bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum
Authors: Nunn D L , Potter B V L , Taylor C W
Published: August 2015

Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues. Here we report that Ins(1,4,5)P3 binds to a single class of high-affinity sites in both permeabilized hepatocytes (KD = 7.8 +/- 1.1 nM) and cerebellar membranes (KD = 6.5 +/- 2.4 nM), and provide evidence that these are unlikely to reflect binding to either of the enzymes known to metabolize Ins(1,4,5)P3. Furthermore, the rank order of potency of synthetic inositol phosphate analogues in displacing specifically bound Ins(1,4,5)P3 is the same as their rank order of potency in stimulating mobilization of intracellular Ca2+ stores, suggesting that the Ins(1,4,5)P3-binding site may be the physiological receptor. Radiation inactivation of the Ins(1,4,5)P3-binding sites of liver and cerebellum reveals that they have similar molecular target sizes: 257 +/- 36 kDa in liver and 258 +/- 20 kDa in cerebellum. We conclude that an Ins(1,4,5)P3-binding protein with a molecular target size of about 260 kDa is probably the receptor that mediates Ca2+ mobilization in hepatocytes, and our limited data provide no evidence to distinguish this from the cerebellar Ins(1,4,5)P3-binding protein.
Investigation of the structural basis of the interaction of calpain II with phospholipid and with carbohydrate
Authors: Crawford C , Brown N R , Willis A C
Published: August 2015

Two forms of pig kidney calpain II were isolated, both of which appeared to contain an intact 80 kDa large subunit, but which showed specific proteolytic degradation at the N-terminal end of the 30 kDa small subunit. The structure of each of these molecules was investigated by amino acid sequence analysis. The forms corresponded to molecules with small subunits starting at residue 38 (degraded calpain A) and at residue 62 (degraded calpain B) of the complete sequence. These molecules were tested for their ability to interact with phosphatidylinositol and with carbohydrate (agarose gel-filtration media). Calpain and degraded calpain A, but not degraded calpain B, would interact with phosphatidylinositol. Thus the sequence (G)17TAMRILG (residues 38-61) is essential for the interaction. Neither calpain nor the degraded forms of the enzyme showed specific interaction with carbohydrate.
Fluoroaluminate mimics agonist application in single rat hepatocytes
Authors: Woods N M , Dixon C J , Cuthbertson K S R , Cobbold P H
Published: August 2015

Single rat hepatocytes microinjected with the photoprotein aequorin were stimulated with glycogenolytic agonists or low concentrations of fluoroaluminate. Both protocols resulted in the generation of oscillations in cytosolic free Ca2+ levels from a resting value of approx. 200 nM and peaking at over 600 nM. However, oscillations induced by receptor-dependent agonists were more regular in both frequency and time course than those induced by direct activation of G-proteins. The role of G-proteins in the generation of repetitive free Ca2+ oscillations is discussed.
The purification and characterization of 3-dehydroquinase from Streptomyces coelicolor
Authors: White P J , Young J , Hunter I S , Nimmo H G , Coggins J R
Published: August 2015

The enzyme 3-dehydroquinase was purified over 4000-fold to homogeneity from Streptomyces coelicolor. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of SDS was 16,000. The native Mr estimated by gel filtration on a Superose 6 column was 209,000, indicating that the enzyme is a large oligomer. The enzyme was found to be extremely thermostable. This stability, along with the structural and kinetic properties of the enzyme, suggest that it is very similar to the quinate-inducible 3-dehydroquinase found in Neurospora crassa and Aspergillus nidulans. This similarity was confirmed by direct N-terminal sequencing.
Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion
Authors: Thams P , Capito K , Hedeskov C J , Kofod H
Published: August 2015

The influence of down-regulation of protein kinase C on glucose-induced insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM) in RPMI 1640 culture medium (8.3 mM-glucose, 0.43 mM-Ca2+) abolished TPA (0.16 microM)-induced insulin secretion and led to a potentiation of phase 1 and a decrease in phase 2 of glucose-induced insulin secretion. Thus, although the total insulin release during 40 min of perfusion with glucose (16.7 mM) (45-85 min) was unaffected, the percentage released during phase 1 (45-55 min) was increased from 12.9 +/- 1.5 (4)% in controls to 35.8 +/- 3.9 (4)% in TPA-treated islets (P less than 0.01), and the percentage released during phase 2 (65-85 min) was decreased from 63.2 +/- 3.9 (4)% to 35.3 +/- 1.4 (4)% (P less than 0.005). In contrast, TPA exposure in TCM 199 medium (5.5 mM-glucose, 1.26 mM-Ca2+) caused a total abolition of both phases 1 and 2 of glucose-induced secretion. However, inclusion of the alpha 2-adrenergic agonists adrenaline (10 microM) or clonidine (10 microM), or lowering of the Ca2+ concentration in TCM 199 during down-regulation, preserved and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1 and inhibited phase 2 of carbamoylcholine (100 microM)-induced insulin secretion at 3.3 mM-glucose, and abolished the potentiating effect of carbamoylcholine (100 microM) at 16.7 mM-glucose. These results substantiate a role for protein kinase C in insulin secretion, and suggest that protein kinase C inhibits phase 1 and stimulates phase 2 of both glucose-induced and carbamoylcholine-induced insulin secretion.
Transition of affinity states for leukotriene B4 receptors in sheep lung membranes
Authors: Votta B , Mong S
Published: August 2015

Leukotriene B4 (LTB4) is a pro-inflammatory arachidonate metabolite. We have characterized the LTB4 receptors in sheep lung membranes and have assessed the contribution of the guanine-nucleotide-binding (G) protein in the regulation of receptor affinity states. Saturation isotherms have demonstrated a single class of LTB4 receptor with a Kd of 0.18 +/- 0.03 nM and a density (Bmax.) of 410 +/- 84 fmol/mg of protein in sheep lung membranes. The effect of the G-protein on receptor affinity was assessed in the presence of non-hydrolysable GTP analogues (e.g. GTP[S]) and in membranes following alkali treatment (pH 12.1) to remove the G-protein. Saturation isotherms produced either in the presence of GTP[S] (Kd.GTP[S] = 0.51 +/- 0.02 nM) or with alkali-treated membranes (Kd.alk. = 0.52 +/- 0.02 nM) demonstrated a 3-fold shift in receptor affinity for [3H]LTB4 binding. In competition experiments, the rank order of affinity of LTB4 analogues was LTB4 greater than 20-OH-LTB4 greater than trans-homo-LTB4 greater than 6-trans-LTB4 greater than 20-COOH-LTB4, using either untreated or alkali-treated membranes, both in the presence and absence of GTP[S]. These findings demonstrate that, in sheep lung membranes, there is only one class of LTB4 receptor. Removal of the G-protein or uncoupling of the receptor from the G-protein shifted the agonist-binding affinity of the receptor by 3-4-fold, without affecting the specificity of the LTB4 receptor in either the high- or the low-affinity state.
Identification of the subunits of bovine NADH dehydrogenase which are encoded by the mitochondrial genome
Authors: Gibb G M , Ragan C I
Published: August 2015

Products of the mitochondrial genome were identified in the bovine kidney cell line NBL-1 by labelling with [35S]methionine in the presence of cycloheximide. Seven proteins were precipitated by an antiserum to bovine heart NADH dehydrogenase, corresponding to the seven mitochondrial gene products identified in the human HeLa cell line. Comparison of these mitochondrial gene products with purified bovine NADH dehydrogenase by SDS/gel electrophoresis revealed that the ND-5 product is probably a previously unidentified protein of apparent Mr 51,000, and the ND-4 product is the protein of apparent Mr 39,000.
Intermediary metabolism in the Swarm rat chondrosarcoma chondrocyte
Authors: Spencer C A , Palmer T N , Mason R M
Published: August 2015

The rat chondrosarcoma chondrocyte has the dual capacity to metabolize glucose (mainly via glycolysis) and glutamine (via an oxidative pathway). Glutamine metabolism, unlike that of glucose, is unable to sustain intracellular ATP concentrations. Glutamine consumption by the chondrosarcoma chondrocyte, however, is significantly in excess of its utilization as an amide-group donor in hexosamine synthesis, implying a novel and major role in cell metabolism.
Fluidity-dependent Mg2+-ATPase activity in membranes from Leishmania donovani promastigotes
Authors: Dutta M , Bandyopadhyay R , Ghosh C , Basu M K
Published: August 2015

The state of the lipid phase of the membrane plays a key role in the exposure of various receptors, antigens and enzymes on the membrane surface. The fluidity of membranes of Leishmania donovani promastigotes was monitored by two independent methods, i.e. influx of sterol from liposomes and removal of phospholipids by treatment with phospholipase C. The altered sterol/phospholipid ratio, in both cases, provided evidence that the activity of the functionally important membrane-bound enzyme Mg2(+)-ATPase is modulated by the state of the lipid phase of the membrane.
The high content of natural suppressor serine tRNA in dystrophic mouse muscle
Authors: Hitaka T , Mizutani T , Watanabe K , Totsuka T
Published: August 2015

In order to gain an insight into the pathogenesis of mouse muscular dystrophy, we investigated the natural suppressor serine tRNA. The natural suppressor seryl-tRNA was distinguished from the other seryl-tRNAs on the basis of its specific property of being converted into phosphoseryl-tRNA by a tRNA kinase. On a wet-weight basis, the content of total tRNA in dystrophic muscles was 47% of that in normal muscles. Although the serine-accepting activities of tRNA were similar in muscles of 3-month-old dystrophic and normal mice, the ratio of [32P]phosphoseryl-tRNA (suppressor tRNA) to the total serine tRNA was significantly enhanced in dystrophic muscles compared with that in normal muscles. This high content of suppressor tRNA in dystrophic muscles was further confirmed by dot-blot hybridization experiments with the DNA probes CGTAGTCGGCAGGAT and CGCCCGAAAGGTGGAA for major tRNA(IGASer) and suppressor tRNA respectively. At the early postnatal age of 3 weeks, when only a week had elapsed since the first manifestation of the dystrophic symptom (hindleg dragging), the ratio of suppressor tRNA to major tRNAs in dystrophic hindleg muscles was abnormally increased. Thereafter it decreased with age in normal mice but remained almost unchanged in dystrophic mice. Consequently, at 3 months old, it was 1.7 times higher in dystrophic than in normal mice. The suppressor tRNA is now accepted to play a role in the synthesis of glutathione peroxidase. The present study showed that the content of this enzyme was abnormally elevated in dystrophic mice. Previously we had demonstrated that the docosahexaenoic (C22:6) acid content in phospholipids was decreased, possibly resulting from the enhanced oxidative milieu caused by the dystrophic condition. Thus far, the findings suggest that an increase in the contents of suppressor tRNA and glutathione peroxidase in dystrophic muscle may have been secondarily induced by such a highly oxidative state in the dystrophic condition. However, it is difficult to exclude the possibility that the natural suppressor tRNA plays a primary role in the pathogenesis of muscular dystrophies.
Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver
Authors: Trinchera M , Ghidoni R
Published: August 2015

In order to clarify the mechanisms of ganglioside biosynthesis and transport we intravenously administered a liposomal dispersion of radiolabelled lactosylceramide (LacCer) to rats and then followed the time course of the individual gangliosides which became radioactive in the Golgi-apparatus and plasma-membrane fractions prepared from the liver. After administration of radiolabelled LacCer the liver retained a substantial amount of radioactivity, which was distributed among an organic phase (mainly residual LacCer), a fraction containing low-Mr substances (mainly 3H2O) and a ganglioside fraction. The hepatocytes were found to provide the bulk of gangliosides biosynthesized from exogenous LacCer. After subcellular fractionation, the total radioactive gangliosides increased in the Golgi apparatus up to 8 h, to then decrease and practically disappear at 24 h; in the plasma membranes they were progressively concentrated, accounting for high absolute values. Ganglioside patterns were greatly modified with time in both the Golgi apparatus and plasma membrane, but without significant differences between them. Biosynthesis in the Golgi apparatus and accumulation in the plasma membrane of each individual ganglioside followed a precursor-product relationship. The obtained results indicated that once a ganglioside is biosynthesized in the Golgi apparatus, it is in part made available for translocation to the plasma membrane, which rapidly occurs, and is in part retained in the Golgi apparatus, where it acts as a precursor for the biosynthesis of more glycosylated gangliosides.
Hydrolysis of fucosyl-GM1 ganglioside by purified pellet-associated human brain and human liver α-l-fucosidases without activator proteins or detergents
Authors: Hopfer R L , Johnson S W , Masserini M , Giuliani A , Alhadeff J A
Published: August 2015

Pellet-associated human brain alpha-L-fucosidase was solubilized with 0.5% (w/v) Triton X-100 and purified by affinity chromatography on agarose-6-aminohexanoyl-fucosamine resin. The procedure resulted in a 290,000-fold purification, a 58% yield and a final specific activity of 11,500 nmol/min per mg of protein. Isoelectric focusing indicated that all six major isoforms (with pI values between 4.1 and 5.3) present in crude brain pellet preparations were purified by the affinity technique. SDS/PAGE indicated the presence of one subunit (54 kDa) and a minor protein band at 67 kDa, which presumably is a contaminant since it was not immunoreactive on Western blotting. The pH optimum of the brain enzyme and its apparent Km for the synthetic substrate 4-methylumbelliferyl alpha-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver alpha-L-fucosidases were both capable of hydrolysing fucosyl-GM1 ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37 degrees C was found at pH 3.4 for both alpha-L-fucosidases, with no activity at pH values above 4.0.
Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein
Authors: Carroll R C , Worthington R E , Boucheix C
Published: August 2015

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5′(6′)-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1′,7(3H)-isobenzofuran]-3′-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab′)2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab′)2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.
19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli
Authors: Sixl F , King R W , Bracken M , Feeney J
Published: August 2015

Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr). The 19F-n.m.r. spectrum of the [5-F-Trp]CRP showed two signals corresponding to the two tryptophan residues, and that of the [3-F-Tyr]CRP showed six signals (two overlapping) corresponding to the six tyrosine residues: these results are as expected for a symmetrical dimer. A comparison of the 19F-n.m.r. spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences. Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects. The 19F-n.m.r. spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP. The 19F-n.m.r. spectrum of the complex of [3-F-Tyr]CRP with tubercidin 3′,5′-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding. [3-F-Tyr]CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5′-AATGTGAGTTAACTCACATT-3′ and excess cyclic AMP gave an 19F-n.m.r. spectrum that was almost identical with that for the [3-F-Tyr]CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues. Proteolysis of [3-F-Tyr]CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain. This fragment contains five of the six tyrosine residues, and its 19F-n.m.r. chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unperturbed by the binding of cyclic nucleotide to the intact [3-F-Tyr]CRP. The similarity of the 19F-n.m.r. chemical shifts in the alpha-fragment and the intact CRP indicates that the alpha-fragment retains the same structure as found in the intact protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Dependence of the P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry. Quantification of selectivity in the catalysed hydrolysis of the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides
Authors: Kowlessur D , Thomas E W , Topham C M , Templeton W , Brocklehurst K
Published: August 2015

1. N-Acetyl-L-phenylalanylglycine 4-nitroanilide and its D-enantiomer were synthesized and characterized and used as substrates with which to evaluate stereochemical selectivity in papain (EC 3.4.22.2)-catalysed hydrolysis. 2. Kinetic analysis at pH 6.0, I 0.1, 8.3% (v/v) NN-dimethylformamide and 25 degrees C by using initial-rate data with [S] much less than Km and weighted non-linear regression provided values of kcat./Km for the catalysed hydrolysis of both enantiomers as (kcat./Km)L = 2040 +/- 48 M-1.S-1 and (kcat./Km)D = 5.9 +/- 0.07 M-1.S-1. These data, taken together with individual values of kcat. and Km for the hydrolysis of the L-enantiomer (a) estimated in the present work as kcat. = 3.2 +/- 1.2 S-1 and Km = 1.5 +/- 0.6 mM and (b) reported by Lowe & Yuthavong [(1971) Biochem. J. 124, 107-115] for the reaction at pH 6.0 in 10% (v/v) NN-dimethylformamide and 35 degrees C, as kcat. = 1.3 +/- 0.2 S-1 and Km = 0.88 +/- 0.1 mM, suggest that (kcat./Km)L congruent to 2000 M-1.S-1 and thus that (kcat./Km)L/(kcat./Km)D congruent to 330.3. Model building indicates that both enantiomeric 4-nitroanilides can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the S2 subsite with preservation of mechanistically relevant hydrogen-bonding interactions and that the main difference is in the positioning of the beta-methylene group. 4. The dependence of P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry for reactions involving derivatives of N-acetylphenylalanine is discussed. The variation in the index of stereochemical selectivity (ratio of the appropriate kinetic or thermodynamic parameter for a given pair of enantiomeric ligands), from 330 for the overall acylation process of the catalytic act, through 40 and 31 for the reaction at electrophilic sulphur in 2-pyridyl disulphides respectively without and with assistance by (His-159)-Im(+)-H, to 5 for the formation of thiohemiacetal adducts by reaction at aldehydic carbon, is interpreted in terms of the extent to which conformational variation of the bound ligand in the catalytic-site region permits the binding mode of the -CH2-Ph group of the D-enantiomer to approach that of the L-enantiomer.
The changes in hepatic enzyme expression caused by selenium deficiency and hypothyroidism in rats are produced by independent mechanisms
Authors: Beckett G J , Nicol F , Proudfoot D , Dyson K , Loucaides G , Arthur J R
Published: August 2015

Selenium (Se) deficiency for 5 weeks in rats produced changes in the activity of a number of hepatic, renal and plasma enzymes. In animals whose food intake was restricted to 75% of normal for 2 weeks, Se deficiency produced significant increases in the activity of hepatic cytosolic ‘malic’ enzyme and mitochondrial alpha-glycerophosphate dehydrogenase (GPD), two enzymes that are particular sensitive to the thyroid-hormone concentrations in tissue. Propylthiouracil-induced hypothyroidism produced significant decreases in ‘malic’ enzyme and GPD activities. The effect of hypothyroidism on the activity of ‘malic’ enzyme, GPD and other enzymes studied in liver and plasma was often opposite to that seen in Se deficiency. Glutathione S-transferase (GST) activity was increased by both Se deficiency and hypothyroidism, but in hypothyroid animals further significant increases in GST were produced by Se deficiency. These data suggest that the changes in enzyme expression observed in Se deficiency are not caused by decreased tissue exposure to thyroid hormones.
High-efficiency transpeptidation catalysed by clostripain and electrostatic effects in substrate specificity
Authors: Yagisawa S , Watanabe S , Takaoka T , Azuma H
Published: August 2015

Clostripain catalyses the transpeptidation between benzoylarginin ethyl ester and amino acid amides, oligopeptides, insulin A- and B-chains and tryptic peptides of myoglobin at millimolar substrate concentrations. The reactions proceed with temporary accumulation of the products, followed by hydrolytic decomposition. The yield was not affected significantly by the type of N-terminal amino acid, but was diminished markedly by the negative charges of the amine components. The yields for natural peptides were linearly related to the charge density of the peptides.
Protein kinase C is involved in desensitization of muscarinic receptors induced by phorbol esters but not by receptor agonists
Authors: Lai W S , Rogers T B , el-Fakahany E E
Published: August 2015

Preincubation with receptor agonists or phorbol esters desensitized muscarinic-receptor-mediated [3H]cyclic GMP responses in mouse neuroblastoma N1E-115 cells. However, desensitization mediated by phorbol esters was heterologous, whereas that effected by receptor agonist was specific towards the muscarinic receptors. In addition, there was no loss of cell surface muscarinic receptors, as measured by the binding of the hydrophilic ligand [3H]N-methylscopolamine, when cells were treated with phorbol esters, but receptor-agonist-induced desensitization was accompanied by a decrease in cell surface receptor density. We examined the role of protein kinase C (PKC) in the desensitization of muscarinic receptors by employing a kinase inhibitor and by down-regulation of PKC by long-term incubation of cells with phorbol esters. Whereas these manoeuvres had marked effects on phorbol-ester-induced desensitization of muscarinic responses, they did not block agonist-induced down-regulation and desensitization of muscarinic receptors. In addition, when phosphoinositide hydrolysis was suppressed, the muscarinic agonist was still capable of mediating receptor sequestration and desensitization. These results suggest that the mechanisms for regulating muscarinic receptor sensitivity could be both PKC-dependent and PKC-independent, being mediated by phorbol esters and receptor agonists respectively.
A point mutation in the cytoplasmic domain of the transferrin receptor inhibits endocytosis
Authors: Alvarez E , Gironès N , Davis R J
Published: August 2015

The rate of receptor-mediated endocytosis of diferric 125I-transferrin by Chinese-hamster ovary cells expressing human transferrin receptors was compared with the rate measured for cells expressing hamster transferrin receptors. It was observed that the rate of endocytosis of the human transferrin receptor was significantly higher than that for the hamster receptor. In order to examine the molecular basis for the difference between the observed rates of endocytosis, a cDNA clone corresponding to the cytoplasmic domain of the hamster receptor was isolated. The predicted primary sequence of the cytoplasmic domain of the hamster transferrin receptor is identical with that of the human receptor, except at position 20, where a tyrosine residue in the human sequence is replaced with a cysteine residue. To test the hypothesis that this structural change in the receptor is related to the difference in the rate of internalization, we used site-directed mutagenesis to examine the effect of the replacement of tyrosine-20 with a cysteine residue in the human transferrin receptor. It was observed that the substitution of tyrosine-20 with cysteine caused a 60% inhibition of the rate of iron accumulation by cells incubated with [59Fe]diferric transferrin. No significant difference between the rate of internalization of the mutant (cysteine-20) human receptor and the hamster receptor was observed. Thus the substitution of tyrosine-20 with a cysteine residue can account for the difference between the rate of endocytosis of the human and hamster transferrin receptors.
Kinetic analysis of duck ε-crystallin, a lens structural protein with lactate dehydrogenase activity
Authors: Chiou S H , Lee H J , Chang G G
Published: August 2015

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.
Structural specificity for prostaglandin effects on hepatocyte glycogenolysis
Authors: Brass E P , Garrity M J
Published: August 2015

Prostaglandins (PGs) are known to have effects on hepatic glucose metabolism. Some actions of PGs in intact liver systems may not involve PG effects directly at the level of the hepatocyte. To define the ability of structurally distinct prostaglandins to affect hepatocyte metabolism directly, the regulation of glycogenolysis was studied in hepatocytes isolated from male Sprague-Dawley rats. PGF and PGB2 inhibited glucagon-stimulated glycogenolysis in the hepatocyte system. Pinane thromboxane A2 (PTA2) and PGD2 had no effect on glucagon-stimulated glycogenolysis. Consistent with their inhibition of glucagon-stimulated glycogenolysis, PGF2 and PGF2 alpha inhibited glucagon-stimulated hepatocyte cyclic AMP accumulation. These actions of PGB2 and PGF2 alpha are identical with those previously reported for PGE2. Additionally, PGE2, PGF2 alpha and PGB2 inhibited glucagon-stimulated adenylate cyclase activity in purified hepatic plasma membranes. In contrast, PGF2 alpha, PGD2 and PTA2 were all without affect on basal rates of hepatocyte glycogenolysis or hepatocyte cyclic AMP content. PGE2 also inhibited glycogenolysis stimulated by the alpha-adrenergic agonist phenylephrine. Exogenous arachidonic acid was not able to reproduce the affects of PGE2 or PGF2 alpha on hepatocyte glycogenolysis, consistent with an extra-hepatocyte source of the prostaglandins in the intact liver. Thus PGE2 and PGF2 alpha act specifically to inhibit glucagon-stimulated adenylate cyclase activity. No prostaglandin tested was found to stimulate glycogenolysis. PGE2 and PGF2 alpha may represent intra-hepatic modulators of hepatocyte glucose metabolism.
A fluorescent residualizing label for studies on protein uptake and catabolism in vivo and in vitro
Authors: Maxwell J L , Terracio L , Borg T K , Baynes J W , Thorpe S R
Published: August 2015

Residualizing labels are tracers which remain in lysosomes after uptake and catabolism of the carrier protein and have been especially useful for studies on the sites of plasma protein degradation. Thus far these labels have contained radioactive reporters such as 3H or 125I. In the present paper we describe a fluorescent residualizing label, NN-dilactitol-N′-fluoresceinylethylenediamine (DLF). Modification of asialofetuin (ASF) or rat serum albumin (RSA) with DLF affected neither their normal kinetics of clearance from the rat circulation nor their normal tissue sites of uptake and degradation. After injection of DLF-ASF, fluorescent degradation products were recovered nearly quantitatively in liver and retained with a half-life of about 2 days. Fluorescent degradation products from DLF-RSA were recovered in skin and muscle, and were localized in fibroblasts by fluorescence microscopy. These results confirm previous studies with radioactive residualizing labels in which fibroblasts in peripheral tissues were identified as primary sites of albumin degradation. Fluorescent catabolites also accumulated in fibroblasts incubated with DLF-RSA in vitro, and residualized with a half-life of about 2 days. Overall, the data establish that DLF functions efficiently as a fluorescent residualizing label both in vivo and in vitro. The advantages of fluorescent, compared with radioactive, residualizing labels should make them valuable tools for studies on protein uptake and catabolism in biological systems.
Guanine nucleotide binding properties of rap1 purified from human neutrophils
Authors: Bokoch G M , Quilliam L A
Published: August 2015

The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5′-[gamma-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein beta/gamma-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.
Insulin-induced phospho-oligosaccharide stimulates amino acid transport in isolated rat hepatocytes
Authors: Varela I , Avila M , Mato J M , Hue L
Published: August 2015

The ability of the insulin-induced phospho-oligosaccharide to stimulate amino acid transport was studied in isolated rat hepatocytes. At low alpha-aminoisobutyric acid concentrations (0.1 mM), both 100 nM-insulin and 10 microM-phospho-oligosaccharide doubled amino acid uptake after 2 h of incubation. This stimulation was prevented by 0.1 mM-cycloheximide or 5 micrograms of actinomycin D/ml, indicating that the phospho-oligosaccharide, like insulin, was acting via the synthesis of a high-affinity transport component. The effects of the phospho-oligosaccharide and of insulin were blocked by Ins2P (2.5 mM), but not by myo-inositol, inositol hexaphosphoric acid or several monosaccharides such as mannose, glucosamine and galactose. Both the temporal effect on amino acid entry and the extent of stimulation of this process by the phospho-oligosaccharide indicate that this molecule mimics, and may mediate, some of the long-term actions of insulin. However, the effects of phospho-oligosaccharide and insulin were not exactly the same, since the effect of insulin, but not of the phospho-oligosaccharide, was additive with that of glucagon.
Recognition of insulin-like-growth-factor-binding proteins in serum and amniotic fluid by an antiserum against a low-molecular-mass insulin-like-growth-factor-inhibitor/binding protein
Authors: Ooi G T , Herington A C
Published: August 2015

An antiserum (R8) raised against a purified specific low-Mr (16,000-18,000) insulin-like-growth-factor (IGF)-inhibitor/binding protein, which is immunologically related to the native growth hormone (GH)-dependent Mr-150,000 IGF-binding protein in serum, has been used to probe a possible additional relationship to the predominant non-GH-dependent IGF-binding protein (Mr approximately 30,000) of human amniotic fluid. Amniotic-fluid fractions and an IGF-inhibitory fraction of serum were analysed by covalent cross-linking, ligand-blotting and immunoblotting techniques. Western blotting of the serum fraction using the R8 antiserum gave five immunoreactive (ir) bands, of which at least three (Mr 38,000, 34,000 and 23,000) possessed IGF-binding activity, as indicated by ligand (125I-IGF-I) blotting. Western blotting of two differently prepared amniotic-fluid fractions, which both showed potent IGF-inhibitory bioactivity, gave several (Mr range 14,500-73,000) ir bands, of which two (Mr 28,000 and 17,000) were most prominent. Ligand-blotting analysis gave a single intensely labelled band at Mr 28,000, consistent with the major presence of the Mr-28,000-30,000 amniotic-fluid IGF-binding protein. Covalent cross-linking of the amniotic-fluid fractions to 125I-IGF-I gave three specifically cross-linked complexes (Mr 36,000, 32,000 and 23,000), which, assuming a 1:1 binding stoichiometry with IGF (Mr 7500), represent binding proteins of Mr 28,500, 24,500 and 15,500 respectively. The Mr-15,500 binding protein, very similar to the Mr-17,000 ir band, in all likelihood represents the IGF-inhibitor protein. Our results indicate that inhibitor-sized binding proteins and IGF-inhibitor bioactivity are present in human amniotic fluid, and that the IGF-inhibitor protein (Mr 16,000) and amniotic-fluid IGF-binding protein (Mr 28,000) are immunologically related. Since the IGF-inhibitor protein is also immunologically and structurally related to the native, GH-dependent, Mr-150,000 binding protein in serum, our data suggest a heretofore-unrecognized immunological similarity between the Mr-150,000 binding protein and the amniotic-fluid binding protein and its serum analogue, the Mr approximately 30,000, non-GH-dependent, binding protein.
Desensitization of prostaglandin F2α-stimulated inositol phosphate generation in NIH-3T3 fibroblasts transformed by overexpression of normal c-Ha-ras-1, c-Ki-ras-2 and c-N-ras genes
Authors: Black F M , Wakelam M J O
Published: August 2015

The stimulation of inositol phosphate generation in control and ras-gene-transformed NIH-3T3 cells by prostaglandin F2 alpha (PGF2 alpha) was investigated. Compared with the control cells, a desensitization of the response was observed in cells transformed by the overexpression of N-, Ha-, or Ki-ras genes. This desensitization was without effect upon the concentration causing half-maximal effect (EC50), dissociation constant (Kd) or number of PGF2 alpha receptors. Inhibition of PG synthesis was without effect upon desensitization, demonstrating that the effect was not agonist-induced. Desensitization could be induced in NIH-3T3 cells by culturing under conditions where the cells were all in the exponential growth phase, or by a 12 h exposure to a C-kinase-activating phorbol ester. These results suggest that desensitization of certain agonist-induced inositol phospholipid responses in ras-transformed cells is a consequence of increased cell proliferation and associated amplification in C-kinase activity and is an indirect consequence of transformation by ras.
Differences between muscarinic-receptor- and Ca2+-induced inositol polyphosphate isomer accumulation in rat cerebral-cortex slices
Authors: Baird J G , Nahorski S R
Published: August 2015

Muscarinic-receptor stimulation or depolarization by elevated K+ leads to increased accumulation of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4 and several degradation products of these polyphosphates separated by h.p.l.c. On the other hand, agents such as ionomycin and maitotoxin, which increase intracellular Ca2+ directly, produce a small accumulation of [3H]Ins(1,4,5)P3 and markedly increase [3H]Ins(1,4)P2, but [3H]Ins(1,3,4,5)P4, [3H]Ins(1,3,4)P3 and [3H]Ins(1,3)P2 are virtually unaffected. Ca2(+)-dependent [3H]inositol polyphosphate metabolism may involve different pools of lipids and/or phosphoinositidases.
Evidence for tripeptide/H+ co-transport in rabbit renal brush-border membrane vesicles
Authors: Tiruppathi C , Kulanthaivel P , Ganapathy V , Leibach F H
Published: August 2015

L-Phe-L-Pro-L-Ala is a tripeptide which is hydrolysable almost exclusively by dipeptidyl peptidase IV in rabbit renal brush-border membrane vesicles. In order to delineate the mechanism of the transport of an intact tripeptide across the brush-border membrane, we studied the characteristics of the uptake of [3H]Phe-Pro-Ala in membrane vesicles in which the activity of dipeptidylpeptidase IV was completely inhibited by treatment with di-isopropyl fluorophosphate. In these vesicles, uptake of radiolabel from the tripeptide was found to be Na(+)-independent, but was greatly stimulated by an inwardly directed H+ gradient. The H(+)-gradient-dependent radiolabel uptake appeared to be an active process, because the time course of uptake exhibited an overshoot phenomenon. The process was also electrogenic, being stimulated by an inside-negative membrane potential. Under the uptake-measurement conditions there was no detectable hydrolysis of [3H]Phe-Pro-Ala in the incubation medium when di-isopropyl fluorophosphate-treated membrane vesicles were used. Analysis of intravesicular contents revealed that the radiolabel inside the vesicles was predominantly (greater than 90%) in the form of intact tripeptide. These data indicate that the uptake of radiolabel from [3H]Phe-Pro-Ala in the presence of an inwardly directed H+ gradient represents almost exclusively uptake of intact tripeptide. Uphill transport of the tripeptide was also demonstrable in the presence of an inwardly directed Na+ or K+ gradient, but only if nigericin was added to the medium. Under these conditions, nigericin, an ionophore for Na+, K+ and H+, was expected to generate a transmembrane H+ gradient. Uptake of Phe-Pro-Ala in the presence of a H+ gradient was inhibited by di- and tri-peptides, but not by free amino acids. It is concluded that tripeptide/H+ co-transport is the mechanism of Phe-Pro-Ala uptake in rabbit renal brush-border membrane vesicles.
N-acetyl-d-glucosamine countertransport in lysosomal membrane vesicles
Authors: Jonas A J , Jobe H
Published: August 2015

Countertransport of GlcNAc was examined in membrane vesicles prepared from rat liver lysosomes which had been lysed by exposure to 5 mM-methionine methyl ester. These vesicles have a random orientation, have intact ATP-dependent acidification and are largely free of soluble hydrolases. Vesicular volume (24.69 +/- 4.51 microliters/mg of protein) was greater than that of lysosomes (3.02 +/- 0.56 microliters/mg of protein), corresponding to a doubling of diameter. Characteristics of GlcNAc transport in vesicles (Km = 1.3 mM) were similar to those observed in intact lysosomes (Km = 4.4 mM). Sulphation or phosphorylation of the substrate resulted in loss of recognition by the carrier. Hydroxyl group orientation at multiple positions did not appear to be critical, whereas orientation of the acetyl group appeared to have a fundamental role in recognition by the carrier. Based on these criteria, phenyl isothiocyanate-GlcNAc (PITC-GlcNAc) was identified as a possible substrate for transport. Under mild conditions, PITC-GlcNAc reversibly inhibited GlcNAc countertransport in lysosomes and vesicles. This and other modified substrates may be of value in identification of the GlcNAc/GalNAc lysosomal transporter. Lysosomal membrane vesicle preparation is a technique that should be useful for the study of other lysosomal transport systems.
Synthesis of retinoids by human retinal epithelium and transfer to rod outer segments
Authors: Das S R , Bhardwaj N , Gouras P
Published: August 2015

The synthesis and release of 11-cis-retinoids by primary cultures of human retinal pigment epithelium (RPE) and the transfer of these retinoids to co-incubated human rod outer segments (ROS) were studied. Monolayers of 2-3-week-old cultured RPE incorporate tritiated all-trans-retinol, esterify it to the corresponding retinyl palmitate, form 11-cis-retinol and 11-cis-retinaldehyde and release retinaldehyde into the culture medium. The ratio of 11-cis to all-trans isomers of retinol, retinyl palmitate and retinaldehyde formed in the cells along with retinaldehyde released and incorporated into the ROS progressively increases, indicating a progressive increase in the concentration of 11-cis isomer from the time it is formed in RPE cells until its transfer to ROS. Incorporation of 11-cis-retinaldehyde into the ROS is directly related to the amount of albumin present in the media, suggesting the transfer of retinoids from RPE to photoreceptor to be a protein-mediated process. Events leading to isomerization, esterification, oxidation and release of retinoids by human RPE and incorporation of retinoids into ROS can therefore be examined in vitro.
Cytosolic copper-binding proteins in rat and mouse hepatocytes incubated continuously with Cu(II)
Authors: Palida F A , Mas A , Arola L , Bethin K , Lonergan P A , Ettinger M J
Published: August 2015

The proteins that bind copper when it first enters cells are likely to play roles in its intracellular distribution and utilization. When hepatocytes were incubated with 64Cu(II), the time-dependence of the subcellular distribution of 64Cu was consistent with one or more cytosolic proteins distributing copper to the mitochondrial and nuclear fractions. Cytosolic copper was reproducibly distributed among four protein fractions from Sephadex G-150 columns at the earliest time (1 min) and at the lowest concentration used [2 microM-64Cu(II)] with both rat and mouse hepatocytes. Copper binding to proteins in these functions was sensitive to copper metabolic status. Hepatocytes from nutritionally copper-deficient rats or neonatal (9-30 days old) developing rats showed an inverse correlation between copper binding to metallothionein and copper binding to proteins in fraction I (approximately 88 kDa apparent) and fraction II (approximately 38 kDa apparent). The distribution of cytosolic 64Cu from the brindled-mouse model of Menkes disease indicated decreased binding by a protein in fraction I. Brindled-mouse hepatocytes also contain decreased levels of a approximately 55 kDa protein or subunit, which most likely represents a liver-specific secondary response to the primary defect. The results are consistent with one or more copper-binding proteins in fractions I and II having significant functions in intracellular copper metabolism.