bluuebunny/crossref_metadata_2025 huggingface dataset:
Gibberellic acid-stimulated α-amylase secretion and phospholipid metabolism in wheat aleurone tissue
Authors: Mirbahar R B , Laidman D L
Published: August 2015

1. Turnovers of [14C]glycerol-labelled phospholipids in wheat aleurone tissue have been measured by using a pulse-decay technique. The most metabolically active phospholipids were phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol. 2. Gibberellic acid action on the tissue led to breakdown of phosphatidylcholine and stimulated turnover of the other phosphatides concomitant with the secretion of alpha-amylase from the tissue. After pulse-labelling of the aleurone tissue with [14C]glycerol, radioactivity was lost from the phospholipids and appeared quantitatively in triacylglycerols, suggesting a stoichiometric metabolism of the former into the latter. Although 1,2-diacylglycerol is an expected intermediate in such a conversion, the patterns of radioactivity in diacylglycerols gave no indication of this. 3. Several aspects of the response of aleurone tissue to gibberellic acid resemble the responses of exocytotic animal tissues to external agonists. In particular, our results and previous reports in the literature suggest that endomembrane flow, exocytosis, phosphatidylinositol turnover and a requirement of Ca2+ for enzyme secretion are common to both plant and animal systems. Although considerable differences also exist between the two, the similarities are sufficient to warrant further consideration that plants and animals might have conserved a similar hormone response-secretion mechanism.
Beryllium toxicity. The selective inhibition of casein kinase 1
Authors: Cummings B , Kaser M R , Wiggins G , Ord M G , Stocken L A
Published: August 2015

1. Cyclic AMP-independent casein kinase 1 in liver cytoplasm and nuclei was inhibited by Be2+ in vitro (Ki 2.5 microM and 29 microM respectively). Casein kinase 2 (phosvitin kinase) and cyclic AMP-dependent protein kinase were unaffected. 2. The inhibition of casein kinase 1 by Be2+ was competitive with respect to the protein substrate; at non-saturating concentrations of casein, inhibition was non-competitive with respect to ATP. 3. In rats given LD50 doses of Be2+ 24 h before death, the activities of cytoplasmic and nuclear casein kinase 1 in livers from partially hepatectomized animals were diminished approx. 50%; with intact rats, nuclear casein kinase 1 was inhibited at concentrations of casein less than the Km.
N-(3-aminopropyl)pyrrolidin-2-one, a product of spermidine catabolism in vivo
Authors: Seiler N , Knödgen B , Haegele K
Published: August 2015

A high-pressure-liquid-chromatographic method suitable for the separation and sensitive detection of putreanine and isoputreanine is described. This method allowed us to study the formation of the metabolites of the oxidative deamination of spermidine and N1-acetylspermidine. Administration of spermidine trishydrochloride to mice causes a time-dependent accumulation of putreanine and N-(3-aminopropyl)pyrrolidin-2-one in various organs. The latter compound yields isoputreanine by hydrolysis. It can be assumed that the analogous lactam. N-(3-acetamidopropyl)pyrrolidin-2-one is formed from N1-acetylspermidine, since hydrolysis of tissue extracts of N1-acetylspermidine-treated mice produced isoputreanine. No putreanine is formed under these conditions. Pretreatment of the animals with 25 mg of aminoguanidine sulphate/kg body wt. completely inhibits the formation of putreanine and of the respective isoputreanine precursor from spermidine and N1-acetylspermidine. This suggests a role for a diamine oxidase-like enzyme in the oxidative deamination of spermidine and N1-acetylspermidine.
The surface glycoproteins of human skin fibroblasts detected after electrophoresis by the binding of peanut (Arachis hypogaea) agglutinin and Ricinus communis (castor-bean) agglutinin I.
Authors: Gordon B B , Pena S D
Published: August 2015

A new methodology was developed to study the cell-surface glycoproteins of cultured human skin fibroblasts. This was based on the binding of a variety of biotinyl-lectins to nitrocellulose electrophoretic transfers of total fibroblast lysates after separation in sodium dodecyl sulphate/polyacrylamide gels, followed by reaction with avidin-biotinyl-peroxidase complexes and detection with 3,3′-diaminobenzidine. The technique proved to be very sensitive and a large number of glycoproteins were detected by binding of concanavalin A and wheat-germ agglutinin. Binding of peanut agglutinin and to a lesser extent of Ricinus communis agglutinin I were found to be dependent on prior removal of sialic acid residues from the glycoproteins. Since by treatment of intact viable cells with neuraminidase only external sialic acid residues were removed, peanut agglutinin and Ricinus communis agglutinin I could thus be utilized for selective detection of cell-surface glycoproteins. Also, because peanut agglutinin was known to bind preferentially to oligosaccharides of the O-glycosidic type, and Ricinus communis agglutinin I to those of the N-glycosidic type, the two lectins were complementary in displaying the surface glycoproteins and in providing information about their oligosaccharide composition.
An efficient method for the isolation and separation of basolateral-membrane and luminal-membrane vesicles from rabbit kidney cortex
Authors: Sheikh M I , Kragh-Hansen U , Jørgensen K E , Røigaard-Petersen H
Published: August 2015

A procedure for isolation and separation of purified luminal-membrane and basolateral-membrane vesicles from adult and newborn rabbit renal cortex by using Ca2+/Mg2+ precipitation, differential centrifugation and a self-orienting Percoll-gradient centrifugation is described. The purity of the membrane-vesicle suspensions was examined by electron microscopy and by measuring the activity of several marker enzymes. The activity of Na+ + K+-stimulated ATPase in the fraction mainly containing adult rabbit basolateral-membrane vesicles was enriched 16-fold, and the activity of alkaline phosphatase in the fraction mainly containing luminal-membrane vesicles was increased 13-fold, compared with the homogenate. Similar results were obtained with kidneys from newborn rabbits. Uptake studies, with a rapid filtration technique and the spectrophotometric method described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J. 208, 359-368], showed that both adult and newborn rabbit luminal-membrane vesicles, in contrast with the basolateral-membrane preparations, possess an Na+-dependent electrogenic transport system for L-proline. Adult rabbit luminal-membrane vesicles take up citrate and L-malate by Na+-dependent electrogenic processes, whereas adult rabbit basolateral membrane vesicles do not exhibit electrogenic uptake of citrate. By contrast, these vesicles show Na+-dependent electrogenic uptake of L-malate.
Effect of corticosterone on myofibrillar protein turnover in diabetic rats as assessed by Nτ-methylhistidine excretion
Authors: Tomas Frank M.
Published: August 2015

The effect of corticosterone on myofibrillar protein breakdown in diabetic rats was investigated in order to assess the possible counteracting effects of the secondary rise in plasma insulin concentrations which normally accompanies such treatment. Nτ-Methylhistidine excretion, an index of myofibrillar protein breakdown, was compared before and after corticosterone treatment (4.0 mg/100 g body wt. per day) of normal control, adrenalectomized, 10-day-streptozotocin-diabetic and adrenalectomized diabetic rats. Diabetic rats received 1.5 units of insulin/100 g body wt. per day throughout the experiment and showed marked hyperglycaemia and glucosuria during corticosterone treatment, whereas non-diabetic rats had only mild hyperglycaemia but elevated insulin concentrations. Corticosterone treatment increased the average rate of myofibrillar protein breakdown by 68% and 95% respectively in non-diabetic and diabetic rats. Net loss of muscle non-collagen protein for the same 7-day period was greater in diabetic than in non-diabetic animals (4.15 versus 2.84% per day), and the calculated average synthesis rates were lowest in diabetic rats. Adrenalectomy had little effect except to decrease slightly the rate of muscle protein breakdown. These results show that the rise in plasma insulin concentrations that accompanies exogenous corticosterone administration to non-diabetic rats diminishes the catabolic effect of this glucocorticoid on muscle. Insulin appears to antagonize the effects of the glucocorticoid by attenuating the increased rates of myofibrillar protein breakdown and, to a lesser extent, by limiting the decrease in synthesis rates.
Sodium-gradient-stimulated transport of l-alanine by plasma-membrane vesicles isolated from liver parenchymal cells of fed and starved rats. Crucial role of the adrenal glucocorticoids
Authors: Quinlan Dennis C. , Todderud C. Gordon , Kelley Darshan S. , Kletzien Rolf F.
Published: August 2015

The ability of liver efficiently to take up amino acids, particularly l-alanine, during starvation was studied in a cell-free system by isolating plasma-membrane vesicles in a transport-competent state from rat liver parenchymal cells. These membrane vesicles have the capacity to accumulate l-alanine against an apparent concentration gradient when exposed to an artificial and transient transmembrane Na+ gradient (extravesicular Na+ concentration greater than inside). The rate of accumulation of l-alanine is dependent on the plasma-membrane vesicle concentration, and the steady-state concentration attained is inversely related to the osmolarity of the medium. The Na+-mediated stimulation is not exhibited if the membrane vesicles are pre-equilibrated with NaCl, if K+ or Li+ are substituted for Na+, or if SO42− replaces Cl− as the counterion. The apparent active transport of l-alanine into the membrane vesicles appears to occur by an electrogenic mechanism: (1) the use of NaSCN significantly heightens the early concentrative phase of transport when compared with the effect of NaCl; (2) an enhanced active transport is also observed when a valinomycin-induced K+ efflux occurs concomitant with Na+ and l-alanine influx. Plasma-membrane vesicles isolated from liver parenchymal cells of a 24 h-starved rat exhibit an initial l-alanine transport rate that is 3–4 times that for membrane vesicles derived from a fed animal. The increased rate of l-alanine transport by plasma-membrane vesicles from starved animals can be obliterated by adrenalectomy and restored by administration of glucocorticoid. These results establish that stimulation of the gluconeogenic pathway by starvation involves a plasma-membrane-localized change affecting l-alanine transport which is regulated in part by the glucocorticoid hormones.
Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C
Authors: Halenda Stephen P. , Rubin Ronald P.
Published: August 2015

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[3H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [32P]Pi, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [14C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca2+ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [32P]Pi and [14C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [32P]Pi labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A2 and C. Whereas mobilization of cellular Ca2+ appears sufficient to cause activation of phospholipase A2 and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca2+ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A2 and C and their specific physiological roles in pancreatic secretion remain to be elucidated.
The effect of adrenalectomy on GDP binding to brown-adipose-tissue mitochondria of obese rats
Authors: Holt Susan , York David A.
Published: August 2015

GDP binding to brown-adipose-tissue mitochondria of young obese Zucker rats (fa/fa) was significantly lower than in lean control rats, as a result of a decrease in the number of binding sites. Adrenalectomy of fa/fa rats restored GDP binding to control values. Corticosterone replacement suppressed GDP binding in adrenalectomized obese rats.
Activation of glycerophosphocholine phosphodiesterase in rat forebrain by Ca2+
Authors: Spanner Sheila , Ansell G. Brian
Published: August 2015

The highest activity of glycerophosphocholine phosphodiesterase (EC 3.1.4.2) in subcellular fractions of rat forebrain was found in the microsomal fraction though significant amounts were found in fractions containing plasma membranes. With the use of Ca2+/EGTA and Ca2+/EDTA buffers it was shown that very low concentrations of free Ca2+(EC50approx. 10−9m) could activate the enzyme.
Certain mouse strains are deficient in a kidney brush-border metallo-endopeptidase activity
Authors: Bond J S , Shannon J D , Beynon R J
Published: August 2015

Preparations of microvilli from kidneys of BALB/c mice contain an alkaline metallo-endopeptidase, meprin (metallo-endopeptidase from renal tissue). Certain genealogically related inbred mice are markedly deficient in meprin activity. The meprin-deficient strains (CBA/J and C3H/HeJ) exhibit normal levels of other brush-border enzymes: alkaline phosphatase, aminopeptidase M and another proteinase, a phosphoramidon-sensitive neutral endopeptidase. Meprin deficiency cannot be attributed to a shift in pH optimum and is unlikely to be due to the presence of endogenous inhibitors.
A simple assay to study protein-mediated lipid exchange by fluorescence polarization
Authors: Xü Y H , Gietzen K , Galla H J , Sackmann E
Published: August 2015

We investigated the protein-mediated phospholipid transfer between small vesicles by fluorescence polarization measurements with diphenylhexatriene as optical probe. Thermotropic-phase-transition curves were taken after mixing two vesicle preparations of lipids exhibiting different gel-to-liquid phase transitions. From the heights of each phase-transition step we were able to follow the lipid transfer process without separating the two vesicle preparations.
Fluorigenic method for the assay of proteinase activity with the use of 4-methylumbelliferyl-casein
Authors: Khalfan H , Abuknesha R , Robinson D
Published: August 2015

A method for the preparation of casein labelled with the 4-methylumbelliferyl fluorophore is described, and the product was used as a fluorigenic macromolecular substrate for a sensitive assay of the activity of proteinases. Nanogram quantities of trypsin, chymotrypsin, elastase and cathepsin D can be detected, but the substrate is unaffected by cathepsin B.
Kinetic study of photochemical and thermal conversion of bilirubin IX α and its photoproducts
Authors: Isobe K , Itoh S , Onishi S , Yamakawa T , Ogino T , Yokoyama T
Published: August 2015

A kinetic study of the photochemical and thermal conversion of photoisomers, especialy peaks 0 and 3 [(EE)-bilirubin IX alpha and (EZ)- and (ZE)-bilirubin IX alpha], under anaerobic conditions, was performed by using reversed-phase high-pressure liquid chromatography. Peaks 0 and 3 are spontaneously, photochemically and thermally converted. Short-term photoirradiation of bilirubin gives a mixture containing not only the geometric isomers (EE)-, (EZ)- and (ZE)-bilirubin IX alpha, but also peak 2, (EZ)-cyclobilirubin IX alpha. On prolonged irradiation, cyclization and 15Z leads to 15E isomerization lead to gradual accumulation of two pairs of photoproducts: (EZ)-cyclobilirubin IX alpha A and B and (EE)-cyclobilirubin IX alpha A and B.
Inhibition of cardiac proteolysis by colchicine. Selective effects on degradation of protein subclasses
Authors: Crie J S , Ord J M , Wakeland J R , Wildenthal K
Published: August 2015

1. The effect of colchicine (2.5 microM) on cardiac protein turnover was tested with foetal mouse hearts in organ culture. 2. Colchicine had no effect on protein synthesis, but inhibited total protein degradation by 12-18%. Lumicolchicine, which lacks colchicine's ability to disaggregate microtubules, but shares its non-specific effects, did not alter protein degradation. 3. The colchicine-induced inhibition of protein degradation was accompanied by significant changes in cardiac lysosomal enzyme activities and distribution. 4. Colchicine inhibited the degradation of organellar proteins, including mitochondrial cytochromes, more than that of cytosolic proteins. 5. Colchicine decreased the rate of myosin degradation and the rate of proteolysis of the total protein pool to a similar extent. Since the regulation of myosin degradation does not involve lysosomes, this suggests that colchicine affects non-lysosomal as well as lysosomal pathways. 6. Release of branched-chain amino acids from colchicine-treated hearts was disproportionately decreased, suggesting that colchicine increased their metabolism. 7. It is concluded that colchicine, via its actions on microtubules, exerts important inhibitory effects on cardiac proteolysis. Colchicine is especially inhibitory to the degradation of organellar proteins, including mitochondrial cytochromes. Its inhibitory effects may be mediated in part via lysosomal mechanisms, but non-lysosomal mechanisms are probably involved as well.
The partial characterization of the binding of avidin-biotin complex to rat liver plasma membrane
Authors: Chalifour L E , Dakshinamurti K
Published: August 2015

Rat liver plasma membrane and rat intestinal mucosa plasma membrane were examined for their ability to bind avidin-[3H]biotin complex. Avidin-[3H]biotin complex bound specifically to liver plasma membrane with a Kd of approx. 35 nM and Bmax. of 136 pmol/mg of membrane protein. The receptor on the liver plasma membrane was exposed by Pronase, sensitive to tryptic hydrolysis and insensitive to neuraminidase. The optimum pH of specific binding was less than 6.0. Simple carbohydrates, mannan and ovalbumin did not inhibit specific binding. Periodate-treated or alpha-mannosidase-treated avidin was able to inhibit binding of either avidin-[3H]biotin complex or untreated avidin to plasma membrane. [3H]Avidin containing methylated lysine residues did not bind to rat liver plasma membrane. There was no specific binding of the avidin-biotin complex to rat intestinal mucosa plasma membrane.
Identification of specific proteins synthesized by type II pneumocytes in primary culture
Authors: Wartell S A , Nafe C K , Watkins C A , Rannels D E
Published: August 2015

Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.
Inhibition of pyruvate carboxylase degradation and total protein breakdown by lysosomotropic agents in 3T3-L1 cells
Authors: Chandler C S , Ballard F J
Published: August 2015

1. Exposure to [3H]biotin during the differentiation of 3T3-L1 cells to adipocytes selectively labelled pyruvate carboxylase (EC 6.4.1.1). A subsequent incubation of labelled cells permitted the measurement of the degradation rate constant of this mitochondrial enzyme. 2. In medium without serum, pyruvate carboxylase was degraded with a half-life of 64 h, considerably longer than that found for average cell protein. The long half-life is commensurate with the enzyme being catabolized when whole mitochondria are destroyed. 3. The breakdown of pyruvate carboxylase was inhibited to a greater extent than the breakdown of total cell protein by insulin, NH4Cl and inhibitors of lysosomal proteinases, suggesting that the enzyme is degraded by the autophagic lysosomal system of the cell. 4. The above evidence implies that whole mitochondria are degraded in lysosomes, a conclusion that agrees with earlier electron-microscopic evidence showing mitochondria within autophagic vacuoles. 5. A second degradative pathway must be invoked to account for the breakdown of mitochondrial proteins of short half-life.
Concanavalin A causes an increase in sodium permeability and intracellular sodium content of pig lymphocytes
Authors: Felber S M , Brand M D
Published: August 2015

1. The 3mV depolarization of pig lymphocytes observed within 2 1/2 min of treatment with concanavalin A [Felber & Brand (1983) Biochem. J. 210, 885-891] is dependent on the presence of high extracellular [Na+]. 2. The concanavalin A-induced changes in membrane potential at high and low extracellular [Na+] are quantitatively explained by an increase in the electrogenic permeability coefficient for Na+ (PNa). This rises from a negligible value in resting cells to around 4% of the permeability coefficient for K+ or Cl- in stimulated cells. 3. Concanavalin A induces a 4mM increase in the Na+ content of pig lymphocytes. This increase in intracellular [Na+] is not due solely to stimulation of electrogenic Na+ influx resulting from the rise in PNa. 4. Thus concanavalin A stimulates both an electrogenic pathway for Na+ influx, resulting in a small depolarization of the plasma membrane, and a non-electrogenic Na+ influx pathway, resulting in a rise in intracellular [Na+].
Ribulose bisphosphate carboxylase/oxygenase in toluene-permeabilized Rhodospirillum rubrum
Authors: Storrø I , McFadden B A
Published: August 2015

Toluene-permeabilized Rhodospirillum rubrum cells were used to study activation of and catalysis by the dual-function enzyme ribulose bisphosphate carboxylase/oxygenase. Incubation with CO2 provided as HCO3-, followed by rapid removal of CO2 at 2 degrees C and subsequent incubation at 30 degrees C before assay, enabled a determination of decay rates of the carboxylase and the oxygenase. Half-times at 30 degrees C with 20 mM-Mg2+ were 10.8 and 3.7 min respectively. Additionally, the concentrations of CO2 required for half-maximal activation were 56 and 72 microM for the oxygenase and the carboxylase respectively. After activation and CO2 removal, inactivation of ribulose bisphosphate oxygenase in the presence of 1 mM- or 20mM-Mn2+ was slower than that with the same concentrations of Co2+ or Mg2+. Only the addition of Mg2+ supported ribulose bisphosphate carboxylase activity, as Mn2+, Co2+ and Ni2+ had no effect. A pH increase after activation in the range 6.8-8.0 decreased the stability of the carboxylase but in the range 7.2-8.0 increased the stability of the oxygenase. With regard to catalysis. Km values for ribulose 1,5-bisphosphate4- were 1.5 and 67 microM for the oxygenase and the carboxylase respectively, and 125 microM for O2. Over a broad range of CO2 concentrations in the activation mixture, the pH optima were 7.8 and 8-9.2 for the carboxylase and the oxygenase respectively. The ratio of specific activities was constant (9:1 for the carboxylase/oxygenase) of ribulose bisphosphate carboxylase/oxygenase in toluene-treated Rsp. rubrum. Below concentrations of 10 microM-CO2 in the activation mixture, this ratio increased.
Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay
Authors: Phillips I R , Shephard E A , Bayney R M , Pike S F , Rabin B R , Heath R , Carter N
Published: August 2015

Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although β-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.
Characterization of phosphate efflux pathways in rat liver mitochondria
Authors: Kaplan R S , Pedersen P L
Published: August 2015

ATP hydrolysis catalysed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, we have attempted to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited, Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). In conclusion, results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalysing Pi influx. Alternatively, a separate Pi efflux carrier may exist.
The effect of [Ca2+] and [H+] on the functional recovery of rat brain synaptosomes from anoxic insult in vitro
Authors: Harvey S A K , Booth R F G , Clark J B
Published: August 2015

The energy status (as measured by the ATP/ADP ratio), oxidative metabolism (14CO2 output) and neurotransmitter synthesis ([14C]acetylcholine production) by rat brain synaptosomes utilizing [U-14C]glucose has been studied. The ability of anoxia in vitro to permanently alter these parameters was investigated with reference to external [Ca2+] and [H+]. It has previously been shown that anoxic damage to synaptosomal preparations is only apparent when their metabolism is stimulated by veratridine [Harvey, Booth & Clark (1982) Biochem. J. 206, 433-439]. It is concluded that low [Ca2+] ameliorates, and high [H+] exacerbates, the damage sustained by veratridine-stimulated anoxic synaptosomes. The combined effects of low pH, anoxia and veratridine stimulation on synaptosomal metabolism most closely approximated to the irreversible damage to brain metabolism observed during acute hypoxia in vivo [Booth, Harvey & Clark (1983) J. Neurochem. 40, 106-110]. Suitably treated synaptosomal preparations may therefore be usefully employed as models to study impaired neurotransmitter synthesis in vivo.
Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions
Authors: Brown K D , Blakeley D M
Published: August 2015

Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step.
Effects of amiprophosmethyl on massive and limited calcium loading of maize mitochondria
Authors: Hertel C , Affolter H , Marme D , Dierks-Ventling C
Published: August 2015

In maize mitochondria the effect of the herbicide amiprophosmethyl was studied on massive and limited Ca2+-loading and on mitochondrial energy transduction. Under massive Ca2+-loading conditions amiprophosmethyl inhibited the Ca2+ transport system directly without a significant effect on the respiratory chain, the membrane potential or Ca2+ efflux. Under limited Ca2+-loading conditions an increased Ca2+ efflux was noted, which could be partially responsible for the inhibition of the net Ca2+ accumulation.
Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles
Authors: Foreman J W , Reynolds R A , Ginkinger K , Segal S
Published: August 2015

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.
Peroxisomal palmitoyl-CoA oxidation in the Zucker rat
Authors: Brady P S , Hoppel C L
Published: August 2015

The effects of 3 or 6 days of starvation on hepatic peroxisomal palmitoyl-CoA oxidation were examined in adult lean and obese Zucker rats. When expressed either per mg of DNA or per total liver, obese rats had almost 2-fold higher oxidation rates than the lean rats. Within 6 days of starvation rates fell by 50% among both phenotypes. When data were expressed per 100 g body wt., lean and obese rats had similar rates, falling from a mean of 0.57 to 0.28 mumol/min per 100 g body wt. within 6 days of starvation. Peroxisomal oxidative changes paralleled mitochondrial beta-oxidative changes.
Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells
Authors: Pearson J D , Slakey L L , Gordon J L
Published: August 2015

ATP (approx. 1-300 microM) induces dose-dependent prostacyclin secretion from perfused columns of microcarrier beads with cultured endothelial monolayers attached. The response is transient, shows little tachyphylaxis and can reach approx. 100 times control values. 2-Methylthio-ATP is more potent, ADP slightly less potent, AMP much less potent and adenosine is ineffective. These results are consistent with the presence of a purinoceptor on endothelium linked to the prostacyclin synthetic pathway.
Expression of insulin receptors and insulin action in cell hybrids and cybrids
Authors: Kahn C R , Lauris V , Fehlmann M , Crettaz M
Published: August 2015

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels. Both parents, the cybrids, and the hybrids, expressed insulin stimulation of alpha-aminoisobutyric acid influx, but the dose-response curves indicated an increased insulin sensitivity in the cells with the higher receptor concentration. Insulin also stimulated 86Rb+ uptake in the hepatoma parent, hybrids and cybrids, but not in the L-cell parent. These data suggest that insulin receptors, like other hepatoma-specific properties, behave as a ‘luxury function’ of the hepatoma cell line and are extinguished when the hepatoma cell is fused with a less differentiated cell type. The biological activities associated with insulin action, on the other hand, are much more complex in their expression and probably the result of the interaction of multiple factors that vary in their expression in cell hybrids and cybrids.
Calcium-dependence of insulin receptor phosphorylation
Authors: Plehwe W E , Williams P F , Caterson I D , Harrison L C , Turtle J R
Published: August 2015

Phosphorylation of the insulin receptor of isolated rat adipocytes in response to insulin has been studied. Immunoprecipitation of adipocyte membrane protein demonstrated increased incorporation of 32P after exposure to insulin for 15 min, but this was dependent on the presence of physiological concentrations of Ca2+ and Mg2+. Autoradiography of solubilized immunoprecipitated membrane protein after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that most of the 32P incorporation occurred in a band corresponding to Mr 95 000, which has been identified previously as the beta-subunit of the insulin receptor. 32P incorporation was inhibited by 2,4-dinitrophenol and trifluoperazine. It is suggested that insulin-receptor phosphorylation is an energy-requiring process that is Ca2+-dependent and may be modulated by calmodulin. Phosphorylation may proceed independently of glucose transport.
Identification of penicillin-binding protein 5a of Bacillus megaterium KM as a dd-carboxypeptidase
Authors: Todd J A , Ellar D J
Published: August 2015

Measurement of the stabilities of DD-carboxypeptidase activity and the penicillin-binding activity of proteins 5 and 5a in membranes isolated from vegetative cells and stage-V forespores suggests that the unique sporulation-specific protein 5a may be a penicillin-sensitive DD-carboxypeptidase.
Studies on the mechanism of the antifungal action of benzoate
Authors: Krebs H A , Wiggins D , Stubbs M , Sols A , Bedoya F
Published: August 2015

A method is described for the determination of the pH of intracellular water based on the distribution of [14C]benzoate (0.01 mM) between intra- and extra-cellular water. Benzoate at higher concentrations (2-10mM) enters the yeast cell in the undissociated form, and its neutralization within the cell can cause a shift of the pH of the intracellular water by more than 1 pH unit. Benzoate causes an accumulation of the two hexose monophosphates of yeast glucose fermentation and a decrease in intermediates beyond phosphofructokinase, suggesting inhibition at this stage. Benzoate also causes a concomitant fall in [ATP]. Phosphofructokinase is inhibited to a greater extent than hexokinase at acid pH. There is a relationship between intracellular pH, phosphofructokinase inhibition and CO2 production, suggesting that the antifungal action of benzoate is caused by an accumulation of benzoate at low external pH, which lowers the intracellular pH into the range where phosphofructokinase is sensitive. The subsequent inhibition of glycolysis causes a fall in [ATP] and thus restricts growth.
Noradrenaline treatment of rats stimulates H2O2 generation in liver mitochondria
Authors: Swaroop A , Patole M S , Puranam R S , Ramasarma T
Published: August 2015

Treatment of rats with noradrenaline stimulated H2O2 generation in liver mitochondria using succinate, choline or glycerol 1-phosphate as substrate. The dehydrogenase activity with either succinate or choline as substrate showed no change, whereas that with glycerol 1-phosphate increased. The effect was obtained with noradrenaline, but not with dihydroxyphenylserine. Phenoxybenzamine and yohimbine, but not propranolol, prevented the response to noradrenaline treatment. Phenylephrine could stimulate H2O2 generation, whereas isoprenaline had only a marginal effect. Theophylline treatment slightly decreased the generation of H2O2 in liver mitochondria, but treatment with pargyline, Ro4-1284 and dibutyryl cyclic AMP had little effect. These studies showed that noradrenaline might possibly be acting through the alpha 2-adrenergic system.
The fate of homologous 125I-labelled immunoglobulin G within rat visceral yolk sacs incubated in vitro
Authors: Weisbecker U , Ibbotson G E , Livesey G , Williams K E
Published: August 2015

When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes.
Purification of the 22000- and 20000-mol.wt. forms of human somatotropin and characterization of their binding to liver and mammary binding sites
Authors: Closset J , Smal J , Gomez F , Hennen G
Published: August 2015

Quantitative data concerning the binding of 22000-mol.wt. human somatotropin and its 20000-mol.wt. variant are described using pregnant-rabbit liver and mammary-gland receptors. The purification and the complete chemical characterization of both human somatotropin and its 20000-mol.wt. variant is also presented. Contamination of the 20000-mol.wt.-variant preparation by 22000-mol.wt. hormone was found to be 0.5% by weight as measured in radioimmunoassay using monoclonal antibody. Labelling of human somatotropin and its 20000-mol.wt. variant using the Iodogen method is described as well as the characterization of the binding to pregnant-rabbit liver and mammary-gland receptor preparations. The maximum binding capacity of the 125I-labelled human somatotropin was between 50 and 60% to liver particulate receptor, whereas that of the 20000-mol.wt. variant was 30%. The specificity of binding of both forms to rabbit hepatic and mammary-gland receptor was found to be similar for both proteins in the same system. The affinity constants and capacity were respectively 0.7 X 10(10)M-1 and 815 fmol/mg of protein for human somatotropin and 0.6 X 10(10)M-1 and 1.250 fmol/mg of protein for the 20000-mol.wt. variant. These data suggest that both proteins behave as partial agonists to the receptors studied.
An intermediary role for the tremorgenic mycotoxin TR-2 in the biosynthesis of verruculogen
Authors: Willingale J , Perera K P W C , Mantle P G
Published: August 2015

14C-Labelled compound TR-2, a tremorgenic mycotoxin, was administered to Penicillium raistrickii in submerged fermentation. Half of the added radiolabel was taken up by the fungus during the 60 h incubation period and the secondary metabolites subsequently isolated, principally verruculogen but also fumitremorgin B, were found to be radiolabelled. The efficiency of biosynthetic incorporation of TR-2 into verruculogen within the mycelium was at least 35%, demonstrating for the first time an intermediary role for TR-2. Fumitremorgin B was also TR-2-derived but may not be an important intermediate in verruculogen biosynthesis.
Pro-(carboxypeptidases A) from whole pig pancreas. Their mass, size, shape and solvation
Authors: Martínez M C , Nieuwenhuysen P , Clauwaert J , Cuchillo C M
Published: August 2015

Sedimentation analysis and light-scattering measurements were made with the two forms of pig pancreas pro-(carboxypeptidase A), in order to determine some of their physical properties. The following values were found (the first value applies to the binary complex and the second one to the monomer). The A 1%/280.1 cm values were 19.9 +/- 0.3 and 16.3 +/- 0.3. The partial specific volumes v -0 were 0.707 +/- 0.016 cm3/g and 0.714 +/- 0.015 cm3/g. The sedimentation coefficients S 0/20,w were 4.90 +/- 0.15S and 3.75 +/- 0.15 S. The diffusion coefficients D 0/20,w were (5.8 +/- 0.1) X 10(-7) cm2/s and (6.95 +/- 0.15) X 10(-7) cm2/s. From these data the following values were calculated. Relative molecular masses Mr were 71 000 +/- 4000 and 46 000 +/- 3000. The frictional ratios f/fmin. were 1.37 +/- 0.06 and 1.31 +/- 0.07; assuming a value for the solvation of the molecules (delta = 0.5 g/g) the asymmetry values range from 3 to 5 for the binary complex and from 2 to 4 for the monomer. The Mr values found in the present work coincide with those found by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate [Martínez, Avilés, SanSegundo & Cuchillo (1981) Biochem. J. 197, 141-147]. Therefore the low values obtained by those authors when using gel-filtration chromatography must be the result of the interaction of the zymogens with the gel matrix, as the asymmetry is too small to justify the large discrepancies found.