bluuebunny/crossref_metadata_2025 huggingface dataset:
The uptake and metabolism of chylomicron-remnant lipids by rat liver parenchymal and non-parenchymal cells in vitro
Authors: Lippiello P M , Sisson P J , Waite M
Published: August 2015

The uptake and metabolism of chylomicron-remnant lipids by individual liver cell types was examined by incubating remnants with monolayer cultures of hepatocytes, Kupffer cells, and endothelial cells from rat liver. Remnants were prepared in vitro from radiolabelled mesenteric-lymph chylomicra, utilizing either purified lipoprotein lipase from bovine milk, or plasma isolated from heparinized rats. The resulting particles contained [3H]phosphatidylcholine and cholesterol, and [14C]oleate in the acylglycerol, phospholipid, fatty-acid and cholesterol-ester fractions. The capacities of the three cell types for uptake of both [3H]lipids and [14C]lipids were determined to be, on a per-cell basis, in the order: Kupffer greater than hepatocytes greater than endothelial. The relative proportions of [3H]phospholipid and total [3H]cholesterol taken up by hepatocytes and non-parenchymal cells remained constant with time. The uptake of [14C]oleoyl lipids by all three cell types was slightly greater than that of the total [3H]cholesterol and [3H]phospholipid components. There was evidence of cholesterol-ester hydrolysis and turnover of [14C]oleate in the phospholipid fraction in hepatocytes and Kupffer cells, but not endothelial cells, over the first 2 h. With both remnant preparations, these observations indicate that significant differences exist between the three major liver cell types with respect to the uptake and metabolism of remnant lipid components.
Antibody-independent interaction between the first component of human complement, C1, and the outer membrane of Escherichia coli D31 m4
Authors: Aubert B , Chesne S , Arlaud G J , Colomb M G
Published: August 2015

The heptoseless mutant of Escherichia coli, E. coli D31 m4, binds C1q and C1 at 0 degrees C and at low ionic strength (I0.07). Under these conditions, the maximum C1q binding averages 3.0 × 10(5) molecules per bacterium, with a Ka of 1.4 × 10(8) M-1. Binding involves the collagen-like region of C1q, as shown by the capacity of C1q pepsin-digest fragments to bind to E. coli D31 m4, and to compete with native C1q. Proenzyme and activated forms of C1 subcomponents C1r and C1s and their Ca2+-dependent association (C1r-C1s)2 do not bind to E. coli D31 m4. In contrast, the C1 complex binds very effectively, with an average fixation of 3.5 × 10(5) molecules per bacterium, and a Ka of 0.25 × 10(8) M-1, both comparable with the values obtained for C1q binding. C1 bound to E. coli D31 m4 undergoes rapid activation at 0 degrees C. The activation process is not affected by C1-inhibitor, and only slightly inhibited by p-nitrophenyl p'-guanidinobenzoate. No turnover of the (C1r-C1s)2 subunit is observed. Once activated, C1 is only partially dissociated by C1-inhibitor. Our observations are in favour of a strong association between C1 and the outer membrane of E. coli D31 m4, involving mainly the collagen-like moiety of C1.
Isolation of pig thyroid lysosomes. Biochemical and morphological characterization
Authors: Alquier C , Guenin P , Munari-Silem Y , Audebet C , Rousset B
Published: August 2015

Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, β-galactosidase and β-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic-pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells.
Physicochemical properties of a novel Mr-21 000 Ca2+-binding protein of bovine brain
Authors: McDonald J R , Walsh M P , McCubbin W D , Oikawa K , Kay C M
Published: August 2015

The physicochemical properties of a novel Mr-21 000 Ca2+-binding protein isolated from bovine brain were investigated. The protein exhibited a partial specific volume of 0.724 ml/g, a degree of hydration of 0.47 g of water/g of protein and a mean residue weight of 119. Sedimentation equilibrium analysis revealed Mr = 22 600 in the absence of Ca2+; Ca2+ binding appeared to induce dimerization of the molecule. Size-exclusion chromatography indicated a compacting of the molecule on binding of Ca2+: the Stokes radius decreased from 2.75 nm in the absence of Ca2+ to 2.56 nm in its presence. Far-u.v.c.d. spectroscopy showed the apoprotein to be composed of 44% α-helix, 18% β-pleated sheet and 38% random coil. Addition of either KCl (0.1 M) plus Mg2+ (1 mM), or Ca2+ (2 mM), changed the conformation to 49% α-helix, 18% β-pleated sheet and 33% random coil. Near-u.v.c.d. and u.v. difference spectroscopy both indicated perturbations in the environments of all three types of aromatic amino acids on binding of Ca2+. Ca2+ binding also resulted in a 30% enhancement in the tryptophan fluorescence emission intensity. Ca2+ titration of the far-u.v.c.d. and fluorescence enhancement provided KD values of 9.91 microM and 4.68 microM respectively. Finally, the protein was shown to bind Zn2+ with KD = 1.44 microM (no Mg2+) and 1.82 microM (+ Mg2+). These observations strongly support the possibility that this novel Ca2+-binding protein resembles calmodulin and related Ca2+-binding proteins and undergoes a conformational change on binding of Ca2+ which reflects a physiological role in Ca2+-mediated regulation of brain function.
13C-n.m.r. study of C hordein
Authors: Tatham A S , Shewry P R , Belton P S
Published: August 2015

The structure of C hordein was studied by a combination of solution and solid-state 13C-n.m.r. spectroscopy. The repetitive primary structure results in simple solution-state n.m.r. spectra, which allow assignment of the majority of the resonances to five major residues. The major resonances, with the exception of the aromatic signals, are also present in the solid-state spectra. The proline residues are in the trans configuration, consistent with an earlier study suggesting a β-turn-rich structure.
Comparison of Ca2+-dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain
Authors: Norling L L , Landt M
Published: August 2015

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.
Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP
Authors: Aiyar V N , Hershfield M S
Published: August 2015

S-Adenosylhomocysteine hydrolase (AdoHcyase) has previously been identified as a cytoplasmic adenosine and cyclic AMP binding protein. In order to examine the relationship between the adenosine and cyclic AMP binding sites on this enzyme we have explored the use of 8-azido analogues of adenosine and cyclic AMP as photoaffinity reagents for covalently labelling AdoHcyase purified from human placenta. 8-Azidoadenosine (8-N3-Ado), like adenosine, inactivated AdoHcyase, and the rate of inactivation was greatly increased by periodate oxidation. In addition, 8-N3-Ado was found to participate in the first step in the catalytic mechanism for AdoHcyase, resulting in conversion of enzyme-bound NAD+ to NADH, although it was not a substrate for the full enzyme-catalysed reaction. Radioactively labelled 8-N3-Ado, its periodate-oxidized derivative and 8-azidoadenosine 3′, 5′-phosphate (8-N3-cAMP) bound specifically to adenosine binding sites on AdoHcyase and, after irradiation, became covalently linked to the enzyme. Photoaffinity-labelled enzyme could be precipitated by monoclonal antibody to human AdoHcyase. Two observations suggested that cyclic AMP and adenosine bind to the same sites on AdoHcyase. First cyclic AMP and adenosine each blocked binding of both radioactively labelled 8-N3-Ado and 8-N3-cAMP, and second, digestion with V8 proteinase generated identical patterns of peptides from AdoHcyase that had been photolabelled with [32P]8-N3-cAMP and [3H]8-N3-Ado. Binding sites for cyclic AMP on AdoHcyase were found to differ functionally and structurally from cyclic AMP binding sites on the R1 regulatory subunit of cyclic AMP-dependent protein kinase.
Nucleoside transport in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. Differences in sensitivity to nitrobenzylthioinosine and thiol reagents
Authors: Belt J A , Noel L D
Published: August 2015

The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p-chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS.
Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Relation to efficacy of agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization
Authors: Evans T , Hepler J R , Masters S B , Brown J H , Harden T K
Published: August 2015

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were ‘full’ agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were ‘partial’ agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells.
Structural differences between plasma-membrane 5′-nucleotidase in different cell types as evidenced by antibodies
Authors: Harb J , Meflah K , Bernard S
Published: August 2015

Antibodies raised against bovine 5′-nucleotidase inhibit this enzyme as well as 5′-nucleotidase from other bovine tissues, showing common structure(s) between these proteins. However, an IgG fraction directed against the glucidic moiety of the liver enzyme did not cross-react with the enzyme from lymphocyte or caudate nuclei, a clear indication that within the same species the 5′-nucleotidase differs from one cell type to another. In addition, immunoblots after electrophoresis show that the previous antibodies recognize 5′-nucleotidase from human, mouse or chicken origin. However, only human 5′-nucleotidase activity can be inhibited by the antibodies. Thus at least three groups of antigenic determinants must exist on the 5′-nucleotidase: one related to the glucidic moiety of the glycoprotein whose binding inhibits the enzyme activity, another related to the catalytic site, as its binding also led to enzyme inhibition, and a last one of structural nature. It seems that the third group of determinant is common to many species, whereas the second one is more restricted.
The inhibition of parathyroid-hormone actions on gluconeogenesis and phosphate transport by 3-mercaptopicolinate in rabbit renal proximal tubules
Authors: Yanagawa N , Jo O D
Published: August 2015

By using a glucose microassay and the technique for isolated renal-tubule perfusion in vitro, the addition of 3-mercaptopicolinate, a gluconeogenesis inhibitor which inhibits phosphoenolpyruvate carboxykinase specifically, was found to abolish the effects of parathyroid hormone on gluconeogenesis and phosphate-transport rate in isolated rabbit renal proximal straight tubules, suggesting that these parathyroid-hormone actions may share some unknown, yet 3-mercaptopicolinate-inhibitable, intracellular processes.
Suppression of high-affinity ligand binding to the major glutathione S-transferase from Galleria mellonella by physiological concentrations of glutathione
Authors: Clark A G , Carrol N
Published: August 2015

The major glutathione S-transferase from larvae of Galleria mellonella binds a number of synthetic triphenylmethane dyes with dissociation constants of the order of 10(-6) M or less. The organ distribution of the enzyme activity does not parallel the uptake of such dyes by the insect's organs in vivo. The affinity of the protein for such dyes is decreased by about an order of magnitude by the presence of glutathione in normal physiological concentration. This appears to be the cause of this protein's lack of efficacy as a ‘ligandin’ in vivo. The dyes appear to be acting as ineffective substrate analogues, binding at the catalytic site and impeding, in a reciprocal fashion, the binding of glutathione. Fluorescence-quenching titration and kinetic experiments together indicate the existence of a single ligand-binding and catalytic site per dimeric enzyme molecule.
Activation of rat liver adenylate cyclase by guanosine 5′-[β,γ-imido]triphosphate and glucagon. Existence of reversibly and irreversibly activated states of the stimulatory GTP-binding protein
Authors: Wong S K F , Martin B R
Published: August 2015

The effects of guanosine 5′-[beta-thio]diphosphate (GDP[S]) on the kinetics of activation of rat liver membrane adenylate cyclase by guanosine 5′-[beta, gamma-imido]triphosphate (p[NH]ppG) were examined. GDP[S] caused immediate inhibition of the activation by p[NH]ppG at all time points tested. Substantial inhibition by GDP[S] was observed even after the time required for the enzyme to reach its steady-state activity, but the extent of inhibition became progressively smaller as the preincubation time with p[NH]ppG increased. The rate at which adenylate cyclase became quasi-irreversibly activated was a strictly first-order process. In the presence of glucagon, the formation of the irreversibly activated state was much slower. A combination of GDP[S] and glucagon could partially reverse the quasi-irreversible activation by p[NH]ppG. Glucagon decreased the lag time required for p[NH]ppG to activate adenylate cyclase and increased the extent of activation by p[NH]ppG. This stimulatory effect of the hormone on top of guanine nucleotide decreased on preincubation with p[NH]ppG, but not with GTP. Our results suggest that the activation of adenylate cyclase by non-hydrolysable GTP analogues is a two-stage process: the formation of a reversibly activated form (G rev) is a rapid process, followed by a much slower formation of the quasi-irreversibly activated form (G irr). Glucagon can stimulate G rev but not G irr, and can partially facilitate the formation of the G rev from the G irr state.
Lipolysis in rat adipocytes during recovery from lactation. Response to noradrenaline and adenosine
Authors: Vernon R G , Finley E
Published: August 2015

The replenishment of lipid reserves of adipocytes following the removal of litters from lactating rats is associated with a 3-fold decrease in both the response of lipolysis to noradrenaline and the maximum rate of lipolysis (measured in the presence of noradrenaline plus adenosine deaminase); these adaptations do not appear to result from the changes in serum prolactin and insulin concentrations that occur on litter removal.
Modification of ovine opsin with the photosensitive hydrophobic probe 1-azido-4-[125I]iodobenzene. Labelling of the chromophore-attachment domain
Authors: Davison M D , Findlay J B C
Published: August 2015

The hydrophobic photosensitive probe 1-azido-4-[125I]iodobenzene (AIB) partitioned preferentially into photoreceptor disc membranes and, upon u.v. irradiation, became covalently bound to opsin and phospholipid. The labelling of both protein and phospholipid was linearly related to AIB concentration. The amount of probe incorporated into protein was not significantly different when membranes were irradiated at −100 degrees, 4 degrees or 25 degrees C, but irreversible aggregation of monomeric opsin was dramatically reduced by performing the photolysis at −100 degrees C. Labelling of opsin after irradiation at −100 degrees or 4 degrees was not significantly reduced by the presence of lysine in the aqueous buffer, indicating that significant amounts of reactive species did not enter the aqueous phase. The incorporation into phospholipid, unlike that into opsin, decreased as the temperature of irradiation increased. Some labelling of opsin occurred on incubation with pre-photoactivated AIB, indicating that reaction may also occur with reactive species of longer lifetimes than the nitrene. Proteolysis of labelled opsin with Staphylococcus aureus V8 proteinase yielded two radiolabelled membrane-bound fragments. The location of the modified sites (cysteine, tryptophan, tyrosine, lysine and histidine residues: all nucleophiles) in the smaller fragment was entirely consistent with putative models for the protein derived from other studies.
Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the β-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain
Authors: Yoshioka N , Atassi M Z
Published: August 2015

A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146.
Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the β-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle
Authors: Horn R S , Lystad E , Adler A , Walaas O
Published: August 2015

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5′-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5′-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5′-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.
Renal hypertrophy in experimental diabetes. Effect of diabetes on the pathways of glucose metabolism: differential response in adult and immature rats
Authors: Sochor M , Kunjara S , Greenbaum A L , McLean P
Published: August 2015

The effect of short-term diabetes, 5 days after the administration of streptozotocin, on renal growth and the activity of alternative pathways of glucose metabolism was studied in immature (21-day-old) rats and in adult rats. The kidney weight increased by 28% in the adult diabetic rats, but by only 10% in the immature diabetic rats, relative to their age-matched control groups. The flux of glucose via the pentose phosphate pathway was increased 2-3-fold in the adult diabetic rats, but was unchanged in the immature diabetic group. Enzymes of this pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) increased by 29% and 77% respectively in adult diabetic rats; in the immature group they showed changes of +5% and +28% respectively. The rate of glucose phosphorylation increased significantly in both groups of diabetic rats; only minor changes were observed in oxidation via the tricarboxylic acid cycle. Increases of 40-50% were found in the activity of enzymes involved in UDP-glucose metabolism (phosphoglucomutase, UDPglucose pyrophosphorylase and UDPglucose dehydrogenase) and in lactate dehydrogenase in both young and adult animals. The results suggest a differential renal response to streptozotocin-diabetes according to the stage of renal growth and development, and it is proposed that the difference is related to the developmental emergence of aldose reductase. Enzymes involved in formation of ribose 5-phosphate and NADPH are strikingly increased in the adult diabetic, whereas metabolic functions dependent on a high ambient glucose concentration, e.g. synthesis of glycogen and glucuronate, are similarly affected in adult and immature diabetic groups, both showing certain aspects of ‘glucose overutilization’.
The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes
Authors: Fotheringham I G , Dacey S A , Taylor P P , Smith T J , Hunter M G , Finlay M E , Primrose S B , Parker D M , Edwards R M
Published: August 2015

In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.
Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni
Authors: Groen B W , van Kleef M A G , Duine J A
Published: August 2015

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name ‘quinohaemoprotein alcohol dehydrogenase’. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.
High-performance liquid chromatography of uroporphyrinogen and coproporphyrinogen isomers with amperometric detection
Authors: Lim C K , Li F , Peters T J
Published: August 2015

A reversed-phase h.p.l.c. system, with an ODS-Hypersil column with acetonitrile or methanol in ammonium acetate buffer as mobile phase, is described for the separation of uro-and copro-porphyrinogen isomers. The porphyrinogens are detected amperometrically with sensitivity comparable with that of the fluorescent detection of porphyrins. The effects of pH, buffer concentration and organic modifiers on retention and resolution were studied. The method is suitable for both analytical and preparative separation of porphyrinogens.
Effect of starvation or treatment with corticosterone on the amount of easily releasable myofilaments in rat skeletal muscles
Authors: Dahlmann B , Rutschmann M , Reinauer H
Published: August 2015

Treatment of isolated myofibrils with an ATP-containing relaxing solution results in the dissociation of a preformed quantity of myofilaments called ‘easily releasable myofilaments’. Van der Westhuyzen, Matsumoto & Etlinger [(1981) J. Biol. Chem. 256, 11791-11797] presented experimental evidence that these myofilaments represent intermediate products in the turnover of myofibrillar proteins. To investigate further this question, we measured the size of the fraction of easily releasable myofilaments in three different species of skeletal muscles from rats subjected to well-defined catabolic conditions, namely starvation or chronic glucocorticoid administration. The results were as follows: (1) The amount of easily releasable myofilaments was transiently increased about 2-3-fold during both experiments, and thus paralleled the known alterations in the rate of overall muscle protein breakdown rather than in those of synthesis. (2) These changes were observed in muscles containing predominantly fast-twitch fibres, but not in slow-twitch soleus muscle, a muscle that is known to be more resistant to catabolic conditions. (3) The starvation-induced increase of the size of the fraction of easily releasable myofilaments could be significantly reduced by treatment of the starving animals with the proteinase inhibitor E-64. These results are compatible with the idea that easily releasable myofilaments are intermediates in the degradative pathway of myofibrillar proteins and that a proteolytic step may be involved in the conversion of myofilaments into easily releasable myofilaments.
The chemical mechanism of sheep liver 6-phosphogluconate dehydrogenase. A Schiff-base intermediate is not involved
Authors: Topham C M , Dalziel K
Published: August 2015

[2-18O]Ribulose 5-phosphate was prepared and shown to be converted enzymically by 6-phosphogluconate dehydrogenase from sheep liver into 6-phosphogluconate with complete retention of the heavy isotope. This finding unequivocally excludes the possibility of a Schiff-base mechanism for the enzyme. The involvement of metal ions has already been excluded, and other possible mechanisms are discussed. The enzyme was purified by an improved large-scale procedure, which is briefly described.
Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.)
Authors: Qu R , Wang S M , Lin Y H , Vance V B , Huang A H
Published: August 2015

Storage lipid bodies, which are prominent organelles present in the storage tissues of most seeds, have not been subjected to intensive biochemical investigation. In the present studies the major proteins in lipid bodies isolated from eleven taxonomically diverse species were shown to be distinctly different, as revealed by SDS/polyacrylamide-gel electrophoresis. The lipid-body membrane of maize (Zea mays L.) contained three major proteins of low Mr (19,500, 18,000 and 16,500), and they were chosen for further study. They all had alkaline pI values and behaved as hydrophobic integral proteins, as shown by their resistance to solubilization after repeated washing, amino acid composition and partitioning in a Triton X-114 system. Labelling in vivo with [35S]methionine and translation in vitro using extracted RNA in a wheat-germ system showed that the proteins were synthesized during seed maturation and not germination. The proteins synthesized in vivo and in vitro exhibited no appreciable difference in their mobilities in two-dimensional gel electrophoresis (isoelectric focusing and molecular sieving). The most abundant protein, that of Mr 16,500, was shown to be synthesized predominantly, if not exclusively, by RNA derived from bound polyribosomes and not from free polyribosomes. The implication of the results on the biosynthesis of the lipid bodies is discussed.
Neural regulation of parvalbumin expression in mammalian skeletal muscle
Authors: Leberer E , Pette D
Published: August 2015

Parvalbumin was purified from rabbit fast skeletal muscle and used to raise antibodies in sheep. Subsequently, a sensitive ‘sandwich’ enzyme-linked immunoadsorbent assay permitted quantification of parvalbumin in homogenates of embryonic, maturing, innervated, denervated and chronically stimulated skeletal muscles of the rabbit. High concentrations of parvalbumin were detected in various adult fast-twitch muscles of the rabbit (700-1200 micrograms/g of muscle), whereas slow-twitch muscles contained negligible concentrations (3-5 micrograms/g of muscle). Parvalbumin was not detectable in embryonic-rabbit muscles (21, 25, 28 days of gestation), either presumptive fast- or slow-twitch. However, parvalbumin concentrations did increase during postnatal development in presumptive fast-twitch muscles. Thus the onset of parvalbumin synthesis appears to be correlated with the neonatal-to-adult transition of motor-neuron activity [Navarrete & Vrbová (1983) Dev. Brain Res. 8, 11-19]. The increase of parvalbumin in maturing, presumptive fast-twitch muscle was suppressed by denervation. In the adult rabbit, denervation of the tibialis anterior muscle caused a reduction of parvalbumin to a level normally found in slow-twitch muscles. In contrast, the already low levels of parvalbumin in maturing and adult slow-twitch soleus muscle were unaffected by denervation. Chronic low-frequency stimulation of adult fast-twitch muscle resulted in a rapid reduction of parvalbumin to a level normally found in slow-twitch muscle. These data support the hypothesis that the expression of parvalbumin is under positive control of fast-type motor-neuron activity.
Purification of the constituent enzyme fractions of mycobacillin synthetase
Authors: Ghosh S K , Mukhopadhyay N K , Majumder S , Bose S K
Published: August 2015

The final purification of the three-fraction enzyme complex mycobacillin synthetase was done by hydroxyapatite column chromatography and sucrose-density-gradient centrifugation; each of the fractions obtained migrates as a single component in SDS/polyacrylamide-gel electrophoresis and gel electrofocusing. The Mr of the enzyme fractions A, B and C by gel filtration is 260 000, 190 000 and 105 000, and that by SDS/polyacrylamide-gel electrophoresis is 252 000, 198 000 and 108 000 respectively. None of the enzyme fractions appears to possess subunit structure.
Regulation of protein phosphorylation in pancreatic acini. Distinct effects of Ca2+ ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate
Authors: Burnham D B , Munowitz P , Hootman S R , Williams J A
Published: August 2015

Regulation of protein phosphorylation in isolated pancreatic acini by the intracellular messengers Ca2+ and diacylglycerol was studied by using the Ca2+ ionophore A23187 and the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate. As assessed by two-dimensional polyacrylamide-gel electrophoresis, the phorbol ester (1 microM) and Ca2+ ionophore (2 microM) altered the phosphorylation of distinct sets of proteins between Mr 83,000 and 23,000 in mouse and guinea-pig acini. The phorbol ester increased the phosphorylation of four proteins, whereas the ionophore increased the phosphorylation of two proteins and, in mouse acini, decreased the phosphorylation of one other protein. In addition, the phorbol ester and ionophore each caused the dephosphorylation of two proteins, of Mr 20,000 and 20,500. Administered together, these agents reproduced the changes in phosphorylation induced by the cholinergic agonist carbamoylcholine. The effects of the phorbol ester and ionophore on acinar amylase release were also studied. In mouse pancreatic acini, a maximally effective concentration of phorbol ester (1 microM) produced a secretory response that was only 28% of that produced by a maximally effective concentration of carbamoylcholine, whereas the ionophore (0.3 microM) stimulated amylase release to two-thirds of the maximal response to carbamoylcholine. In contrast, in guinea-pig acini, the phorbol ester and carbamoylcholine evoked similar maximal secretory responses, whereas the maximal secretory response to the ionophore was only 35% of that to carbamoylcholine. Combination of phorbol ester and ionophore resulted in a modest synergistic effect on amylase release in both species. It is concluded that cholinergic agonists act via both diacylglycerol and Ca2+ to regulate pancreatic protein phosphorylation, but that synergism between these intracellular messengers is of limited importance in stimulating enzyme secretion.
Caerulein and carbamoylcholine stimulate pancreatic amylase release at resting cytosolic free Ca2+
Authors: Bruzzone R , Pozzan T , Wollheim C B
Published: August 2015

Cytosolic free calcium concentrations ([Ca2+]i) and amylase secretion were measured in isolated rat pancreatic acini loaded with the intracellularly trapped fluorescent indicator quin2. Both caerulein and carbamoylcholine caused a rapid increase in [Ca2+]i, with a maximal 3-fold increase at 10(-9) M-caerulein and 10(-4) M-carbamoylcholine. However, caerulein (10(-12) M and 10(-11) M) as well as carbamoylcholine (10(-7) M) caused a significant stimulation of amylase release, while not inducing any detectable rise in [Ca2+]i. Changes in [Ca2+]i after addition of either secretagogue were transient and did not last more than 2-3 min. By contrast, when amylase secretion was monitored as a function of time, two distinct secretory phases could be observed upon addition of either carbamoylcholine (10(-5) M) or caerulein (10(-10) M). An initial, rapid phase (0-5 min) which caused a 6-7-fold increase above basal, followed by a sustained (5-30 min), but less marked, secretory rate (2-3-fold above basal). Addition of atropine (10(-4) M) 5 min after carbamoylcholine (10(-5) M) (i.e. after termination of the rise in [Ca2+]i and of the first secretory phase) did not cause any significant change in [Ca2+]i, while significantly inhibiting amylase secretion from 5 to 30 min to the same rate observed in the absence of the secretagogue. These results show that caerulein and carbamoylcholine, two agents thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, are capable of eliciting amylase secretion independently of a concomitant rise in [Ca2+]i. Furthermore, with both secretagogues the rise in [Ca2+]i, when observed, was only transient, while the stimulation of amylase release was sustained.
Alterations by peroxisome proliferators of acyl composition of hepatic phosphatidylcholine in rats, mice and guinea-pigs. Role of stearoyl-CoA desaturase
Authors: Kawashima Y , Hirose A , Kozuka H
Published: August 2015

Rats, mice and guinea-pigs were administered p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2′-(decamethylenedithio)diethanol (tiadenol). The treatments of rats and mice with either clofibric acid or tiadenol increased markedly the activities of stearoyl-CoA desaturase, palmitoyl-CoA chain elongation, 1-acylglycerophosphate (1-acyl-GP) acyltransferase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, but not 2-acylglycerophosphocholine (2-acyl-GPC) acyltransferase in liver microsomes. The treatment of guinea-pigs with clofibric acid did not cause any change in the activities of these enzymes. The treatment of guinea-pigs with tiadenol caused a slight, but significant, increase in the activities of 1-acyl-GP acyltransferase and 1-acyl-GPC acyltransferase. The treatment of rats and mice with either clofibric acid or tiadenol increased markedly the proportion of 18:1 and decreased greatly the proportion of 18:0 in liver microsomal phosphatidylcholine. However, there is a considerable difference in the effects of the two peroxisome proliferators on the composition of polyunsaturated fatty acids in phosphatidylcholine between rats and mice. The treatment of guinea-pigs with either of the two peroxisome proliferators caused no change in acyl composition of phosphatidylcholine. The possible role of stearoyl-CoA desaturation in the regulation of acyl composition of phosphatidylcholine was discussed.
The fate of 14C derived from radioactively labelled dietary precursors in young rats of the Zucker strain (Fa/- and fa/fa)
Authors: Haggarty P , Reeds P J , Fletcher J M , Wahle K W J
Published: August 2015

The metabolic fate of 14C derived from radioactively labelled dietary precursors was determined in immature (18- and 25-day-old) lean and obese Zucker rats. This included measurement of 14C incorporated into body lipid, non-essential amino acids and expired CO2. Before weaning (18 days) there was no phenotypic difference between the fates of [14C]palmitate and [14C]-glucose. However, after weaning (25 days) all the precursors studied exhibited an increase in the fraction incorporated into lipid in the obese rat as compared with the lean animal. This was reflected in the fate of acetyl-CoA in the tricarboxylic acid cycle. There was little phenotypic difference in the fraction of leucine or valine catabolized. The results presented here suggest that the high rate of lipogenesis found in the obese rat is supported by carbon from all the dietary precursors studied. It is also argued that the decreased protein deposition found in the obese rat is not caused by the high rate of lipogenesis removing precursors for protein synthesis, as has been suggested elsewhere [Cleary, Vasselli & Greenwood (1980) Am. J. Physiol. 238, E284-E292].
The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats
Authors: Jepson M M , Pell J M , Bates P C , Millward D J
Published: August 2015

The response of muscle and liver protein metabolism to either a single or three successive daily injections of an endotoxin (Escherichia coli lipopolysaccharide, serotype 0127 B8; 1 mg/ml, 0.3 mg/100 g body wt.) was studied in vivo in the fed rat, and at 24 and 30 h after endotoxin treatment during fasting. In the fed rats there was a catabolic response in muscle, owing to a 60-100% increase in muscle protein degradation rate, and a 52% fall in the synthesis rate. Although there was a 20% decrease in food intake, the decrease in protein synthesis was to some extent independent of this, since rats treated with endotoxin and fasted also showed a lower rate of muscle protein synthesis, which was in excess of the decrease caused by fasting alone. The mechanism of this decreased protein synthesis involved decreased translational activity, since in both fed and fasted rats there was a decreased rate of synthesis per unit of RNA. This occurred despite the fact that insulin concentrations were either maintained or increased, in the fasted rats, to those observed in fed rats. In the liver total protein mass was increased in the fed rats by 16% at 24 h, and the fractional synthesis rate at that time was increased by 35%. In rats fasted after endotoxin treatment the liver protein mass was not decreased as it was in the control fasted rats, and the fractional synthesis rate was increased by 22%. In both cases the increased synthesis rate reflected an elevated hepatic RNA concentration. The extent of this increase in hepatic protein synthesis was sufficient at one point to compensate for the fall in estimated muscle protein synthesis, so that the sum total in the two tissues was maintained.