rev. Plant Physiol. 33, 97-132] in this tissue. At 2 h after the application of [1-14C]acetate, more than 85% of the radioactivity at the sn-2 position of each chloroplast lipid was in 16-carbon fatty acids. However, after 60 h, molecular species containing labelled C18 fatty acids at position sn-2 and presumably derived from microsomal PC made a large contribution (20-70%) to each chloroplast lipid except phosphatidylglycerol. These findings are consistent with the contention that the chain length of the fatty acid at the sn-2 position of glycerol is an accurate predictor of whether a particular lipid molecule has been synthesized by the prokaryotic or eukaryotic pathway. At 30 min after the start of [1-14C]acetate labelling, only 12.3% of the radioactivity in PC was in saturated fatty acids, but the proportion increased steadily to 24.3% after 142 h. It is suggested that steps involved in the conversion of PC to chloroplast lipids on the eukaryotic pathway discriminate against palmitate-containing species. The step involved does not appear to be transfer of PC to the chloroplast because extrachloroplastic and chloroplast membranes purified from Arabidopsis mesophyll protoplasts each contained PC with a fatty acid composition similar to that of the same lipid from leaves. Positional analysis of unlabelled lipids, together with the information summarized above, is used to construct a quantitative scheme of the fluxes through the prokaryotic and eukaryotic pathways during lipid synthesis in Arabidopsis. This scheme shows that 38% of the fatty acids synthesized de novo in the chloroplast enter the prokaryotic pathway in the chloroplast envelope. Of the 62% which are exported as acyl-CoA species to enter the eukaryotic pathway, 56% (34% of the total) are returned to complete synthesis of the chloroplast's complement of glycerolipids.
A study of the metabolism of [U-14C]3-methyl-2-oxopentanoate by rat liver mitochondria using h.p.l.c. with continuous on-line monitoring of radioactive intact acyl-coenzyme A intermediates
Authors: Causey A G , Middleton B , Bartlett K
Published: August 2015

A reverse-phase h.p.l.c. system for the resolution of the acyl-CoA intermediates of the degradation of 3-methyl-2-oxopentanoate is described. The validation of a method for the measurement of radioactive intermediates produced by the incubation of [U-14C]3-methyl-2-oxopentanoate with rat liver mitochondrial fractions is described. The absence of bicarbonate caused the accumulation of [14C]propionyl-CoA. The accumulation of [14C]2-methylbutyryl-CoA was observed in incubations with mitochondrial fractions derived from riboflavin-deficient animals. Studies of the accumulation of labelled intermediates with time suggest that there is regulation of the pathway of isoleucine degradation at methylmalonyl-CoA mutase, as suggested for valine [Corkey, Martin-Requero, Walajtys-Rode, Williams & Williamson (1982) J. Biol. Chem. 257, 9668-9676]. These studies demonstrate that h.p.l.c. with on-line continuous radiochemical detection is a powerful method for the investigation of the control of intermediary metabolism.
Purification and characterization of human hepatic cysteine-conjugate β-lyase
Authors: Tomisawa H , Ichihara S , Fukazawa H , Ichimoto N , Tateishi M , Yamamoto I
Published: August 2015

Cysteine-conjugate beta-lyase (EC 4.4.1.13) was purified about 880-fold from human liver obtained post mortem. The purification procedure included (NH4)2SO4 precipitation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and chromatofocusing. The purified enzyme cleaves the C-S bond of several S-aryl-L-cysteines to yield equimolar amounts of thiols, pyruvic acid and ammonia via an alpha beta-elimination reaction. The Mr of the enzyme was estimated to be 88,000 by gel filtration. The enzyme is thermolabile, has a pH optimum of 8.5, and an apparent Km of 0.7 mM towards S-(p-bromophenyl)-L-cysteine. The enzyme requires pyridoxal 5′-phosphate as a cofactor, and hence the enzyme activity was completely abolished by hydroxylamine. No effect of EDTA or thiol-blocking reagents was observed on the activity of the enzyme.
A flow-dialysis method for obtaining relative measures of association constants in calmodulin-metal-ion systems
Authors: Buccigross J M , O'Donnell C L , Nelson D J
Published: August 2015

A flow-dialysis apparatus suitable for the study of high-affinity metal-binding proteins has been utilized to study calmodulin-metal exchange as a measure of relative calmodulin-metal association constants. Calmodulin labelled with radioactive 153Gd was dialysed against buffer containing various competing metal ions. The rate of label exchange was monitored by a gamma-ray scintillation detector. Competing metals used were Ca2+ and Cd2+, and the lanthanides Gd3+, Eu3+, La3+ and Lu3+. All exchange processes were first-order, and two categories of metal were found: Ca2+ and Cd2+ in one, the lanthanides comprising the other. In addition calmodulin-metal complexes with radioactive 109Cd and 45Ca released the bound label without any competing metal being added to the buffer. The kinetics of this metal loss can be described by two consecutive first-order processes, and the fraction of label associated with each rate can be determined. Studies of phosphodiesterase activation by calmodulin show Cd2+ and calmodulin to cause 80% of the maximum activation found when Ca2+ and calmodulin are used.
Proteoglycans from pig aorta. Comparative study of their interactions with lipoproteins
Authors: Wegrowski J , Moczar M , Robert L
Published: August 2015

Different proteoglycans (PGs) were isolated from pig aorta for aggregation studies with hyaluronic acid and human low-density lipoproteins (LDL). Extraction of the intima-media with 4M-guanidinium chloride and digestion of the residue with collagenase solubilized 91% of aortic hexuronic acid content. From the guanidinium chloride extract two PGs were isolated by ion-exchange and gel-permeation chromatography: proteochondroitin sulphate (PGI) with a protein-core apparent Mr of 250 000 and proteodermatan-chondroitin sulphate (PGII) with a protein-core apparent Mr of 55 000. Only PGI forms high-Mr aggregates with hyaluronic acid. From the collagenase digest two other PGs were isolated: proteoheparan sulphate and proteochondroitin sulphate (PGIII and PGIV respectively). PGIV had a smaller hydrodynamic size than PGI. PGI and PGII formed insoluble complexes with human LDL in the presence of Ca2+. PGIII or PGIV did not form precipitates with the LDL. PGI and PGII, but neither PGIII nor PGIV, were bound to LDL-Sepharose. The main peaks of PGI and PGII were eluted from LDL-Sepharose with 60 mM- and 90 mM-NaCl respectively. The results indicate that aortic PGs have different interacting potentials with lipoproteins, depending on their Mr and their glycosaminoglycan composition.
Pig heart fumarase really does exhibit negative kinetic co-operativity at a constant ionic strength
Authors: Hasinoff B B , Davey J P
Published: August 2015

The kinetics of the action of fumarase on L-malate and fumarate were investigated at constant ionic strength. This was done to evaluate reports that fumarase follows simple Michaelis-Menten kinetics. However, when pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.
Inositol phospholipid arachidonic acid metabolism in GH3 pituitary cells
Authors: Dudley D T , Spector A A
Published: August 2015

Inositol phospholipids in cultured GH3 cells, a prolactin secreting, thyrotropin-releasing hormone (TRH) sensitive rat pituitary cell line, exhibit a preferential selectivity for incorporating arachidonic acid. Fatty acid composition data show that all inositol phospholipids are enriched in stearic and arachidonic acids to a much greater degree than other cellular phospholipids. Incubation of GH3 cells with radioactive stearate, oleate, arachidonate, eicosapentaenoate or docosahexaenoate also showed that much more stearate and arachidonate were incorporated into inositol phospholipids. In short term incubations with tracer amounts of radioactive arachidonate, incorporation was initially into phosphatidylinositol (PtdIns), with phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being labelled at later times. During longer incubations, all of the inositol phospholipids reach equilibrium at about 10 h, and the resulting specific activities of the three fractions were similar. These findings suggest that arachidonate is incorporated initially into PtdIns and that PtdIns is then phosphorylated. There was no release of either arachidonate or eicosanoid products when GH3 cells were incubated with TRH. However, TRH stimulation of 32P-labelled GH3 cells resulted in rapid breakdown of PtdIns(4,5)P2 and PtdIns4P, with concomitant increases in [32P]phosphatidic acid and [32P]PtdIns. When the [32P]PtdIns was further analysed by argentation chromatography to separate PtdIns molecular species, it was found that tetraenoic (stearate/arachidonate) species accounted for 80% of the stimulated labelling. The selectivity for arachidonate incorporation into inositol phospholipids coupled with turnover of the arachidonate-containing molecular species suggests that inositol phospholipids containing arachidonic acid or the diacylglycerol resulting therefrom may play a vital cellular role in GH3 cells. This role may involve the operation of the PtdIns cycle itself rather than a stimulated release of arachidonate for eicosanoid formation.
The influence of poly(ethylene glycol) 6000 on spermine-induced aggregation of liposomes
Authors: Tadolini B , Varani E , Cabrini L
Published: August 2015

Poly(ethylene glycol) 6000 affected the aggregation of mixed liposomes induced by spermine. It lowered the concentration of spermine causing 50% maximal aggregation, accelerated the rate and increased the extent of aggregation. The effect was inversely proportional to the density of the acidic phospholipid in the vesicles. These effects were not due either to poly(ethylene glycol) 6000-induced permanent structural modification of the liposome or increased binding of spermine to the vesicles. These findings are discussed in relation to a decreased hydration force caused by the ability of poly(ethylene glycol) 6000 to alter the water of hydration of the phospholipid polar groups in the liposome.
Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta
Authors: Tinker D , Geller J , Romero N , Cross C E , Rucker R B
Published: August 2015

The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.
Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya)
Authors: McKee R A , Adams S , Matthews J A , Smith C J , Smith H
Published: August 2015

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3′ untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.
The action of human articular-cartilage metalloproteinase on proteoglycan and link protein. Similarities between products of degradation in situ and in vitro
Authors: Campbell I K , Roughley P J , Mort J S
Published: August 2015

Interleukin 1 stimulation of human articular cartilage in organ culture produced the concomitant release of proteoglycan fragments and latent metalloproteinase. The released fragments ranged in size from that of almost intact proteoglycan subunits to the product of limiting digestion generated by the activated metalloproteinase. None of the fragments possessed the ability to interact with hyaluronic acid. Analysis of proteoglycan aggregate digested with the activated metalloproteinase showed that isolated hyaluronic acid-binding regions were produced from the proteoglycan subunits, and that the two higher-Mr link-protein components (Mr 48,000 and 44,000) were converted into the lowest-Mr component (Mr 41,000). Link protein extracted from cartilage under stimulation with interleukin 1 showed a similar conversion. These results suggest that interleukin 1 stimulates the release of latent metalloproteinase from chondrocytes and that a proportion of the enzyme is activated in situ in the cartilage matrix. The mode of action of the activated enzyme is compatible with a role in the changes in proteoglycan structure seen in aging.
Binding of zinc ions to heparin. Analysis by equilibrium dialysis suggests the occurrence of two, entropy-driven, processes
Authors: Woodhead N E , Long W F , Williamson F B
Published: August 2015

Analysis by equilibrium dialysis of the binding of Zn2+ to heparin suggested that two interactions, one of high affinity and one of low affinity, occur. The stoichiometry of binding in both cases is about one Zn2+ ion bound per average heparin disaccharide unit. Both types of interaction appear to be entropy-driven.
Increased mouse epidermal ornithine decarboxylase activity by the tumour promoter 12-O-tetradecanoylphorbol 13-acetate involves increased amounts of both enzyme protein and messenger RNA
Authors: Verma A K , Erickson D , Dolnick B J
Published: August 2015

Evidence was sought that the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse epidermal ornithine decarboxylase (ODC, EC 4.1.1.17) activity involves both increased ODC mRNA and ODC protein. Application of 10 nmol of TPA to mouse skin led to a dramatic increase in soluble epidermal ODC activity which paralleled an increase in amount of enzymically active ODC protein as determined by gel electrophoresis of immunoprecipitated difluoromethyl[3H]ornithine-bound ODC. Application of TPA to mouse skin also resulted in an increase in ODC mRNA measured by dot-blot analysis using a radiolabelled cDNA probe. ODC mRNA induction preceded the increase in ODC activity by TPA. TPA-increased ODC mRNA displayed a single major band of 2.1 kilobases in size identified by the Northern blotting procedure.
The decrease in phosphatidylinositol 4,5-bisphosphate in ADP-stimulated washed rabbit platelets is not primarily due to phospholipase C activation
Authors: Vickers J D , Kinlough-Rathbone R L , Mustard J F
Published: August 2015

Addition of 10 micron-ADP to washed rabbit platelets caused platelet shape change and aggregation without release of the contents of the amine-storage granules, and caused a transient decrease (8.8% at 10 s) in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2). By 20 s the decrease in PIP2 was no longer apparent, but by 60 s the amount of PIP2 was again decreased. Addition of thrombin (1 unit/ml), which causes platelet shape change, aggregation and the release of the contents of the amine-storage granules, caused a decrease in the amount of PIP2 (8.0% at 10 s); at 60 s the amount of PIP2 was not significantly different from that in controls. In platelets prelabelled with [3H]glycerol, the specific radioactivity of PIP2 was increased at 10 s in ADP-stimulated platelets, and unchanged in thrombin-stimulated platelets. In platelets prelabelled with [3H]inositol and incubated with 20 mM-Li+ to inhibit the degradation of the inositol phosphates to inositol, there was no increase in the labelling of inositol trisphosphate (IP3) upon stimulation with ADP. In contrast, stimulation with thrombin caused a significant increase in the labelling of IP3 at 10 s. These differences in the changes in polyphosphoinositide metabolism in ADP- and thrombin-stimulated platelets are consistent with the hypothesis that the decrease in PIP2 in ADP-stimulated platelets may be due not to degradation of PIP2 by phospholipase C, but rather to a shift in the equilibrium between PIP2 and phosphatidylinositol 4-phosphate (PIP). Increases in the labelling of phosphatidic acid at 10 s and of inositol bisphosphate and inositol phosphate after 20 s are consistent with phospholipase C being stimulated through some other mechanism that leads to the degradation of PIP and phosphatidylinositol; one possibility is that ADP causes an increase in cytoplasmic Ca2+.
The cloning and expression of the aroL gene from Escherichia coli K12. Purification and complete amino acid sequence of shikimate kinase II, the aroL-gene product
Authors: Millar G , Lewendon A , Hunter M G , Coggins J R
Published: August 2015

The aroL gene encoding the enzyme shikimate kinase II was cloned from Escherichia coli K12. Construction of over-expressing strains permitted for the first time the purification to homogeneity of a monofunctional shikimate kinase. The complete amino acid sequence of shikimate kinase II was determined by a combined nucleotide and direct amino acid sequencing strategy. E. coli shikimate kinase II is a monomeric enzyme containing 173 amino acid residues with a calculated Mr 18,937. The amino acid sequence contains a region homologous with other kinases and ATP-requiring enzymes. Evidence is presented suggesting that the transcriptional start site of the aroL gene is located within a potential operator site.
Reversible deactivation of β-lactamase by quinacillin. Extent of the conformational change in the isolated transitory complex
Authors: Persaud K C , Pain R H , Virden R
Published: August 2015

Conditions have been established where the deactivation of the beta-lactamase from Staphylococcus aureus PC1 by the penicillin substrate, quinacillin, is close to complete but fully reversible. The temperature-dependence of the rate of re-activation indicated a half-life of about 170 min for the deactivated state at 0 degrees C. Measurement of the relative viscosity of mixtures of enzyme and quinacillin at 8.4 degrees C ruled out any significant difference in shape or solvation between the deactivated and the normal enzyme. C.d. measurements of the deactivated protein, separated from excess quinacillin, showed that the quinacillin side-chain chromophore was bound in an asymmetric environment. The ellipticity associated with the bound quinacillin chromophore decreased with the same first-order rate constant as that for reappearance of enzyme activity. These findings support the accumulation of a deactivated state that contains bound quinacillin or a derivative. Quinacillin caused a 3-fold increase in the rate of 3H exchange-out (at a rate that was low compared with that for the substantially unfolded or expanded protein). However, there was rapid exchange-out of about 50 3H atoms on addition of 1 M-urea to the deactivated enzyme, whereas the same concentration had no effect on the exchange-out of 3H from native enzyme. The interpretation that quinacillin increases the susceptibility of the native state to unfolding in the presence of urea is supported by the demonstration that SO4(2)- ions decreased the rate and extent of deactivation but had no effect on the rate of re-activation, as predicted from the observation that SO4(2)- ions, in competition with urea, stabilize the native state relative to the partially unfolded state H [Mitchinson & Pain (1985) J. Mol. Biol. 184, 331-342].
Transcription of genomic bovine and Xenopus laevis DNA species by RNA polymerase III in HeLa-cell cytosol extracts
Authors: Furth J J , Su C Y
Published: August 2015

RNA transcribed from genomic Xenopus laevis DNA by RNA polymerase III in HeLa-cell extract was found in discrete size classes and was transcribed from at least two different Xenopus repeat DNA species. Very little 5S ribosomal RNA was transcribed, contrasting with results obtained on transcription of genomic Xenopus DNA by Xenopus extract [Bogenhagen et al. (1982) Cell 28, 413-421]. The low transcription was not due to an inability to use 5S rDNA templates, since the cloned Xenopus 5S ribosomal gene and pseudogene were effective templates for RNA polymerase III in HeLa extract. RNA transcribed from genomic bovine DNA by RNA polymerase III in HeLa-cell cytosol extract consisted of 120-nucleotide RNA and a larger amount of heterogeneously sized RNA (180-650 nucleotides). Only a small portion of the 120-nucleotide RNA was 5S rRNA. Most of the 120-nucleotide RNA and the larger RNA species were transcribed from one bovine repeat DNA. Genes for 5S rRNA and bovine repeat DNA were transcribed roughly in proportion to their frequency in Bos, contrasting with results in a homologous system in which transcription of repeat genes is repressed [Furth (1985) Biochem. Biophys. Res. Commun. 131, 551-556]. Bovine 5S rRNA genes appear to be concentrated on one DNA fragment obtained by restriction-enzyme-HindIII digestion of genomic bovine DNA.
N-alkylation of exogenous haem analogues caused by drugs in isolated hepatocytes. Structural isomerism and chirality of the resulting porphyrins
Authors: De Matteis F , Harvey C , Martin S R
Published: August 2015

Isolated rat hepatocytes incubated with two suicide substrates of cytochrome P-450, 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine(4-ethyl-DD C), convert exogenous mesohaem and deuterohaem into N-alkylated mesoporphyrins and deuteroporphyrins respectively. The N-alkylated mesoporphyrins can be separated by h.p.l.c. from the corresponding N-alkylated protoporphyrins originating from endogenous haem; in this way the contribution of both endogenous and exogenous pools of haem can be studied in the same experiment. N-Alkylated mesoporphyrin exhibits chiral properties, and its isomeric composition and/or amount are dependent on the particular cytochrome P-450 enzyme predominating in the cell. These findings provide additional and more direct evidence that exchangeable haem is taken up by cytochrome P-450 before being N-alkylated.
1-Naphthylisothiocyanate-induced permeability of hepatic tight junctions to proteins
Authors: Kan K S , Coleman R
Published: August 2015

We have studied the early action of 1-naphthylisothiocyanate (ANIT) in relation to its effect on the permeability barrier formed by hepatic tight junctions. Materials having different Mr values [inulin (5000), horseradish peroxidase (HRP) (40,000), ovalbumin (also 40,000) and pig gamma-globulin (IgG) (160,000)] were individually pulsed, within 1 min, into perfused rat livers operating under single-pass conditions. In untreated rats, a small peak of HRP and ovalbumin and a comparatively larger peak of inulin were observed in the bile at 7 min. In rats treated with ANIT, with increasing duration of ANIT treatment the inulin peak increased proportionally, whereas the HRP and ovalbumin peaks remained unchanged until after 10 h of ANIT exposure; gamma-globulin was not detected in the 7 min bile sample until after 14 h of ANIT treatment. Bile flow in all rats remained approximately the same until after 14 h of ANIT pretreatment, when substantial bile-flow reduction was observed. Phenobarbitone pretreatment increased the effect of ANIT and massively elevated the first HRP peak; it also shortened the time (to 4 h) at which the increase in permeability to this protein was observed. In contrast, the first HRP peak was virtually abolished in rats that had received the mixed-function-oxidase inhibitor SKF 525A. These experiments suggest that (i) ANIT progressively increased the permeability of the junctional barrier before the reduction in bile flow, (ii) the ANIT-increased permeability change seems to be inversely dependent upon the Mr of the infused proteins, and (iii) metabolites of ANIT were involved in the development of the junctional permeability change.
Comparison of small proteoglycans from skin fibroblasts and vascular smooth-muscle cells
Authors: Rauch U , Glössl J , Kresse H
Published: August 2015

Physicochemical and chemical properties of small proteoglycans containing galactosaminoglycan chains from cultured human skin fibroblasts and human smooth-muscle cells were compared to determine the extent of structural similarity. The proteoglycan secreted by smooth-muscle cells was of larger molecular size and of higher buoyant density, due to longer glycosaminoglycan chains, than the secretion product of skin fibroblasts. Additionally, both proteoglycans differed in the ratio of iduronic acid and glucuronic acid residues. On the other hand, degradation of secreted [3H]leucine-labelled proteoglycans with chondroitin ABC lyase followed by SDS/polyacrylamide-gel electrophoresis resulted in the appearance of core protein bands of identical size (Mr 48,000 and 45,000, depending on the number of asparagine-bound oligosaccharides). An Mr value of 40,000 was determined for the core protein of cells pretreated with tunicamycin. An antibody against the core protein from fibroblast secretions was cross-reactive with the core protein from smooth-muscle cells. Core protein accumulating intracellularly after treatment with carbonyl cyanide m-chlorophenylhydrazone exhibited, on reduction and alkylation, an isoelectric point of 7.8 in both cell types. Limited proteolysis by staphylococcal V8 serine proteinase or endoproteinase Lys-C led in both instances to the formation of peptides of identical size. Peptides bearing asparagine-bound oligosaccharides were free of glycosaminoglycan chains. Similar peptide patterns were obtained when 125I-labelled core proteins were digested with either trypsin or chymotrypsin. Thus small proteoglycans from fibroblasts and smooth-muscle cells can be differentiated by their glycosaminoglycan moieties but not by the nature of their core proteins.
Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450
Authors: Wyman J F , Gollan J L , Settle W , Farrell G C , Correia M A
Published: August 2015

After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglobin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that haemoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of [3H]haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Our findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic ‘free’ haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.
Metabolism of glucose, glutamine, long-chain fatty acids and ketone bodies by murine macrophages
Authors: Newsholme P , Curi R , Gordon S , Newsholme E A
Published: August 2015

Maximum activities of some key enzymes of metabolism were studied in elicited (inflammatory) macrophages of the mouse and lymph-node lymphocytes of the rat. The activity of hexokinase in the macrophage is very high, as high as that in any other major tissue of the body, and higher than that of phosphorylase or 6-phosphofructokinase, suggesting that glucose is a more important fuel than glycogen and that the pentose phosphate pathway is also important in these cells. The latter suggestion is supported by the high activities of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. However, the rate of glucose utilization by ‘resting’ macrophages incubated in vitro is less than the 10% of the activity of 6-phosphofructokinase: this suggests that the rate of glycolysis is increased dramatically during phagocytosis or increased secretory activity. The macrophages possess higher activities of citrate synthase and oxoglutarate dehydrogenase than do lymphocytes, suggesting that the tricarboxylic acid cycle may be important in energy generation in these cells. The activity of 3-oxoacid CoA-transferase is higher in the macrophage, but that of 3-hydroxybutyrate dehydrogenase is very much lower than those in the lymphocytes. The activity of carnitine palmitoyltransferase is higher in macrophages, suggesting that fatty acids as well as acetoacetate could provide acetyl-CoA as substrate for the tricarboxylic acid cycle. No detectable rate of acetoacetate or 3-hydroxybutyrate utilization was observed during incubation of resting macrophages, but that of oleate was 1.0 nmol/h per mg of protein or about 2.2% of the activity of palmitoyltransferase. The activity of glutaminase is about 4-fold higher in macrophages than in lymphocytes, which suggests that the rate of glutamine utilization could be very high. The rate of utilization of glutamine by resting incubated macrophages was similar to that reported for rat lymphocytes, but was considerably lower than the activity of glutaminase.
Purification and characterization of quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41
Authors: Dokter P , Frank J , Duine J A
Published: August 2015

Quinoprotein glucose dehydrogenase (EC 1.1.99.17) from Acinetobacter calcoaceticus L.M.D. 79.41 was purified to homogeneity. It is a basic protein with an isoelectric point of 9.5 and an Mr of 94,000. Denaturation yields two molecules of PQQ/molecule and a protein with an Mr of 48000, indicating that the enzyme consists of two subunits, which are probably identical because even numbers of aromatic amino acids were found. The oxidized enzyme form has an absorption maximum at 350 nm, and the reduced form, obtained after the addition of glucose, at 338 nm. Since double-reciprocal plots of initial reaction rates with various concentrations of glucose or electron acceptor show parallel lines, and substrate inhibition is observed for glucose as well as for electron acceptor at high concentrations, a ping-pong kinetic behaviour with the two reactants exists. From the plots, Km values for glucose and Wurster's Blue of 22 mM and 0.78 mM respectively, and a Vmax. of 7.730 mumol of glucose oxidized/min per mg of protein were derived. The enzyme shows a broad substrate specificity for aldose sugars. Cationic electron acceptors are active in the assay, anionic acceptors are not. A pH optimum of 9.0 was found with Wurster's Blue and 6.0 with 2,6-dichlorophenol-indophenol. Two types of quinoprotein glucose dehydrogenases seem to exist: type I enzymes are acidic proteins from which PQQ can be removed by dialysis against EDTA-containing buffers (examples are found in Escherichia coli, Klebsiella aerogenes and Pseudomonas sp.); type II enzymes are basic proteins from which PQQ is not removed by dialysis against EDTA-containing buffers (examples are found in A. calcoaceticus and Gluconobacter oxydans).
Squid neurofilaments. Phosphorylation and Ca2+-dependent proteolysis in situ
Authors: Brown A , Eagles P A M
Published: August 2015

Three major polypeptides co-purify with neurofilaments from squid (Loligo forbesi) axoplasm: P60 (apparent Mr 60,000), P200 (apparent Mr 200,000) and Band 1 (apparent Mr 400,000). Anti-IFA, a monoclonal antibody that recognizes an epitope common to all classes of intermediate filaments, binds to P200 and P60. When axoplasm is incubated with [32P]Pi, the major phosphorylated polypeptides are P200 and Band 1. We have investigated Ca2+-dependent proteolysis of [32P]phosphorylated axoplasm in order to localize the major sites of phosphorylation and to probe the arrangement of the polypeptides in the filament. The proteinase preferentially cleaves P200 and Band 1, liberating the phosphorylated domains. Analysis of proteolysed filaments by electron microscopy and gel electrophoresis shows that most of P200 and Band 1 can be cleaved while still maintaining intact filaments. We suggest that P200 is initially cleaved within a single highly sensitive region, generating two major fragments called P100p (apparent Mr 100,000) and P110s (apparent Mr 110,000). P100p contains the Anti-IFA epitope and co-sediments with filaments, whereas P110s is highly phosphorylated and does not sediment with filaments. Band 1 is cleaved to produce a soluble high-Mr fragment that is phosphorylated and that represents a major portion of the undigested component, whereas P60 is relatively resistant to limited proteolysis. Thus proteolysis appears to define two major filament domains: a conserved core that forms the backbone of the filament, and a highly phosphorylated peripheral region that is not essential for filament integrity.
Effect of intracellular alkalinization on pancreatic islet calcium uptake and insulin secretion
Authors: Lindström P , Sehlin J
Published: August 2015

Microdissected beta-cell-rich pancreatic islets of ob/ob mice were used in studies of the relationship between intracellular pH (pHi) and 45Ca2+ uptake and insulin release. Stepwise increases in extracellular pH (pHo) from 6.80 to 8.00 resulted in a parallel, although less pronounced, elevation of pHi from 7.24 to 7.69. Experimental conditions that alkalinize the islet cell interior, i.e. addition of 5 mM-NH4+, sudden withdrawal of extracellular bicarbonate buffer or increase in pHo, induced insulin secretion in the absence of other types of secretory stimulation (1 mM-D-glucose). Intracellular acidification by lowering pHo below 7.40 or sudden addition of bicarbonate buffer did not induce insulin secretion. The removal of extracellular bicarbonate buffer, increase in pHo from 7.40 to 8.00, or the addition of 5 mM-L-5-hydroxytryptophan or 5 mM-NH4+, which all alkalinize the islet cells and induce insulin secretion, also increased the La3+-non-displaceable 45Ca2+ uptake in the presence of 1 mM-D-glucose. The results suggest that intracellular alkalinization in beta-cells can trigger insulin secretion. Taken together with the fact that D-glucose increases pHi in the islet cells, the results also point to the possibility that alkalinization may be a link in the stimulus-secretion coupling sequence in beta-cells.
Evidence for conservation of dietary lipid in the rat during lactation and the immediate period after removal of the litter. Decreased oxidation of oral [1-14C]triolein
Authors: Oller do Nascimento C M , Williamson D H
Published: August 2015

Production of 14CO2 from an oral load of [1-14C]triolein was greatly decreased (70%) in lactating rats or immediately after (24-48 h) removal of the litter, compared with virgin rats. This decreased oxidation of dietary lipid was accompanied by accumulation of 14C-labelled lipid in lactating mammary gland or adipose tissue (after litter removal). No difference in 14CO2 production between lactating and virgin rats was observed when [1-14C]octanoate was administered. It is concluded that a major factor in this conservation of dietary triacylglycerol is the relative activity of lipoprotein lipase in the tissues.
Secretin induces rapid increases in inositol trisphosphate, cytosolic Ca2+ and diacylglycerol as well as cyclic AMP in rat pancreatic acini
Authors: Trimble E R , Bruzzone R , Biden T J , Farese R V
Published: August 2015

Previous studies have shown that the dose-response relationship for secretin-stimulated cyclic AMP accumulation is different from that for secretin-stimulated enzyme secretion in the rat exocrine pancreas. Here we show that secretin concentrations of 10(-10) M and higher stimulated a rise in cyclic AMP levels, with maximum effect on cyclic AMP accumulation being achieved already with 10(-8) M-secretin. However, at this concentration of secretin, enzyme secretion rates were approximately half-maximal. Unexpectedly, at concentrations of secretin greater than 10(-8) M there was evidence suggestive of phosphatidylinositol bisphosphate hydrolysis with rapid increases in inositol trisphosphate, cytosolic free calcium and diacylglycerol content of rat pancreatic acini. Furthermore, there was a dose-response relationship among secretin concentration (in the range 10(-8) M-2 × 10(-6) M), increases in inositol trisphosphate and increases in cytosolic free calcium ([Ca2+]i). Contrary to what has been previously believed, these results clearly indicate that in rat pancreatic acini secretin not only stimulates cyclic AMP accumulation but also raises inositol trisphosphate, [Ca2+]i and diacylglycerol. Thus, two second messenger systems may play a role in the regulation of secretin-induced amylase release.
Primary structure of human complement component C2 Homology to two unrelated protein families
Authors: Bentley D R
Published: August 2015

The primary structure of the second component of human complement (C2) was determined by cDNA cloning and sequence analysis. C2 has 39% identity with the functionally analogous protein Factor B. The C-terminal half of C2a is homologous to the catalytic domains of other serine proteinases. C2b contains three direct repeats of approx. 60 amino acid residues. They are homologous to repeats in Factor B, C4b-binding protein and Factor H, suggesting a functional significance of the repeat in C4b and C3b binding. The repeats are also found in the non-complement proteins beta 2-glycoprotein I and interleukin-2 receptor, and this repeat family may be widespread.
Mannitol-1-phosphate dehydrogenase of Escherichia coli Chemical properties and binding of substrates
Authors: Chase T
Published: August 2015

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5′-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5′-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.