bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative analysis of proton-linked transport systems. Glutamate transport in Staphylococcus aureus
Authors: Mitchell W J , Booth I R , Hamilton W A
Published: August 2015

1. The magnitude of the protonmotive force in respiring Staphylococcus aureus was measured over the range of extracellular pH from 5.6 to 7.8. 2. The membrane potential remains constant at 150 mV, inside-negative, but the pH gradient decreases from 2.1 units, inside-alkaline, at pH 5.6 to zero at pH 7.5 and above. 3. The accumulation of glutamate in the soluble cell pool is pH-independent at a value equivalent to 100 mV. 4. The results of experiments studying co-transport of protons are consistent with a proton/glutamate stoichiometry of 2 and electrogenic transport across the pH range examined. 5. The amount of glutamate uptake is the result of a kinetic steady state between influx and efflux pathways. 6. Evidence is presented for the regulation of this kinetic steady state by the response of the initial rate of uptake to changes in the protonmotive force.
Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms
Authors: Guzelian Philip S. , Swisher Robert W.
Published: August 2015

Degradation of intrinsic hepatic [14C]haem was analysed as 14CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16h after pulse-labelling with 5-amino[5-14C]laevulinic acid, a precursor that labels bridge carbons of haem in non-erythroid tissues. NADPH-catalysed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [14C]haem (largely cytochrome P-450 haem), but little 14CO formation. No additional 14CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [14C]haem. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl2 or phenyl-t-butylnitrone(a spin-trapping agent for free radicals), it was concluded that carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide, may promote loss of cytochrome P-450 haem through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic haem, catabolism of [14C]methaemalbumin by microsomal haem oxygenase in vitro produced equimolar quantities of 14CO and bilirubin, although these catabolites reflected only 18% of the degraded [14C]haem. This value was increased to 100% by addition of MnCl2, which suggests that lipid peroxidation may be involved in degradation of exogenous haem to products other than CO. Phenyl-t-butylnitrone completely blocked haem oxygenase activity, which suggests that hydroxy free radicals may represent a species of active oxygen used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [14C]haem was decreased and haem oxygenase activity was unchanged; however, 14CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [14C]haem and 14CO excretion, one may infer that an important fraction of hepatic [14C]haem in normal rats is degraded by endogenous pathways not involving CO. We conclude that carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 haem through mechanisms that do not yield CO as an end product, and that are insensitive to cycloheximide and independent of haem oxygenase activity.
Acetate is the preferred substrate for long-chain fatty acid synthesis in isolated spinach chloroplasts
Authors: Roughan P G , Holland R , Slack C R
Published: August 2015

1. Commercially available [2-14C]pyruvate and [2-14C]malonate were found to contain 3-6% (w/w) of [14C]acetate. 2. The contaminating [14C]acetate was efficiently utilized for fatty acid synthesis by isolated chloroplasts, whereas the parent materials were poorer substrates. 3. Maximum incorporation rates of the different substrates examined were (ng-atoms of C/h per mg of chlorophyll): [1-14C]acetate, 2676; [2-14C]pyruvate, 810; H14CO3-, 355; [2-14C]malonate, 19. 4. Products of CO2 fixation were probably not a significant carbon source for fatty acid synthesis in the presence of exogenous acetate.
Identification of albumin as the plasma carrier for zinc absorption by perfused rat intestine
Authors: Smith K T , Failla M L , Cousins R J
Published: August 2015

The isolated vascularly perfused rat intestine exhibits an obligatory need for a protein carrier in order to absorb zinc. Therefore this system is ideal for use as a model to identify the plasma carrier during zinc absorption. Affinity chromatography on Blue Sepharose CL-6B was employed to separate the major serum zinc-binding proteins in the portal effluent of the perfused intestine. It was found that 94% of newly absorbed 65Zn was transported in the portal serum-containing perfusate as an albumin-65Zn complex. The identity of albumin as the plasma carrier was confirmed by polyacrylamide-slab-gel electrophoresis. This evidence suggests that albumin is the plasma protein that is involved in removal of zinc from intestinal-mucosal cells and subsequent transport of the metal in portal blood to the liver.
Effect of methylamalonic acid on gluconeogenesis in isolated rat and guinea-pig hepatocytes
Authors: Arinze I J , Waters D , Donaldson M K
Published: August 2015

Gluconeogenesis from pyruvate, alanine, lactate and propionate was inhibited by methylmalonate in both rat and guinea-pig hepatocytes. The effect was dose-dependent. Gluconeogenesis from glycerol and xylitol was not affected.
Investigation of human erythrocyte superoxide dismutase by 1H nuclear-magnetic-resonance spectroscopy
Authors: Hill H A O , Lee W K , Bannister J V , Bannister W H
Published: August 2015

The 170MHZ 1 H n.m.r. spectra of the Cu(II)/Zn(II), Cu(I)/Zn(II) and apo- forms of human erythrocyte superoxide dismutase (EC 1.15.1.1) are reported. Resonances are assigned to the C-2 and C-4 protons of histidine residues in the active site, and it is suggested that five or six histidine residues serve as ligands to the metal ions in each subunit of the enzyme. The remaining assigned resonances are associated with histidine-41, N-terminal N-acetyl group, histidine- 108 and cysteine- 109. A comparison of the n.m.r. spectra of human and bovine superoxide dismutases suggests significant structural homology.
Electrophoretically distinct forms of uridine kinase in the rat. Tissue distribution and age-dependence
Authors: Absil J , Tuilie M , Roux J M
Published: August 2015

By the use of polyacrylamide-gel electrophoresis, uridine kinase from foetal rat liver was separated into four types designated I, II, III and IV in decreasing order of mobility towards the anode. The most anodic (type I) was found only in rapidly growing tissues, such as foetal liver and brain, postnatal spleen and tumour cells. In adult tissues, types II, III and IV were found in the kidney, and types III and IV in the spleen and the liver, whereas type IV was the sole form of uridine kinase present in the brain.
DNA-binding specificity of a chromatin non-histone protein fraction of HeLa cells
Authors: Dunn J H J , Lyall R M , Briggs R C , Campbell A M , Hnilica L S
Published: August 2015

The DNA-binding site of a previously characterized non-histone chromosomal protein antigen(s) from HeLa cells was investigated for its species specificity. Treatment with large amounts of micrococcal nuclease abolishes immunoactivity, which can then be recovered by the subsequent addition of human or HeLa DNA to reconstitute the immune complex. Neither rat nor calif DNA exhibits this property, but DNA from monkey cells gives considerable activity. The antigen is not, however, detectable in monkey chromatin.
A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas
Authors: Giles I G , Poat P C
Published: August 2015

1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired.
Partial deglycosylation of blood-group-specific glycoproteins
Authors: Donald A S R
Published: August 2015

The time course for the partial deglycosylation of blood-group-specific glycoproteins from human ovarian-cyst fluids with 0.25 M-H2SO4/acetic acid and 6 M-HCl in methanol was studied. Either reagent readily removed about 80% of the carbohydrate from the glycoproteins to leave non-diffusible glycopeptides that contain N-acetylgalactosamine as the predominant sugar. Some changes in amino acid distribution were observed during the deglycosylation, which were attributed to an accelerated break-up of the nonglycosylated regions of the parent glycoprotein. The N-acetylgalactosaminyl-peptides isolated were judged to be polydisperse by gel filtration, and ion-exchange chromatography divided the glycopeptide population into several fractions with differing amino acid compositions. A Lumbricus terrestris hexosaminidase preparation was successful in removing almost all the remaining sugar from the glycopeptides, but caused further rupture of the peptide. When a per O-acetylated glycoprotein was treated with the H2SO4/acetic acid reagent the glycopeptide contained, in addition to N-acetylgalactosamine, about 50% of the sialic acid present in the parent glycoprotein, indicating that most of this sugar is located near the peptide end of the carbohydrate chains.
Calcium ion-regulated thin filaments from vascular smooth muscle
Authors: Marston S B , Trevett R M , Walters M
Published: August 2015

Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 × 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g.
A biochemical comparison of normal human liver and hepatocellular carcinoma ferritins
Authors: Bullock S , Bomford A , Williams R
Published: August 2015

1. The iron contents, gel migration rates and isoelectric-focusing patterns of normal liver and hepatocellular carcinoma ferritins from the same patients were compared. 2. Sucrose-density-gradient centrifugation showed that the number of iron atoms per ferritin molecule was decreased to approximately half in carcinoma tissue when compared with normal liver. 3. On electrophoresis, hepatocellular carcinoma ferritin migrates faster and is therefore more negatively charged than normal liver ferritin, thus refuting the general view that the more negatively charged a ferritin molecule the greater its iron content. 4. Comparison of tumour and normal liver ferritin subunit compositions on acid/urea/polyacrylamide gels showed hepatocellular carcinoma ferritin to contain an additional, more negatively charged, subunit to normal liver ferritin. 5. Isoelectric focusing showed that hepatocellular carcinoma tissue contains isoferritins with isoelectric points intermediate between the ranges of normal liver and normal heart isoferritins.
The development of a radioimmunoassay for Tamm–Horsfall glycoprotein in serum
Authors: Dawnay Anne , McLean Charles , Cattell William R.
Published: August 2015

The Tamm–Horsfall glycoprotein prepared by salt precipitation from urine was found to comprise a heterogeneous collection of aggregates. These could be disaggregated with 8m-urea, following which chromatography on a column of Bio-Gel A.15m yielded a homogeneous glycoprotein of mol.wt. 73000 together with several unidentified impurities. Gel filtration of normal plasma showed the glycoprotein to exist predominantly in a form that is eluted identically with the purified preparation. In one case, material of higher molecular weight was also detected. The purified glycoprotein was used to develop a rapid specific radioimmunoassay for its measurement in human serum or plasma by the use of the Tamm–Horsfall glycoprotein, labelled with 125I by the chloramine-t method as the tracer, an antiserum raised in rabbits, and separation of the bound and free fractions by a second antibody covalently linked to magnetizable particles. Parallelism was demonstrated between the standard preparation and samples. Recovery of added standard to serum varied between 99 and 109%. Total assay time was less than 4h with an intra-assay and inter-assay coefficient of variation of less than 10%. There were no significant differences in the ranges covered with regard to either age or sex, and no circadian rhythm was observed in normal subjects. A physiological range of 70–540ng/ml was established based on serum samples from 95 subjects with normal renal function, as defined by their serum creatinine and urea concentrations. No Tamm–Horsfall glycoprotein was detected in the serum of six anephric patients.
Kinetics of suicide substrates
Authors: Waley S G
Published: August 2015

When suicide substrates inactivate enzymes during catalysis, formation of product and inactivation of enzyme proceed concurrently. The steady-state hypothesis is applicable when catalytic quantities of enzyme are used. Equations for the rate of inactivation have been derived and integrated to obtain equations describing progress curves.
Maleylacetate reductase from Trichosporon cutaneum
Authors: Gaal A B , Neujahr H Y
Published: August 2015

The enzyme catalysing the reduction of maleylacetate to 3-oxoadipate was purified 150-fold from Trichosporon cutaneum, induced for aromatic metabolisms by growth with resorcinol as a major carbon source. The enzyme separated upon electrofocusing into three species with PI values 4.6, 5.1 and 5.6. They had similar catalytic properties and the same molecular weight.
Polyribosomes in isolated liver cells. Preparative procedures, effects of incubation and correlation with protein synthesis
Authors: Dickson A J , Pogson C I
Published: August 2015

Methods have been derived which permit the isolation of undergraded polyribosomes from isolated rat liver cells. Under the conditions used the polyribosome profile of hepatocytes immediately after isolation was essentially identical with that from intact liver. However, during incubation of cells in complex physiological media there was a progressive dissociation of polyribosomes. The addition of a variety of factors that produce reaggregation of polyribosomes in rat liver in vivo did not prevent dissociation during cell incubations. Although large polyribosomes were lost most rapidly, the albumin-synthesizing capacity of isolated cells was not selectively lost when compared with total protein synthesis. The significance of these results for the use of isolated hepatocytes in the study of liver protein synthesis is discussed.
Sodium ion/L-lactate co-transport in rabbit small-intestinal brush-border-membrane vesicles
Authors: Hildmann B , Storelli C , Haase W , Barac-Nieto M , Murer H
Published: August 2015

Uptake of L-lactate into rabbit jejunal brush-border-membrane vesicles prepared by a Ca2+-precipitation procedure was studied by a rapid filtration technique with L-[14C]-lactate as tracer. Transport of L-lactate into an intravesicular (osmotically reactive) space could be established. An inwardly directed NaCl gradient (outside 21 mM/inside 0mM) stimulated the uptake of L-lactate at 15 s 2-4-fold compared with that observed with an equal KCl gradient. A transient accumulation of L-lactate inside the vesicles (overshoot) was observed in the presence of an NaCl gradient. Gradients of LiCl, RbCl, CsCl or choline chloride were not able to replace NaCl in the stimulation of L-lactate uptake. L-Lactate uptake was saturable only in the presence of Na+. D-Lactate, DL-thiolactate (2-DL-mercaptopropionate), pyruvate and propionate inhibited the Na+-stimulated L-lactate uptake; D-lactate, thiolactate and pyruvate provoked trans-stimulation of L-lactate uptake. Artificially imposed diffusion potentials (inside negative) did not exert any effect on the Na+-dependent L-lactate uptake. The results are consistent with the existence of an electroneutral Na+/L-lactate co-transport system in the brush border of rabbit small intestine.
The identification of the folate conjugates found in rat liver 48 h after the administration of radioactively labelled folate tracers
Authors: Connor M J , Blair J A
Published: August 2015

About 70% of the radioactivity retained in the livers of rats dosed 48 h earlier with radioactively labelled folate was incorporated into two folate conjugates. The major derivative was purified and isolated by Sephadex G-15, DEAE-cellulose and DEAE-Sephadex ion-exchange column chromatography and paper chromatography. It was identified as 10-formylpteroylpentaglutamate by a combination of spectral, microbiological, chemical and chromatographic techniques. The minor conjugate, though less well characterized, exhibited similar properties and was assigned the structure 10-formylpteroyltetraglutamate. 10-Formylpteroylpentaglutamate (2.0nmol/g) and 10-formylpteroyltetraglutamate (0.25nmol/g) comprised about 20% of the total endogenous hepatic folate as determined by microbiological assay (Lactobacillus casei after conjugase treatment.
Mechanisms of loss of latency of lysosomal enzymes. Effects of incubation on the properties of lysosomal membranes
Authors: Ruth R C , Weglicki W B
Published: August 2015

1. The effects of sucrose and KCl on the loss of latency of lysosomal enzymes caused by incubation at 37 degrees C, pH 7.4, were examined by using Triton-filled lysosomes from rat liver and two fractions from livers of rats not injected with Triton. 2. After incubation, the percentage free activity of lysosomal enzymes was measured before and after cooling to 0 degrees C in order to determine the amount of latency lost at 37 degrees C without cooling and the additional amount lost on cooling the incubated lysosomes to 0 degrees C. 3. The latency that is lost without cooling is first decreased and then increased by increasing the osmotic strength of the incubation medium with KCl, or with sucrose in the presence of KCl. However, if the osmotic strength is increased with sucrose alone, loss of latency is decreased up to 0.25M-sucrose, but is increased only slightly at higher sucrose concentrations. Apparently the lysosome is permeated by hyperosmolar KCl but not by sucrose during incubation. 4. If the osmotic strength of the assay medium is increased with KCl, the loss of latency caused by incubation for 60 min in hyperosmolar KCl is repressed. Thus it appears that a KCl-permeated lysosome can be obtained which is relatively stable until exposure to lower osmolarities. 5. The loss of latency caused by cooling incubated lysosomes to 0 degrees C is largely eliminated if the osmotic strength of the medium in which the lysosomes are cooled is raised sufficiently with either sucrose or KCl. 6. Osmotic-fragility curves were obtained after incubation for 1 and 60 min at iso-osmoticity (0.2M-KCl or 0.25 M-sucrose). Although little loss of latency occurs at iso-osmoticity, lysosomes incubated for 60 min display greatly increased fragility on exposure to hypo-osmolar KCl, hypo-osmolar sucrose or hyperosmolar KCl. 7. It is suggested that permeability to KCl at 37 degrees C and the increase in fragility on exposure to hypo-osmolar conditions are both consequences of injury, probably from enzymic action, sustained by the lysosomal membrane during incubation at 37 degrees C.
The inhibition of macrophage protein turnover by a selective inhibitor of thiol proteinases
Authors: Shaw E , Dean R T
Published: August 2015

1. A new inhibitor of thiol proteinases, benzyloxycarbonylphenylalanylalanine diazomethyl ketone (benzyloxycarbonylphenylalanylalanyldiazomethane, Z-Phe-Ala-CHN2) was added to cultured mouse peritoneal macrophages prelabelled with [14C]leucine. The degradation of protein was studied under conditions of basal proteolysis in the presence of 10% pig serum. After a lag of about 6 h a time- and dose-dependent inhibition of protein degradation was observed, up to a maximum of about 40%. 2. The inhibitor entered the cells with kinetics consistent with entry by pinocytosis, giving access to the lysosomal system. 3. Intracellular cathepsin B was almost completely inactivated after 90 min of exposure of the culture to 0.1 mm-inhibitor. 4. The inhibition of proteolysis and of cathepsin B was reversed virtually completely within 24 h, when the inhibitor was removed from the medium. Since the inhibitor forms a covalent bond with the enzyme, the recovery of cathepsin B activity presumably reflects production of new molecules of active enzyme. 5. The inhibitory effects of pepstatin, the carboxyl proteinase inhibitor, were under some circumstances additive with those Z-Phe-Ala-CHN2, and were also largely reversible. 6. It is concluded that thiol proteinases play a major role in lysosomal proteolysis in cultured macrophages.
Inhibition of protein synthesis in vitro by proteins from the seeds of Momordica charantia (bitter pear melon)
Authors: Barbieri L , Zamboni M , Lorenzoni E , Montanaro L , Sperti S , Stirpe F
Published: August 2015

1. A haemagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated Sepharose 6B. It has mol.wt. 115 000 and consists of four subunits, of mol.wts. 30 500, 29 000, 28 500 and 27 000. 2. The lectin inhibits protein synthesis by a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of approx. 5 micrograms/ml. Protein synthesis by Yoshida ascites cells is partially inhibited by the lectin at a concentration of 100 micrograms/ml. 3. From the same seeds another protein was purified which has mol.wt. 23 000 and is a very potent inhibitor of protein synthesis in the lysate system, with an ID50 of 1.8 ng/ml. This inhibitor has no effect on protein synthesis by Yoshida cells, and has no haemagglutinating properties. 4. Artemia salina ribosomes preincubated with the lectin or with the inhibitor lose their capacity to perform protein synthesis. The proteins seem to act catalytically, since they inactivate a molar excess of ribosomes. 5. The lectin and the inhibitor are somewhat toxic to mice, the LD50 being 316 and 340 micrograms/100 g body wt. respectively.
Effect of temperature on the reversal of the calcium ion pump in sarcoplasmic reticulum
Authors: Vale M G P , Carvalho A P
Published: August 2015

Sarcoplasmic-reticulum vesicles were actively loaded with Ca2+ in the presence of phosphate, and the ADP-induced Ca2+ efflux and ATP synthesis were measured as a function of temperature. Arrhenius plots show break points for both processes at about 18 and 37 degrees C. Between 18 and 37 degrees C, Ca2+ efflux and ATP synthesis occur with an activation energy of 67.2-71.4 kJ/mol, whereas it is about 189-210 kJ/mol for temperatures below 18 degrees C. Above 37 degrees C, the rates of ADP-induced Ca2+ release and of ATP synthesis sharply decline until the temperature reaches about 42 degrees C. Above this temperature, the Ca2+ efflux increases again even in absence of ADP, although the synthesis of ATP is inhibited, which reflects leakiness of the vesicles. The results show that the transition temperatures for ADP-induced Ca2+ efflux and for ATP synthesis resemble those for active Ca2+ uptake, which indicates that the same coupling mechanism is involved during the inward and outward Ca2+ translocations across the membrane.
Regulation of tyrosine aminotransferase in foetal rat liver
Authors: Andersson S M , Räihä N C , Ohisalo J J
Published: August 2015

A specific tyrosine aminotransferase, separate from the aspartate aminotransferases, is present in low concentration in foetal rat liver at the 21st day of gestation. Intraperitoneal injections of tyrosine methyl ester into the foetuses in utero increase the activity 2-fold, whereas glucose injections decrease it. Tyrosine, dexamethasone and dibutyryl cyclic AMP induce the enzyme activity in organ culture to the same extent as in adult rat liver in vivo.
Modulation of cyclic AMP-dependent protein kinase by vasopressin and calcitonin in cultured porcine renal LLC-PK1 cells
Authors: Ausiello Dennis A. , Hall Dale H. , Dayer Jean-Michel
Published: August 2015

We have previously demonstrated that a cultured porcine kidney cell, LLC-PK1, maintains the characteristics of a polar renal epithelial cell in culture, and responds to salmon calcitonin and [arginine]vasopressin by increasing cyclic AMP content. To demonstrate the usefulness of this cell line as a model for the study of the biochemical events distal to cyclic AMP production, the activation of cyclic AMP-dependent protein kinase was examined. Intact cells in monolayer demonstrated progressive increases in cyclic AMP content and activation of protein kinase in response to [arginine]vasopressin (2–200nm) and salmon calcitonin (0.03–30nm) with both hormones fully activating the enzyme at a cell cyclic AMP content of 35pmol/mg of protein. Of the total cyclic AMP-dependent protein kinase activity, 80% was found in the 27000g supernatant fraction of sonicated cell material, and this soluble protein kinase could be fully activated by hormone. Conversely, the 27000g pellet contained a significant proportion of cyclic AMP-independent protein kinase and only 20% of total cell cyclic AMP-dependent protein kinase; the latter showed little response to hormone. On the basis of DEAE-cellulose chromatography, type II protein kinase was the predominant isoenzyme in both soluble and particulate fractions of the LLC-PK1 cells and the soluble fractions of rat and guinea-pig renal medulla. Thus, the LLC-PK1 cell line can serve as a model for hormonal modulation of protein kinase and as a potential source for defining the endogenous substrates for these enzymes.
Development of NADPH-producing pathways in rat heart
Authors: Andrés A , Satrústegui J , Machado A
Published: August 2015

The behaviours of the principal NADPH-producing enzymes (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, cytoplasmic and mitochondrial ‘malic’ enzyme and NAPD+-dependent isocitrate dehydrogenase) were studied during the development of rat heart and compared with those in brain and liver. 1. The enzymes belonging to the pentose phosphate pathway exhibit lower activities in heart than in other tissues throughout development. 2. The pattern of induction of heart cytoplasmic and mitochondrial ‘malic’ enzymes does not parallel that found in liver. Heart mitochondrial enzyme is slowly induced from birth onwards. 3. NADP+-dependent isocitrate dehydrogenase has similar activities in all tissues in 18-day foetuses. 4. Heart mitochondrial NADP+-dependent isocitrate dehydrogenase is greatly induced in the adult, where it attains a 10-fold higher activity than in liver. 5. The physiological functions of mitochondrial ‘malic’ enzyme and NADP+-dependent isocitrate dehydrogenase are discussed.
Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes
Authors: Newby A C
Published: August 2015

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable (less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.
Lipogenic enzymes in rat maternal adipose tissue in the perinatal period
Authors: Sinnett-Smith P A , Vernon R G , Mayer R J
Published: August 2015

1. The specific activities of fatty acid synthetase, acetyl-CoA carboxylase and pyruvate dehydrogenase were measured in rat adipose-tissue extracts in pregnancy and lactation. Fatty acid synthetase specific activity correlates very closely with the rate of fatty acid synthesis, the enzyme specific activity decreasing after mid-pregnancy in a manner very similar to the rate of fatty acid synthesis. Acetyl-CoA carboxylase specific activity also decreases dramatically after mid-pregnancy. Initial pyruvate dehydrogenase specific activity shows a decrease between 2 days pre partum and 2 days post partum, but total enzyme activity shows no significant change in the same period. 2. Immunotitrations of fatty acid synthetase and pyruvate dehydrogenase activities were carried out; the titrations showed that the change in the fatty acid synthetase activity is due to a change in the enzyme amount; the amount of pyruvate dyhydrogenase does not change. Therefore the decrease in fatty acid biosynthesis in subcutaneous and parametrial adipose tissue in late pregnancy and early lactation is associated with a decrease in the amount of at least one of the enzymes involved in fatty acid biosynthesis. The correlation of these events with known hormonal changes is discussed.
Effect of tunicamycin and cycloheximide on the secretion of acid hydrolases from I-cell cultured fibroblasts
Authors: Miller A L , Kress B C , Lewis L , Stein R , Kinnon C
Published: August 2015

I-cell cultures fibroblasts secrete excessive amounts of N-acetyl-beta-D-hexosaminidase and alpha-L-fucosidase into the culture media as compared with normal fibroblasts. Addition of tunicamycin or cyd [14C]leucine (40-50%) into trichloroacetic acid-precipitable material decreased the secretion of these I-cell hydrolases to normal values within 24 h, but had no effect on the secretion of acid hydrolases from normal fibroblasts. These results indicate that I-cell cultured fibroblasts secrete at least two types of acid hydrolases: one is tunicamycin- and cycloheximide-sensitive and constitutes the greater proportion of the secreted hydrolases, and a smaller proportion is insensitive to tunicamycin and cycloheximide, similar t9 the acid hydrolases secreted by normal cultured fibroblasts.
Substrate kinetics of the alternative oxidase of Neurospora crassa
Authors: Vanderleyden J , Peeters C , Verachtert H , Bertrand H
Published: August 2015

The kinetics of the succinate oxidation by cyanide-sensitive and cyanide-insensitive submitochondrial particles of Neurospora crassa cells suggest that both respiratory pathways use the same complex II. This is confirmed by comparing the kinetics of the reductase activities of the isolated succinate-ubiquinone oxidoreductase (complex II) of cyanide-sensitive and cyanide-insensitive cells respectively. No alternative-oxidase activity was found to be associated with the isolated complex II of cyanide-insensitive cells.
Low-level chemiluminescence of hydroperoxide-supplemented cytochrome c
Authors: Cadenas Enrique , Boveris Alberto , Chance Britton
Published: August 2015

Ferricytochrome c showed low-level chemiluminescence, with a light-emission measured of about 1×103–3×103 counts/s, when supplemented with organic hydroperoxides. Tertiary hydroperoxides (cumene hydroperoxide and t-butyl hydroperoxide) showed a saturation behaviour at about 5mm-hydroperoxide, whereas primary hydroperoxides showed a quadratic dependence on the hydroperoxide concentration. Chemiluminescence depended linearly on cytochrome c concentration, and optimal light-emission was observed at [t-butyl hydroperoxide]/[ferricytochrome c] ratios of 160–500. Hydroperoxide-supplemented ferricytochrome c consumed O2 at a rate of 1.0μmol/min per μmol of cytochrome c; the rate of O2 uptake was linearly related to the concentration of cytochrome c. The Soret absorption band of ferricytochrome c decreased about 64% after incubation with t-butyl hydroperoxide, whereas the 530nm band was almost totally abolished. Light-emission was (a) inhibited competitively by cyanide. (b) inhibited by singlet-oxygen quenchers (e.g. β-carotene), scavengers (e.g. dimethylfuran) and traps (e.g. histidine and tryptophan) and (c) increased by singlet-oxygen-chemiluminescence enhancer 1,4-diazabicyclo[2.2.2]-octane. Superoxide dismutase had no effect on the present system. The participation of free radicals is suggested by the effect of the radical trap 2,5-di-t-butylquinol. Singlet-oxygen dimol emission seems to be mainly responsible for the observed light-emission; a mechanism that can account for the major part of the present experimental observations is proposed.
The evaluation of initial rates in the presence of background noise
Authors: Jauker F
Published: August 2015

A three-parameter model was used to test a curvilinear regression for its gamma-intercept. It was applied in the kinetic analysis of a model reaction and of real experiments. The model reaction consisted of a dialysis experiment with increasing amounts of background added at t0. In the presence of this background, initial rates of solute exchange were determined precisely enough to be in agreement with Fick's law of diffusion. The biological experiments were concerned with the saturation kinetics of the acid-soluble radioactivity of cells exposed to 3H-labelled amino acids. Two models of regression were applied, facilitated and physical diffusion, both consistent with the assumption of a substantial amount of background. This background was assumed to be caused by adsorption of 3H-labelled amino acids to cell-surface proteins. Without further consideration it caused a systematic overestimation of the initial rates.
Characteristics of a copper-dependent cross-linking reaction between two forms of cytochrome P-450 in rabbit-liver microsomal membranes
Authors: McIntosh P R , Freedman R B
Published: August 2015

1. In liver microsomal membranes from adult rabbits treated with beta-naphthoflavone, reaction with Cu2+ salts plus 1,10-phenanthroline leads to the cross-linking of the two specifically beta-naphthoflavone-inducible cytochrome P-450 species, form 4 and form 6, to form homo- and hetero-dimer species. 2. The cross-linking is not reversed by treatment with 2-mercaptoethanol, so that it can be observed conveniently and specifically on conventional reducing sodium dodecyl sulphate/polyacrylamide gels. 3. The reaction occurs rapidly, and significant cross-linking is observed after 30s at all temperatures from −10 to 40 degrees C. 4. The cross-linking can be brought about by Cu2+ alone at concentrations greater than 0.5 mM, but not by 1,10-phenanthroline alone; at low Cu2+ concentrations, 1,10-phenanthroline enhances the cross-linking reaction, but high concentrations of 1,10-phenanthroline are inhibitory; the optimal molar ratio of Cu2+ to 1,10-phenanthroline is 4:1.5. The effect of Cu2+ is not mimicked by Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Zn2+ or Ag+; Cu+ is probably also ineffective. 6. The cross-linking reaction is inhibited by the prior addition of high concentrations of EDTA or thiol compounds, by sodium dodecyl sulphate at greater than or equal to 0.1% and by sodium deoxycholate and non-ionic detergents at greater than or equal to 1%; the reaction cannot be reversed by incubation with EDTA or with thiol compounds after reaction with cupric phenanthroline; the cross-linking reaction is not inhibited by prior treatment of microsomal membranes with N-ethylmaleimide. 7. The chemical nature of the cross-linking reaction is unknown, but it is most unlikely that it involves the formation of intermolecular disulphide bonds. 8. The great specificity of the reaction makes it a promising tool for the study of molecular interactions between cytochrome P-450 species in intact microsomal membranes.
A mass-spectrometric method for the estimation of the ratio of γ-carboxyglutamic acid to glutamic acid at specific sites in proteins. Application to the N-terminal region of bovine prothrombin
Authors: Rose K , Priddle J D , Offord R E , Esnouf M P
Published: August 2015

When a polypeptide containing gamma-carboxyglutamic acid is decarboxylated in 2H2O, residue of (gamma gamma-2H2)glutamic acid are formed. Subsequent proteolytic digestion produces peptides which contain at each site 2H2-substituted and unsubstituted glutamic acid in the same ratio as existed for gramma-carboxy-substitution. The peptides may be identified and this ratio determined by combined gas chromatography–mass spectrometry. We also discuss decarboxylation in 3H2O followed by amino-acid analysis and Edman degradation.
Retention of the 4-pro-R hydrogen atom of mevalonate at C-2,2′ of bacterioruberin in Halobacterium halobium
Authors: Swift I E , Milborrow B V
Published: August 2015

Intact cells of Halobacterium halobium fed with (3R,4R)-[2-14C,4-3H1]mevalonic acid were found to incorporate label into acyclic C40 and C50 carotenoids, of which bacterioruberin was the most abundant. The 14C/3H ratios of the isolated carotenoids demonstrated that the 4-pro-R hydrogen atom of mevalonic acid was retained at the C-2 and C-2′ positions of bacterioruberin. Diphenylamine was found to inhibit the production of bacterioruberin.
Isolation of the smallest component of silk protein
Authors: Tokutake S
Published: August 2015

Silk proteins were solubilized from cocoons with ethylenediamine/cupric hydroxide solution. A series of polymers of the smallest component, detected by polyacrylamide-gel electrophoresis, could be converted into the smallest component by reduction and aminoethylation. Fibroin and sericin fractions were separated by precipitation of sericin at pH 5.5. On gel electrophoresis, sericin showed distinct bands but fibroin did not. The components of fibroin and sericin were fractionated by gel filtration on Sepharose 6B. The smallest component in the sericin fraction was purified by rechromatography and showed a single band on gel electrophoresis. Its mol. wt. was 24 000, and its amino acid composition was determined.
A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin
Authors: Mauldin Douglas , Roufogalis Basil D.
Published: August 2015

Treatment of extensively washed erythrocyte membranes with 0.1mm-EDTA decreased their Mg2+-dependent, Ca2+-stimulated ATPase [(Mg2++Ca2+)-ATPase] activity. An activator released by this treatment restored the (Mg2++Ca2+)-ATPase to its original value in a Ca2+-dependent manner. This activator was different from calmodulin, as determined by a number of criteria. It was retained on an Amicon XM-100 ultrafiltration membrane (molecular-weight cut-off 100000); it appeared in the void volume of Sephadex G-100 and G-75 columns; it was not retained on a DEAE-cellulose ion-exchange column at ionic strengths similar to those used to retain calmodulin; and it maximally activated (Mg2++Ca2+)-ATPase activity less than calmodulin and at a higher Ca2+ concentration. Like calmodulin, the activator is heat-stable. The activator fraction isolated on a 2.5–15% sucrose gradient in 0.16m-KCl showed a single band of mol.wt. 63000 and no calmodulin on 10%-polyacrylamide/sodium dodecyl sulphate gels. A trace amount of calmodulin was detected in the activator fraction by radioimmunoassay (approx. 10pg/ml of ‘ghosts’), but this amount was insufficient to account for the (Mg2++Ca2+)-ATPase activation. Furthermore, calmodulin-binding protein failed to inhibit (Mg2++Ca2+)-ATPase activity by more than 10–20% in the membrane preparations from which the activator was extracted. It was concluded that erythrocyte membranes contain a (Mg2++Ca2+)-ATPase activator that may attenuate the activation of the Ca2+-transport ATPase by calmodulin.
Chemical identity of tryptensin with angiotensin
Authors: Arakawa K , Yuki M , Ikeda M
Published: August 2015

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.
The action of chloride peroxidase on 4-chloroaniline. N-oxidation and ring halogenation
Authors: Corbett M D , Chipko B R , Batchelor A O
Published: August 2015

Chloride peroxidase catalyses both the ring halogenation and N-oxidation reactions of 4-chloroaniline by H2O2 and either KCl or KBr. In the absence of any halide salt only the N-oxidation reaction was observed, with the resulting conversion of 4-chloroaniline into 4-chloronitrosobenzene. The N-oxidation reaction proceeded even more rapidly in the presence of Cl- or Br-, in spite of the fact that ring halogenation was also a rapid reaction. The enhancement of N-oxidation was highly dependent on the pH of the media and displayed an optimum in the region of pH 3.5-4.0. No rate enhancement was observed above pH 5.5. KF partially inhibited the rate of N-oxidation in a pH-dependent manner. On the basis of calculated catalytic-centre activity the N-oxidation reaction was the major reaction at pH 3.5 or higher, whereas the ring-halogenation reaction became the major reaction below pH 3.5. In the presence of high concentrations of 4-chloroaniline relative to H2O2 the reaction intermediate, 4-chlorophenylhydroxylamine, was detected for the first time in a chloride peroxidase-catalysed reaction with this arylamine substrate. These findings were interpreted on the basis of current knowledge concerning the mechanism of action of chloride peroxidase.
Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria
Authors: Cadenas Enrique , Boveris Alberto
Published: August 2015

Rat and pigeon heart mitochondria supplemented with antimycin produce 0.3–1.0nmol of H2O2/min per mg of protein. These rates are stimulated up to 13-fold by addition of protophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chloromethoxyphenylhydrazone and pentachlorophenol). Ionophores, such as valinomycin and gramicidin, and Ca2+ also markedly stimulated H2O2 production by rat heart mitochondria. The enhancement of H2O2 generation in antimycin-supplemented mitochondria and the increased O2 uptake of the State 4-to-State 3 transition showed similar protophore, ionophore and Ca2+ concentration dependencies. Thenoyltrifluoroacetone and N-bromosuccinimide, which inhibit succinate–ubiquinone reductase activity, also decreased mitochondrial H2O2 production. Addition of cyanide to antimycin-supplemented beef heart submitochondrial particles inhibited the generation of O2−, the precursor of mitochondrial H2O2. This effect was parallel to the increase in cytochrome c reduction and it is interpreted as indicating the necessity of cytochrome c13+ to oxidize ubiquinol to ubisemiquinone, whose autoxidation yields O2−. The effect of protophores, ionophores and Ca2+ is analysed in relation to the propositions of a cyclic mechanism for the interaction of ubiquinone with succinate dehydrogenase and cytochromes b and c1 [Wikstrom & Berden (1972) Biochim. Biophys. Acta283, 403–420; Mitchell (1976) J. Theor. Biol.62, 337–367]. A collapse in membrane potential, increasing the rate of ubisemiquinone formation and O2− production, is proposed as the molecular mechanism for the enhancement of H2O2 formation rates observed on addition of protophores, ionophores and Ca2+.