bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for the sequential assembly of cytochrome oxidase subunits in rat liver mitochondria
Authors: Wielburski A , Nelson B D
Published: August 2015

The assembly of cytochrome oxidase was studied in isolated rat liver mitochondria and isolated rat hepatocytes labelled in vitro with L-[35S]methionine. This was achieved by studying the temporal association of radioactive subunits which are immunoabsorbed with antibodies against subunits I, II and the holoenzyme. Antibodies against the holoenzyme were shown to be highly specific for subunit V. The results show that subunit I appears in the holoenzyme late in the assembly process. No radioactive subunit I is absorbed with antiserum against subunit II or the holoenzyme (subunit V) after a 30 min pulse in either isolated mitochondria or hepatocytes. However, both antisera absorb radioactive subunits I after a 150 min chase in isolated hepatocytes. This was confirmed using antibodies against subunit I, which absorbed only radioactive subunit I after a 30 min pulse but absorbed radioactive subunits I-III and VI after a 150 min chase. Thus, the late assembly of radioactive subunit I is explained by a temporal sequence in the assembly process and not by the presence of a large, non-radioactive pool of subunit I. Using the above approach and the three specific antisera, the following temporal sequence in the assembly of cytochrome oxidase was established. Subunits II and III assemble rapidly with each other or with cytoplasmically translated subunit VI. This complex of three peptides in turn assembles slowly with subunit I or with the other cytoplasmically translated subunits. The early association of subunit VI with the mitochondrially translated subunits II and III suggests a possible role of the former in integration of the holoenzyme.
Regulation of the gastric microsomal (H+ + K+)-transporting ATPase system by the endogenous activator. Effect of phospholipase A2 treatment
Authors: Ray T K , Nandi J
Published: August 2015

Pig gastric microsomal (H+ + K+)-stimulated ATPase activity was nearly abolished within 10 min of digestion with phospholipase A2 at room temperature. The enzyme activity could be largely restored by a cytosolic activator protein partially purified from the gastric cells. The K+ sensitivity and turnover of 32P-labelled intermediates produced by the control and the activator-reconstituted microsomal (H+ + K+)-stimulated ATPase were closely similar but were widely different to those from treated membranes without activator reconstitution. The data suggest an essential requirement for the endogenous activator for gastric (H+ + K+)-stimulated ATPase function.
Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non-hepatic tissues of the rat
Authors: McGarry J D , Mills S E , Long C S , Foster D W
Published: August 2015

The requirement for carnitine and the malonyl-CoA sensitivity of carnitine palmitoyl-transferase I (EC 2.3.1.21) were measured in isolated mitochondria from eight tissues of animal or human origin using fixed concentrations of palmitoyl-CoA (50 microM) and albumin (147 microM). The Km for carnitine spanned a 20-fold range, rising from about 35 microM in adult rat and human foetal liver to 700 microM in dog heart. Intermediate values of increasing magnitude were found for rat heart, guinea pig liver and skeletal muscle of rat, dog and man. Conversely, the concentration of malonyl-CoA required for 50% suppression of enzyme activity fell from the region of 2-3 microM in human and rat liver to only 20 nM in tissues displaying the highest Km for carnitine. Thus, the requirement for carnitine and sensitivity to malonyl-CoA appeared to be inversely related. The Km of carnitine palmitoyltransferase I for palmitoyl-CoA was similar in tissues showing large differences in requirement for carnitine. Other experiments established that, in addition to liver, heart and skeletal muscle of fed rats contain significant quantities of malonyl-CoA and that in all three tissues the level falls with starvation. Although its intracellular location in heart and skeletal muscle is not known, the possibility is raised that malonyl-CoA (or a related compound) could, under certain circumstances, interact with carnitine palmitoyltransferase I in non-hepatic tissues and thereby exert control over long chain fatty acid oxidation.
Interaction of malonyl-CoA and related compounds with mitochondria from different rat tissues. Relationship between ligand binding and inhibition of carnitine palmitoyltransferase I
Authors: Mills S E , Foster D W , McGarry J D
Published: August 2015

The sensitivity of carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) to inhibition by malonyl-CoA and related compounds was examined in isolated mitochondria from liver, heart and skeletal muscle of the rat. In all three tissues the same order of inhibitory potency emerged: malonyl-CoA much greater than succinyl-CoA greater than methylmalonyl-CoA much greater than propionyl-CoA greater than acetyl-CoA. For any given agent, suppression of CPT I activity was much greater in skeletal muscle than in liver, with the heart enzyme having intermediate sensitivity. With skeletal-muscle mitochondria a high-affinity binding site for [14C]malonyl-CoA was readily demonstrable (Kd approx. 25 nM). The ability of other CoA esters to compete with [14C]malonyl-CoA for binding to the membrane paralleled their capacity to inhibit CPT I. Palmitoyl-CoA also competitively inhibited [14C]malonyl-CoA binding, in keeping with its known ability to overcome malonyl-CoA suppression of CPT I. For reasons not yet clear, free CoA displayed anomalous behaviour in that its competition for [14C]malonyl-CoA binding was disproportionately greater than its inhibition of CPT I. Three major conclusions are drawn. First, malonyl-CoA is not the only physiological compound capable of suppressing CPT I, since chemically related compounds, known to exist in cells, also share this property, particularly in tissues where the enzyme shows the greatest sensitivity to malonyl-CoA. Second, malonyl-CoA and its analogues appear to interact with the same site on the mitochondrial membrane, as may palmitoyl-CoA. Third, the degree of site occupancy by inhibitors governs the activity of CPT I.
Segregation into separate rouleaux of erythrocytes from different species. Evidence against the agglomerin hypothesis of rouleaux formation
Authors: Forsdyke D R , Ford P M
Published: August 2015

Erythrocytes from one species were labelled with fluorescein isothiocyanate and mixed with unlabelled erythrocytes from another species. Albumin polymers were added to generate rouleaux. The species of origin of erythrocytes in rouleaux was determined by fluorescence microscopy. Erythrocytes from different species segregated into independent rouleaux. However, fluorescent and non-fluorescent erythrocytes from one individual were mixed randomly in rouleaux. These results confirm, using a novel experimental approach, previous observations of Sewchand & Canham [(1976) Can. J. Physiol. Pharmacol. 54, 437-442]. Since rouleaugenic agents are not species-specific, under the ‘agglomerin’ hypothesis of rouleau formation they would be expected to form bridges between cells from different species. It follows that either the agglomerin hypothesis is incorrect, or additional species-specific surface components are involved in the aggregation of agglomerin-cross-bridged cells.
Monocytes and platelets share the glycoproteins IIb and IIIa that are absent from both cells in Glanzmann's thrombasthenia type I
Authors: Gogstad G , Hetland Ø , Solum N O , Prydz H
Published: August 2015

By means of an antiserum specific to the complex of the platelet membrane glycoproteins IIb and IIIa we demonstrate here that monocytes and purified monocyte membranes share these glycoproteins with platelets. The monocyte glycoprotein IIb-IIIa complex showed complete immunological identity with the platelet counterpart and, furthermore, dissociated after EDTA treatment exactly as did the platelet complex. In Glanzmann's thrombasthenia type I, monocytes as well as platelets lack this antigen completely.
Duration of the calcium signal in the mitogenic stimulation of thymocytes
Authors: Hesketh T R , Bavetta S , Smith G A , Metcalfe J C
Published: August 2015

An increase in the free cytoplasmic Ca2+ concentration in thymocytes can be detected by the fluorescent indicator quin 2 within a few seconds of the addition of concanavalin A and the response is quantified from the increased proportion of quin 2 in the cells chelated by Ca2+ (‘% Ca-quin 2’). The % Ca-quin 2 in untreated cells is 53 +/- 6%, increases to 64 +/- 7% immediately after the addition of concanavalin A and declines spontaneously over 24 h back to the level in untreated cells (53 +/- 6%). The increase in % Ca-quin 2 in response to concanavalin A is completely blocked when 50 mM-alpha-methyl D-mannoside is added before concanavalin A and completely reversed when the competing sugar is added immediately after the mitogen. Addition of alpha-methyl D-mannoside at increasing intervals after concanavalin A addition causes a progressively smaller decrease in % Ca-quin 2 and has a negligible effect after 24 h, when the % Ca-quin 2 is the same as that in untreated cells. The decline in the calcium signal defined by these experiments has a similar time course to cap formation by concanavalin A on the cells. It is concluded that the calcium signal lasts only while concanavalin A is bound to the cell surface and is terminated either by capping or by the addition of alpha-methyl D-mannoside.
Time course of the effect of catabolic doses of corticosterone on protein turnover in rat skeletal muscle and liver
Authors: Odedra B R , Bates P C , Millward D J
Published: August 2015

The time course of the response of protein synthesis in muscle and liver to catabolic doses of corticosterone (10 mg/day per 100 g body wt.) was studied in vivo in growing rats over a 12-day period. The rate of protein synthesis in muscle and liver and the rate of actomyosin synthesis in muscle were measured by the phenylalanine-flooding technique, and 3-methylhistidine (N tau-methylhistidine) synthesis was measured by injection of labelled histidine. 3-Methylhistidine concentrations in tissue free pools and urinary excretion were also measured to compare directly with the rate of muscle protein degradation determined as the difference between synthesis and growth each day during the treatment. The overall rate of protein synthesis in muscle fell gradually over the first 4 days, reaching a rate after 5 days that was 36% of the initial rate, and this lower rate was then maintained for the following week. This decrease in the overall rate was accompanied with changes in the relative rate of synthesis in muscle proteins, since during the first 4 days there was a disproportionate decrease in the rate of actomyosin synthesis, and specifically 3-methylhistidine synthesis. In the latter case the synthesis rate was decreased to only 4% of its initial rate after 4 days. These changes in protein synthesis in muscle were accompanied by a transient increase in the rate of protein degradation, which was more than doubled on days 2 and 3 of treatment but which returned to the original rate on day 5, and a similar pattern of response was indicated by urinary 3-methylhistidine excretion, which also exhibited a transient increase. Thus in this case 3-methylhistidine excretion and measured rates of protein degradation in muscle do correlate. The transient effects of the glucocorticoids on degradation compared with the sustained effect on synthesis suggest that these two responses are achieved by different mechanisms. The hepatic size and protein mass were increased by the treatment, and protein synthesis was well maintained until after 12 days, when the rate was suppressed. Although the fractional synthesis rate was transiently increased for 24 h, it is argued that the enlarged liver most likely reflects a decrease in protein degradation resulting from the increased amino acid supply to the liver. This would result from the cessation of muscle growth while dietary supply was maintained.
Alterations in phosphofructokinase isoenzymes during early human development. Establishment of adult organ-specific patterns
Authors: Davidson M , Collins M , Byrne J , Vora S
Published: August 2015

Human 6-phosphofructokinase (EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M), liver (L) and platelet (P) subunits, which are under separate genetic control. In the adult, the proportion of these subunits in different organs reflects the relative activity of glycolysis versus gluconeogenesis. To elucidate the developmental basis for the observed distribution, we investigated the isoenzymic transitions of phosphofructokinase in human foetuses (12-40 weeks' gestation) by using high-resolution chromatography and monoclonal antibodies. We studied skeletal muscle, heart, liver and brain because these organs show very different glycolytic fluxes and isoenzymic patterns in adult individuals. Our results demonstrate that there is no unique ‘foetal’ form of phosphofructokinase in humans, but all three loci are variably expressed in all foetal organs during early gestation. As development proceeds, muscle and liver isoenzyme patterns show dramatic changes, with disappearance of P and L subunits in muscle and transient reappearance of M and P subunits in liver; in contrast, phosphofructokinase isoenzymes change little in brain and heart. Most changes occur at mid-gestation and near term, and adult isoenzyme patterns are expressed at birth, indicating that organ differentiation is complete. These studies show that phosphofructokinase undergoes changes of isoenzyme patterns similar to, but not identical with, those of other multilocus isoenzyme systems of glycolysis. The observed changes probably reflect changing patterns of gene expression, with repression of some loci and activation of others.
Muscle glutamine production in diabetic ketoacidotic rats
Authors: Goldstein L , Perlman D F , McLaughlin P M , King P A , Cha C J
Published: August 2015

The mechanism of activation of glutamine production by the hindlimb during diabetic ketoacidosis (DKA) was investigated in rats. Muscle glutamine production was estimated to account for over 90% of the total glutamine produced by the hindlimb. DKA produced significant increases in the concentrations of NH4+ and IMP in hindlimb muscles, suggesting that AMP deaminase is activated by DKA. NH4Cl- and HCl-induced acidosis did not produce these changes, indicating either that acidosis itself is not the stimulus for increased AMP deaminase activity or that the more severe degree of acidosis accompanying DKA is necessary for activation. Muscle glutamine concentrations were depressed in DKA. Experiments with isolated epitrochlearis muscle showed that the transport and permeability properties of the muscle cells (as judged by uptake and release of alpha-aminoisobutyrate and glutamine) were not altered by DKA. However, glutamine uptake by muscle cells was significantly inhibited by L-leucine, the concentration of which, along with other branched-chain amino acids, is markedly elevated in DKA.
Low-spin ferric forms of cytochrome a3 in mixed-ligand and partially reduced cyanide-bound derivatives of cytochrome c oxidase
Authors: Hill B C , Brittain T , Eglinton D G , Gadsby P M A , Greenwood C , Nicholls P , Peterson J , Thomson A J , Woon T C
Published: August 2015

Optical-absorption-, e.p.r.- and m.c.d. (magnetic-circular-dichroism)-spectroscopic measurements were made on liganded derivatives of oxidized and partially reduced cytochrome c oxidase. When NO was added to oxidized cyanide-bound cytochrome c oxidase, no changes occurred in the optical-absorption difference spectrum. In contrast, NO induced reduction of cytochrome a3 and formation of the nitrosylferrohaem species when the oxidized resting enzyme was the starting material. E.p.r. spectroscopy of the NO-treated oxidized cyanide-bound enzyme revealed the presence of a low-spin haem signal at g = 3.40, whereas the g = 3.02 and g = 2.0 signals of the oxidized enzyme remained unchanged. Both haem groups in this species are e.p.r.-detectable simultaneously. Examination of an identical sample by m.c.d. spectroscopy in the near-i.r. region identified two distinct low-spin species at 1565 and 1785 nm. Irradiation with white light of the NO-treated cyanide-bound sample at 10K resulted in the disappearance of the g = 3.40 e.p.r. signal and the m.c.d. signal at 1785 nm, whereas a band at 1950nm increased in intensity. When the photolysed sample was warmed to 50K and held in the dark for 15 min, the original spectrum returned. Magnetization studies of the 1785nm m.c.d. band support the assignment of this signal to the same metal centre that gives rise to the g = 3.40 e.p.r. signal. The effect of NO on the oxidized cyanide-bound enzyme was compared with that obtained when the oxidized cyanide-bound species was taken to the partially reduced state. Cytochrome a3 is e.p.r.-detectable with a g-value of 3.58 [Johnson, Eglinton, Gooding, Greenwood & Thomson (1981) Biochem. J. 193, 699-708]. Its near-i.r. m.c.d. spectrum shifts from 1950nm in the oxidized cyanide-bound enzyme to 1545nm on addition of reductant. A scheme is advanced for the structure of the cytochrome a3-CuB site that allows for cyanide binding to Fea3 and NO binding to CuB. Cyanide is the bridging ligand in the ferromagnetically coupled cytochrome a3-CuB pair of oxidized cyanide-bound cytochrome c oxidase. The bridged structure and the magnetic interaction are broken when the enzyme is partially reduced. However, when NO binds to CuB the cyanide bridge remains intact, but now the odd spins of NO and CuB are magnetically coupled.
The effect of pH on the exchangeability with deuterium of protons coupled to molybdenum(V) in the active and the desulpho forms of xanthine oxidase
Authors: Malthouse J P G , Bray R C
Published: August 2015

The effect of pH variation on the exchangeability with deuterium of protons strongly coupled to Mo(V) in the active and desulpho forms of xanthine oxidase was studied by e.p.r. and rapid freezing, in extension of the work of Gutteridge, Tanner & Bray [Biochem. J. (1978) 175, 887-897]. Above neutrality, exchange rates increased with increasing pH. Detailed studies were made on the desulpho enzyme under a variety of conditions, and exchange rate constants at 22 degrees C ranged from 0.16s -1 at pH 6.6 to 1.6s -1 at pH 11.3. The mechanism of proton exchange in the enzyme is discussed. The interpretation by the above workers that the strongly coupled proton of the active enzyme is on sulphur and that of the desulpho enzyme is on oxygen remains valid (and is in agreement with other work), as do their proposals for the structures of the protonated and deprotonated species. However, pK values cannot be calculated from the exchange data. It is likely that the relatively low rates of exchange observed are due to the difference of structure between the protonated and the deprotonated forms. In the case of the desulpho enzyme, an exchange mechanism, which involves the proton exchanging both as such and along with oxygen in the form of a hydroxyl ion, is discussed.
A new mass-spectrometric C-terminal sequencing technique finds a similarity between γ-interferon and α2-interferon and identifies a proteolytically clipped γ-interferon that retains full antiviral activity
Authors: Rose K , Simona M G , Offord R E , Prior C P , Otto B , Thatcher D R
Published: August 2015

A novel mass-spectrometric technique is described that permits the identification of the C-terminal peptide of a protein. The technique involves the incorporation of 18O into all alpha-carboxy groups liberated during enzyme-catalysed partial hydrolysis of the protein, followed by mass spectrometry to identify as the C-terminal peptide the only peptide that did not incorporate any 18O. The technique has been used to identify the true C-terminal tryptic peptide of a bacterially produced gamma-interferon and to distinguish it from a peptide produced by anomalous tryptic cleavage. It was found that a closely similar sequence segment of bacterially produced alpha 2-interferon undergoes an analogous cleavage. The technique was also used to identify the C-terminus of a clipped gamma-interferon that retains full antiviral activity.
The optical properties of CuA in bovine cytochrome c oxidase determined by low-temperature magnetic-circular-dichroism spectroscopy
Authors: Greenwood C , Hill B C , Barber D , Eglinton D G , Thomson A J
Published: August 2015

The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.
Purification to homogeneity of pig leucocyte catabolin, a protein that causes cartilage resorption in vitro
Authors: Saklatvala J , Curry V A , Sarsfield S J
Published: August 2015

Catabolin, a protein that causes proteoglycan resorption in explants of living cartilage, was purified to homogeneity from culture medium conditioned by culturing buffy-coat leucocytes from 60 litres of pig blood in the presence of concanavalin A. The purification steps were (1) gel filtration of concentrated medium, (2) chromatofocusing, (3) hydroxyapatite chromatography, (4) anion-exchange chromatography (Mono Q), (5) reversed-phase high-pressure liquid chromatography (h.p.l.c.) (Zorbax ODS). These achieved approx. 9000-fold purification from the starting material. The purified protein when reduced ran as a single band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis with Mr 21000. On isoelectric focusing its pI was 4.8-5.0, and there was evidence of micro-heterogeneity. The protein co-migrated with active material on h.p.l.c., isoelectric focusing and SDS gels (15 and 12.5% acrylamide) under both reducing and non-reducing conditions. The pure protein caused proteoglycan release from cultured bovine nasal cartilage at 20pM. Its possible identity with interleukin 1 is discussed.
Purification and properties of two lectins from the latex of the euphorbiaceous plants Hura crepitans L. (sand-box tree) and Euphorbia characias L. (Mediterranean spurge)
Authors: Barbieri L , Falasca A , Franceschi C , Licastro F , Rossi C A , Stirpe F
Published: August 2015

1. From the latex of two members of the plant family Euphorbiaceae, Hura crepitans L. (sand-box tree) and Euphorbia characias L. (Mediterranean spurge), two lectins were purified by affinity chromatography on acid-treated Sepharose 6B followed by elution with D-galactose. 2. The lectin from E. characias is a single molecular species with Mr 80 000, made up of two identical subunits with Mr 40 000, and is a glycoprotein containing 11% carbohydrate. 3. The lectin from H. creptians appears as a mixture of three isolectins with Mr 140 000, consisting of four different subunits with Mr values 37 500, 35 500, 31 000, and 29 000. 4. Both lectins have haemagglutinating activity, with no specificity for human blood groups. The haemagglutinating activity is inhibited by D-galactose and by galactose-containing oligosaccharides. 5. The lectin from H. crepitans is mitogenic to human T-, but not to B-, lymphocytes. The latex of E. characias is mitogenic to T- and, to a lesser extent, to B-, lymphocytes, but the purified E. characias lectin has no mitogenic activity. 6. The lectin from H. crepitans, but not that from E. characias, inhibits protein synthesis by a rabbit reticulocyte lysate.
Regulation of activity of purified guanylate cyclase from liver that is unresponsive to nitric oxide
Authors: Tsai S C , Adamik R , Manganiello V C , Vaughan M
Published: August 2015

Guanylate cyclase was purified from rat liver supernatant. Electrophoresis under denaturing conditions revealed one major peptide of Mr approx. 69 000. On the basis of the Stokes radius (4.7 nm) and S20,w (6.4S), the calculated Mr value of the native enzyme was 133 000, i.e. it is apparently a homodimer. Kinetics of inactivation by diamide (which was reversible with dithiothreitol) suggested that oxidation of a single class of thiol sites was involved. In the absence of other additions, cyclase activity assayed with Mn2+ was over 7 times that assayed with Mg2+; maximal effects were observed with approx. 5 mM of each (with 1 mM-GTP). The purified enzyme was markedly activated by nitrosylhaemoglobin. Relative activation was much greater in assays with Mg2+ than with Mn2+, although maximal activities were similar. When assayed with Mg2+, the enzyme exhibited a single Km (0.35 mM) for GTP; with Mn2+, plots of 1/v versus 1/[S] were non-linear. Activator or nitrosylhaemoglobin increased Vmax, but did not alter Km in the presence of either Mg2+ or Mn2+. The enzyme was inhibited by Na3VO4, Na2WO4 and Na2B4O7. Reduction from VV to VIV abolished the inhibitory effect of vanadate. Na2B4O7 (2 mM) inhibited activity with Mn2+, but not with Mg2+. In assays with Mg2+, but not with Mn2+, FMN, NAD+ and NADH (each 0.5 mM) inhibited activation by protoporphyrin IX and nitrosylhaemoglobin. Rotenone (0.6 mM) inhibited activity with protoporphyrin IX to a greater extent than with nitrosylhaemoglobin. Methylene Blue (1 mM) inhibited activation by nitrosylhaemoglobin, protoporphyrin IX and activator. It appears that this enzyme purified from rat liver lacks haem (and perhaps other components) required for activation by NO, and it should be particularly useful for elucidating the mechanism of action of NO, protoporphyrin IX and other activators.
Identification and metabolism of polyphosphoinositides in isolated islets of Langerhans
Authors: Laychock S G
Published: August 2015

Isolated islets were incubated with [32P]P1 and radiolabelling of polyphosphoinositides were determined. Labelling equilibrium was approached after 45 min, with a half-time of 15 min. D-Glucose decreased the amount of [32P]PO4 in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) within 0.5 min, and loss of radiolabel was still evident at 1 min. [32P]PO4 levels in polyphosphoinositides returned to basal levels within 5 min. Neither D-galactose nor D-glucose after pretreatment of islets with mannoheptulose elicited the polyphosphoinositide effect. The glucose-stimulated breakdown of polyphosphoinositides was inhibited by EGTA; re-addition of Ca2+ partially restored the glucose effect. Ionomycin and tolbutamide promoted the rapid breakdown of PtdIns(4,5)P2, whereas the breakdown of PtdIns4P was less rapid and of a lesser magnitude. The results suggest that the Ca2+-dependent breakdown of polyphosphoinositides in an early metabolic event during the initiation of insulin release.
Hormone-stimulated lipolysis in cardiac myocytes
Authors: Palmer W K , Kane T A
Published: August 2015

Type L hormone-sensitive lipase (HSL) activity was increased approx. 35% above control in cardiac myocytes incubated for 15 min with 5 nM-adrenaline. Concomitantly. adrenaline-stimulated myocytes had a lower triacylglycerol content, released more non-esterified fatty acid and had a higher intracellular concentration of cyclic AMP than did myocytes incubated without hormone. The lipase activity measured in adrenaline-stimulated and non-stimulated myocytes was stable in acetone/diethyl ether, stimulated by serum and inhibited by NaCl. These properties are consistent with the type L designation of this HSL. The finding that type L HSL is stimulated by adrenaline indicates that the enzyme that is being activated is found in the cell and not associated with an extracellular compartment of the myocardium.
The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers
Authors: Papa S , Guerrieri F , Izzo G
Published: August 2015

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active ‘pulsed’ state and the first one or two turnovers.
The α-adrenergic-mediated activation of the cardiac mitochondrial Ca2+ uniporter and its role in the control of intramitochondrial Ca2+in vivo
Authors: Crompton M , Kessar P , Al-Nasser I
Published: August 2015

Administration of methoxamine (10 microM, 2 min) to perfused rat hearts increased the rate at which subsequently isolated mitochondria accumulated Ca2+. Methoxamine did not change significantly the development of delta phi with time or the basal rates of Ca2+ flux on inhibition of the uniporter with Ruthenium Red. With 200 microM-Pi, the rates of Ca2+ uptake at constant delta phi were unaffected by the small variations in endogenous [Pi] between mitochondrial preparations, and were also unaffected by changes in internal Ca2+ over the approximate range 8-43 nmol of Ca2+/mg. At low internal Ca2+ (about 8 nmol/mg of protein) the rates of Ca2+ uptake at constant delta phi were unaffected by addition of 200 microM-Pi. Under these conditions, the uniporter activity and the uniporter conductance were increased by 38-40% by methoxamine pretreatment. The endogenous Ca2+ content of mitochondria from control heart was about 1.8 nmol of Ca2+/mg of protein. Perfusion with agonist increased the Ca2+ content as follows: 10 microM-methoxamine (2 min), 48%; 1 microM-isoprenaline (2 min), 100%; 1 microM-adrenaline (2 min), 140%. The implications of the data for the adrenergic control of oxidative metabolism by intramitochondrial Ca2+ is discussed.
Effect of starvation and refeeding on amino acid uptake by mammary gland of the lactating rat. Role of ketone bodies
Authors: Viña J R , Puertes I R , Montoro J B , Viña J
Published: August 2015

Arteriovenous differences of amino acids across the mammary glands of lactating rats are diminished when the rats are starved for 24 h. When 24 h-starved rats were refed for 2 1/2 h, the arteriovenous differences of amino acids returned to values similar to those found in well-fed rats. In order to find a possible explanation for these rapid changes, we tested the effect of ketone bodies on amino acid uptake by the gland. At 5 min after injection of acetoacetate to fed rats, when the total concentration of ketone bodies in blood was similar to that found in starvation, the uptake of amino acids by the mammary gland was similar to that found after starvation, i.e. lower than in fed rats. However, 30 min after administration of acetoacetate, when the arterial concentration of ketone bodies had returned to values similar to those in fed rats, the arteriovenous differences of amino acids were similar to those found in fed rats. We conclude that the changes in blood ketone bodies may be responsible, at least in part, for the changes in amino acid uptake that occur in starvation and in the starvation—refeeding transition.
Regio- and stereo-selective metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans
Authors: Cerniglia C E , Fu P P , Yang S K
Published: August 2015

Metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans was studied. C. elegans metabolized 4-methylbenz[a]anthracene primarily at the methyl group, this being followed by further metabolism at the 8,9- and 10,11-positions to form trans-8,9-dihydro-8,9-dihydroxy-4-hydroxymethylbenz[a]anthracene and trans-10,11-dihydro-10,11-dihydroxy-4-hydroxymethylbenz[a]anthracene. There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4-positions) or at the ‘K’ region (5,6-positions). The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by the application of u.v.-visible-absorption-, 1H-n.m.r.- and mass-spectral techniques. The 4-hydroxymethylbenz[a]anthracene trans-8,9- and −10,11-dihydrodiols were optically active. Comparison of the c.d. spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans with those of the corresponding benz[a]anthracene trans-dihydrodiols formed by rat liver microsomal fraction indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans- 10,11-dihydrodiol formed by C. elegans have S,S absolute stereochemistries, which are opposite to those of the predominantly 8R,9R- and 10R,11R-dihydrodiols formed by the microsomal fraction. Incubation of C. elegans with 4-methylbenz[a]anthracene under 18O2 and subsequent mass-spectral analysis of the metabolites indicated that hydroxylation of the methyl group and the formation of trans-dihydrodiols are catalysed by cytochrome P-450 mono-oxygenase and epoxide hydrolase enzyme systems. The results indicate that the fungal mono-oxygenase-epoxide hydrolase enzyme systems are highly stereo- and regio-selective in the metabolism of 4-methylbenz[a]anthracene.
Biosynthesis in vitro of complement subcomponents C1q, C1s and C1̅ inhibitor by resting and stimulated human monocytes
Authors: Bensa J C , Reboul A , Colomb M G
Published: August 2015

The capacity of cultured human monocytes to synthesize and to secrete the subcomponents of C1 and C1 inhibitor was examined. Non-stimulated monocytes secreted C1q and C1s from day 5 of culture. C1s reached a plateau immediately at its maximum level, whereas C1q secretion increased progressively until the end of the second week. Between day 12 and day 25, C1q secretion remained nearly constant (1-15 fmol/day per microgram of DNA, depending on the donor), whereas C1s secretion decreased and even in some cases stopped. C1r and C1 inhibitor were not secreted in detectable amounts by these resting cells. Stimulation of monocytes by yeasts, immunoglobulin G-opsonized sheep red blood cells or latex beads did not modify consistently C1q and C1s secretion. Activation by conditioned media from mitogen-, antigen- or allogeneic-stimulated lymphocyte cultures increased C1q production from 2 to 7 times and re-activated C1s secretion. Under the same conditions of activation, C1 inhibitor was secreted (up to 300 fmol/day per microgram of DNA) and C1r became detectable in culture supernatants. Isolated human monocytes are thus able to synthesize the whole C1 subcomponents; C1, if assembled, could be protected from non-immunological activation by locally produced C1 inhibitor. Activated monocytes appear to be a good tool for studying the assembly of C1 subcomponents and the role of C1 inhibitor in this process.
Slow passive diffusion of NAD+ between intact isolated plant mitochondria and suspending medium
Authors: Neuburger M , Douce R
Published: August 2015

Isolated potato (Solanum tuberosum) tuber mitochondria purified by isopycnic centrifugation in density gradients of Percoll were found to be highly intact, to be devoid of extramitochondrial contaminations and to retain a high rate of O2 consumption. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD+ content by passive diffusion. This led to a slow decrease of oxoglutarate-dependent O2 consumption by isolated mitochondria. Addition of NAD+ to the medium restored the initial State-3 rate of oxoglutarate oxidation. The rate of NAD+ accumulation in the matrix space was concentration-dependent, exhibited Michaelis-Menten kinetics and was strongly inhibited by the analogue N-4-azido-2-nitrophenyl-4-aminobutyryl-NAD+.
The effect of starvation and refeeding on lipogenic enzymes in mammary glands and livers of lactating rats
Authors: Grigor M R , Gain K R
Published: August 2015

Lactating rats were starved for 48 h and refed a high-carbohydrate diet for a further 48 h. Starvation stops milk secretion, which resumes shortly after refeeding. Three lipogenic enzymes, fatty acid synthase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and ‘malic’ enzyme (EC 1.1.1.40) all decrease in the mammary gland during starvation and are restored to the pre-starvation levels 48 h after refeeding. The same enzymes in liver also decrease during starvation, but increase to values significantly higher than those for the normal fed rats after refeeding the high-carbohydrate diet. For the fatty acid synthase these values were four times the pre-starvation values. Serum insulin and prolactin concentrations also increased upon refeeding the high-carbohydrate diet.
Glutamine synthetase activity of muscle in acidosis
Authors: King P A , Goldstein L , Newsholme E A
Published: August 2015

Metabolic acidosis stimulates the rate of glutamine release from muscle, and this in turn is used by the kidney in acid-base balance. NH4Cl, HCl or diabetic ketoacidosis increases the maximum activity of glutamine synthetase in skeletal muscle. Starvation and administration of adrenal steroids also increase the activity of the enzyme in muscle.
Effect of 1,3-diaminopropane on ornithine decarboxylase enzyme protein in thioacetamide-treated rat liver
Authors: Seely J E , Pegg A E
Published: August 2015

A radioimmunoassay for ornithine decarboxylase was used to study the regulation of this enzyme in rat liver. The antiserum used reacts with ornithine decarboxylase from mouse, human or rat cells. Rat liver ornithine decarboxylase enzyme activity and enzyme protein (as determined by radioimmunoassay) were measured in thioacetamide-treated rats at various times after administration of 1,3-diaminopropane. Enzyme activity declined rapidly after 1,3-diaminopropane treatment as did the amount of enzyme protein, although the disappearance of enzyme activity slightly preceded the loss of immunoreactive protein. The loss of enzyme protein after cycloheximide treatment also occurred rapidly, but was significantly slower than that seen with 1,3-diaminopropane. When 1,3-diaminopropane and cycloheximide were injected simultaneously, the rate of disappearance of enzyme activity and enzyme protein was the same as that seen with cycloheximide alone. These results show that the rapid loss in enzyme activity after 1,3-diaminopropane treatment is primarily due to a loss in enzyme protein and that protein synthesis is needed in order for 1,3-diaminopropane to exert its full effect. A macromolecular inhibitor of ornithine decarboxylase that has been termed antizyme is induced in response to 1,3-diaminopropane, but our results indicate that the loss of enzyme activity is not due to the accumulation of inactive ornithine decarboxylase-antizyme complexes. It is possible that the antizyme enhances the degradation of the enzyme protein. Control experiments demonstrated that the antiserum used would have detected any inactive antizyme-ornithine decarboxylase complexes present in liver since addition of antizyme to ornithine decarboxylase in vitro did not affect the amount of ornithine decarboxylase detected in our radioimmunoassay. Anti-(ornithine decarboxylase) antibodies may be useful in the purification of antizyme since the antizyme-ornithine decarboxylase complex can be immunoprecipitated, and antizyme released from the precipitate with 0.3 M-NaCl.
Effects of exogenous amines on mammalian cells, with particular reference to membrane flow
Authors: Dean R T , Jessup W , Roberts C R
Published: August 2015

We have reviewed the evidence that amines accumulate in intracellular vesicles of low pH, such as lysosomes and endosomes. There is consequent elevation of intravesicular pH, and inhibition of receptor-ligand dissociation often results from this pH change. We have argued that the capacity for fusion of such vesicles is also reduced by the high pH. We suggest that the variety of effects of amines on membrane flow and macromolecular transport we describe are at least partly due to such reduced fusion (Figs. 1 and 2). We propose that an internal low pH may facilitate heterologous vesicle-vesicle and vesicle-plasma membrane fusion. There is some evidence that clathrin can accelerate phospholipid vesicle fusion in vitro at low pH (Blumenthal et al., 1983) but no direct evidence on the role of intravesicular pH. This idea is consistent not only with the preceding discussion, but also with the fact that the intracellular membrane-bound compartments least involved in fusion events (e.g. mitochondria) are of neutral or alkaline internal pH. Membrane fusion is certainly required for the formation of vesicles at the periphery of the Golgi apparatus, and possibly earlier in the transport and processing of biosynthetic products in the Golgi (Bergeron et al., 1982). Thus the accumulation of amines in the Golgi may be responsible for several effects on the flow of macromolecules along their translocation pathways. The status of the plasma membrane in this view is complex. It might be argued that the pH dictating the fusion step in endocytosis is that of the extracellular fluid, in which case the inhibitory effects of amines on this process are not explained. However, the rapidity of acidification of the newly formed endocytic vesicles allows the possibility that plasma membrane invaginations might temporarily sequester areas which are of lower pH than that of the bulk extracellular fluid even before fusion, since the proton pumping enzyme(s) are probably present on the plasma membrane. Were this the case, then an acid pH could again be a factor determining membrane fusion at the plasma membrane. The inhibition of endocytosis by weak bases thus may again reflect elevation of pH in a sequestered compartment. From the data on the dependence of response on the concentration of amines, we anticipate that most responses involving membrane flow will be biphasic, with inhibitory effects at low amine concentration, giving way to stimulatory ones at higher concentrations. We suggest that the reported dichotomy between different amines in intracellular membrane fusion systems (D'Arcy Hart, 1982) may result from this concentration dependence.(ABSTRACT TRUNCATED AT 400 WORDS)
A study of the maloalcoholic fermentation pathway in Schizosaccharomyces pombe
Authors: Maconi E , Manachini P L , Aragozzini F , Gennari C , Ricca G S
Published: August 2015

The pathway of the maloalcoholic fermentation in Schizosaccharomyces pombe was investigated by a 1H-, 2H- and 13C-n.m.r.-spectroscopic study of hydrogen and deuterium distribution on the ethanol produced by S. pombe from L-malic acid in 2H2O and from L-[2-2H]malic acid. Our findings rule out a double-decarboxylation mechanism and agree with a pathway that involves acetaldehyde as intermediate.
Formation and reduction of a ‘peroxy’ intermediate of cytochrome c oxidase by hydrogen peroxide
Authors: Wrigglesworth J M
Published: August 2015

In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a ‘peroxy’ intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called ‘pulsed’ or ‘oxygenated’ ferric form of the enzyme.
Organic phosphates increase the solubility of avian haemoglobin D and embryonic chicken haemoglobin
Authors: Baumann R , Goldbach E , Haller E A , Wright P G
Published: August 2015

The isolated minor haemoglobin fractions (haemoglobin D) of ostrich, chicken and duck haemoglobin, which constitute about 30% of total intracellular haemoglobin, form crystalline aggregates upon deoxygenation at physiological temperature, ionic strength and pH and at haemoglobin concentrations even well below those present in the red cell. The aggregation is reversed by oxygenation, and can be inhibited by addition of organic phosphates or the corresponding major haemoglobin fraction in a stoichiometric ratio of 1:1. Embryonic haemoglobin from chicken has similar characteristics with respect to its solubility. The results indicate close functional homology of alpha D and embryonic pi-chains as well as a novel role for organic phosphates in the regulation of haemoglobin function.