bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficiency of 5′-deoxy-5′-methylthioadenosine phosphorylase activity in malignancy. Absence of the protein in human enzyme-deficient cell lines
Authors: Della Ragione F , Oliva A , Palumbo R , Russo G L , Gragnaniello V , Zappia V
Published: August 2015

The absence of 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAase) activity in malignant cells, and the putative localization of its gene, suggest that this enzyme deficiency might be due to a genomic alteration also involving a tumour-suppressor gene. We studied the possible occurrence of inactive forms of the protein in two MTAase-negative cell lines, namely K562 and Jurkat, by immunochemical methods. Two highly specific antisera, directed against different epitopes of the phosphorylase [Della Ragione, Oliva, Gragnaniello, Russo, Palumbo & Zappia (1990) J. Biol. Chem. 265, 6241-6246], were used to carry out immunotitration and immunoblotting analyses, as well as to investigate the biosynthesis of the enzyme. No MTAase protein was detected by Western-blotting technique performed under conditions where all the phosphorylase-positive samples gave a clear band at the MTAase subunit molecular mass. No cross-reacting material was observed by a sensitive immunotitration method which permitted the detection of as low as 0.5 ng of protein. Moreover, the results obtained by [35S]methionine-labelling experiments ruled out phosphorylase biosynthesis in the negative cell lines. Altogether, these data suggest that an alteration at the gene level hampering the specific mRNA biosynthesis or resulting in an untranslatable mRNA is the cause of the enzyme deficiency in the MTAase-negative cell lines studied.
Generation of forms of fragment E with differing thrombin-binding properties during digestion of fibrinogen by plasmin
Authors: Goodwin C A , Kakkar V V , Scully M F
Published: August 2015

Two principal forms of fragment E are generated upon digestion of fibrinogen by plasmin, according to the concentration of enzyme used. At a high concentration of plasmin (above 10 micrograms/ml), a form lacking fibrinopeptide A (FpA) at the N-terminus of the A alpha-chain was generated. This form of fragment E caused a dose-dependent increase in thrombin clotting times but had no measurable inhibitory activity towards thrombin cleavage of D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide. At a low concentration of plasmin (less than 1 microgram/ml), fragment E containing 35-40% of the original amount of FpA was present in the terminal digest. The FpA-containing form of fragment E inhibited thrombin cleavage of fibrinogen, inhibited amidolytic activity and bound to the enzyme with an affinity 3-fold tighter than fibrinogen itself (Kd 4.1 +/- 0.3 microM as opposed to 12.7 +/- 1.8 microM). During digestion of fibrinogen at low plasmin concentration, up to 65% of the FpA was cleaved just subsequent to the progressive release of B beta-(1-42)-peptide, and the Arg-16-Gly-17 bond of the A alpha-chain became relatively stable towards plasmin action when present in fragment E (and possibly fragment Y). It is proposed that both forms of fragment E can inhibit clotting by binding to the fibrin(ogen)-recognition site (anion-binding exosite) of thrombin. The FpA-containing form of fragment E can also inhibit binding that occurs distal to the P1 site and thereby interfere with amidolysis of the peptide substrate. Our finding of a lability of the Arg-16-Gly-17 bond in the early phase of digestion may provide an alternative explanation of the increased FpA concentrations observed during thrombolytic therapy.
Characterization of a new kallikrein-like enzyme (KLP-S3) of the rat submandibular gland
Authors: Berg T , Schøyen H , Wassdal I , Hull R , Gerskowitch V P , Toft K
Published: August 2015

The submandibular gland of the rat contains several enzymes belonging to the kallikrein family. These include tissue kallikrein, antigen gamma (T-kininogenase), esterase B and tonin. In the present study, a new member of this family, which we have named KLP-S3, was identified and purified from the submandibular gland. KLP-S3 was classified as a kallikrein-like enzyme on the basis of its immunological similarity to other kallikrein-like enzymes and its showing 70% and 73% identity in partial amino acid sequence with tissue kallikrein and tonin respectively. Furthermore, the 44 sequenced amino acid residues showed complete correspondence to the mRNA S3 of the kallikrein gene family, which was the rationale for the name kallikrein-like protein (KLP) S3. KLP-S3 consisted of three isoenzymes with pI 6.75, 6.90 and 6.95, which significantly differed from those of other kallikrein-like enzymes. In conjunction with its immunological relationship to kallikrein, this parameter (pI) was considered robust enough to identify the enzyme during purification, since a specific physiological substrate for KLP-S3 has yet to be identified. In SDS/PAGE the three isoenzymes ran as one band with a molecular mass of 25,800 Da, which after reduction with 2-mercaptoethanol was split into two chains with molecular masses of 16,500 and 13,300 Da. In common with other kallikrein-like enzymes, KLP-S3 was inhibited by phenylmethanesulphonyl fluoride, and was thus classified as a serine protease. It was also inhibited by soya-bean trypsin inhibitor but not by aprotinin. It showed weak reactivity against the chromogenic substrates S2288, S2266, S2366 and S2302 (D-Ile-Pro-Arg 4-nitroanilide, D-Val-Leu-Arg 4-nitroanilide, Glu-Pro-Arg 4-nitroanilide and D-Pro-Phe-Arg 4-nitroanilide respectively) and did not cleave rat T-kininogen or dog high-molecular-mass/low-molecular-mass kininogen. Its specific angiotensin II-generating activity (angiotensin I as substrate) was 0.04% of that of rat tonin. KLP-S3 (1-100 nM) induced a statistically significant angiotensin-independent contraction of isolated rat aorta rings. The maximum contraction was 15% of the response to the alpha-adrenoceptor agonist phenylephrine (1 microM). The concentration of KLP-S3 in the rat submandibular gland was by single radial immunodiffusion estimated to be 47 +/- 3 micrograms/mg of protein.
Characterization and cellular distribution of acidic peptide and oligosaccharide metal-binding compounds from kidneys
Authors: Predki P F , Whitfield D M , Sarkar B
Published: August 2015

Two low-molecular-mass Ni-binding fractions first isolated from human kidneys [Templeton & Sarkar (1985) Biochem. J. 230, 35-42] are further characterized. Both components are acidic and are readily separated from each other by gel chromatography on Bio-Gel P-2. After equilibration with 63Ni the largest complex constitutes about 30% of the radioactive 63Ni and is an approx. 3.5 kDa peptide and the smallest species comprise short oligosaccharides containing 70% of the radioactivity. Both of these components are found in human, bovine and porcine kidneys as well as in a porcine proximal tubule-like cell line LLC-PK1. There is a small variation in amino acid composition between species. The oligosaccharides are reducing sugars and contain sulphate, glucosamine, glucuronic acid and iduronic acid with two to four overall negative charges. The monosaccharide composition was determined by h.p.l.c. with pulsed amperometric detection of the acid hydrolysates and by gas chromatography. In the LLC-PK1 cell line the acidic peptide is both intracellular and extracellular, whereas the oligosaccharides are only intracellular. The concentration of extracellular peptide, as measured by 63Ni binding, is found to increase after exposure of the cells to low micromolar concentrations of Ni, whereas the oligosaccharide concentrations, also measured by 63Ni binding, remain constant. The oligosaccharide component is decreased by 40% in the presence of NH4Cl, suggesting that is derived from degradation of internalized heparan sulphate.
Modulation by betaine of cellular responses to osmotic stress
Authors: Petronini P G , De Angelis E M , Borghetti P , Borghetti A F , Wheeler K P
Published: August 2015

Various solutes were tested to see if they could modify the responses of SV-3T3 cells to hyperosmotic (0.5 osM) conditions, which cause an inhibition of general cell protein synthesis and of the rate of cell proliferation, coupled with an induction of amino acid transport activity. The added solutes were glycerol, proline, taurine, betaine, dimethylglycine and sarcosine. Of these, betaine produced the most dramatic and consistent effects. Addition of 10-25 mM-betaine to the hyperosmotic medium largely prevented the 90% inhibition of cell proliferation that occurred in its absence. Whether it was added initially or after the cells were exposed to hyperosmotic medium, 25 mM-betaine also converted a 50% recovery of the rate of protein synthesis into 100%. Similarly, the same concentrations of betaine prevented a 30% decrease in cell volume and decreased the induction of amino acid transport via system A by 73%. Lower concentrations of betaine produced smaller but still significant changes in these functional responses. With chick-embryo fibroblasts, under identical hyperosmotic conditions, 25 mM-betaine completely counteracted a 75% inhibition of the rate of protein synthesis. At present it is not clear how betaine modulates these effects of hyperosmolarity on cell functions.
Modulation of 5′-nucleotidase activity in plasma membranes and intact cells by the extracellular matrix proteins laminin and fibronectin
Authors: Olmo N , Turnay J , Risse G , Deutzmann R , von der Mark K , Lizarbe M A
Published: August 2015

Modulation of 5′-nucleotidase activity by the extracellular matrix proteins fibronectin, laminin and their fragments has been studied in plasma membrane preparations as well as in intact BCS-TC2 and Rugli cells. The ectoenzyme on plasma membranes is activated by laminin; fibronectin inhibits the AMPase activity on BCS-TC2 plasma membranes but no inhibitory effect is found in plasma membrane preparations from Rugli cells. These effects are dependent on the preincubation time and protein concentration. When the effect of the extracellular matrix proteins is studied on intact cells, both BCS-TC2 and Rugli cells show similar behaviour. A decrease in the enzyme activity is observed in the presence of fibronectin. The AMPase inhibitory activity is located on its 40 kDa fragment. No inhibitory activity is found in other fibronectin fragments, including the 140 kDa fragment which contains the RGDS cell-adhesion sequence. Laminin and its E1-4 and E8 fragments are able to activate the ecto-5′-nucleotidase activity of both BCS-TC2 and Rugli cells. The effect of the E1-4 fragment on intact cells is greater than that observed for the E8 fragment and uncleaved laminin. Our results suggest a bifunctional role for 5′-nucleotidase as ectoenzyme and cell receptor for extracellular matrix proteins.
In defence of the general validity of the Cha method of deriving rate equations. The importance of explicit recognition of the thermodynamic box in enzyme kinetics
Authors: Topham C M , Brocklehurst K
Published: August 2015

1. The suggestion by Segel & Martin [(1988) J. Theor. Biol. 135, 445-453] that the well-known schematic method for the derivation of rate equations for combined quasi-equilibrium-steady-state models proposed by Cha [(1968) J. Biol. Chem. 243, 820-825] may not be generally applicable was shown to be incorrect. By contrast, Cha's method was shown (a) to yield correct initial-rate equations that are exact and (b) not to require any constraints on the relative magnitudes of rate constants for slow steps outside the quasi-equilibrium segments of the kinetic model, including those suggested by Segel & Martin. 2. Examination of residual King-Altman patterns for the general modifier model of Botts & Morales [(1953) Trans. Faraday Soc. 49, 696-707] revealed the reasons for the erroneous conclusions reached by Segel & Martin. The errors arise from the failure to take account of fluxes in parallel pathways that connect the two isolated groups of enzyme species existing in quasi-equilibrium with the modifier. 3. A similar failure to take explicit account of parallel pathways in a thermodynamic box that delayed proper appreciation of the form of pH-dependence of kcat/Km is briefly discussed.
Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues
Authors: Buck M R , Karustis D G , Day N A , Honn K V , Sloane B F
Published: August 2015

Our laboratory has previously demonstrated that increased malignancy of several histological types of human and animal tumours is associated with increases in their cathepsin B activity, particularly cathepsin B activity associated with plasma-membrane/endosomal vesicles or shed vesicles. Here we report that cathepsin B from normal or tumour tissues degrades purified extracellular-matrix components, type IV collagen, laminin and fibronectin, at both acid pH and neutral pH. The number and sizes of degradation products were analysed by SDS/PAGE. Cathepsin B from both sources exhibited similar activities towards, and similar patterns of cleavage of, the extracellular-matrix proteins. At neutral pH, cathepsin B from both sources appeared to undergo autodegradation, a process that was decreased in the presence of alternative substrates such as the extracellular-matrix proteins. Cathepsin B readily degraded type IV collagen at 25 degrees C, indicating activity towards native type IV collagen. Fibronectin degradation products of 100-200 kDa and of 18 and 22 kDa were observed. A single 70 kDa fragment was released from laminin under non-reducing conditions and multiple fragments ranging from 45 to 200 kDa under reducing conditions. These results suggest that cathepsin B at or near the surface of malignant tumour cells may play a functional role in the focal dissolution of extracellular matrices.
Tissue distribution of enzymic methylation of glutathione S-transferase and its effects on catalytic activity. Methylation of glutathione S-transferase 11-11 inhibits conjugating activity towards 1-chloro-2,4-dinitrobenzene
Authors: Johnson J A , Finn K A , Siegel F L
Published: August 2015

Glutathione S-transferases (GSTs) were isolated from rat liver, lung, heart, kidney, testis and brain by coupled affinity chromatography and subunits were resolved by reverse-phase h.p.l.c. The reverse-phase h.p.l.c. technique was improved from our previously published work [Johnson, Neal, Collins & Siegel (1990) Biochem. J. 270, 483-489] by changing from a C4 to a C18 wide-pore reverse-phase column; this resulted in baseline or near-baseline resolution of all GST subunits. There were significant tissue-dependent differences in the expression of GST subunits and the level of GST subunits present was quantitatively determined for each of the tissues. The extent of methylation of GSTs in vitro and distribution of GST methyltransferase (GST-MT) was determined in cytosolic fractions from each of these tissues. Purified GST isoenzymes were methylated with partially purified liver GST-MT. Methylation of Mu class subunits 3 and 4, the preferred substrates of methylation in liver, was substoichiometric in all tissues. The extent of methylation of subunit 3 ranged from 0.13% to 0.94% and subunit 4 from 0.03% to 0.60%. Methylation of Alpha class subunits was either not detectable or 5-10-fold less than that of Mu class subunits 3 and 4. Pi class subunit 7 was methylated to a greater extent than the Alpha class subunits but less than Mu class isoenzymes. A notable exception to this low level of methylation was GST 11-11, found mainly in testis and brain. Methylation of subunit 11 reached 21.9% (219 pmol of methyl group/nmol of subunit 11) when this isoenzyme was incubated with partially purified liver GST methyltransferase. Methylation of GST 11-11 was found to inhibit the conjugating activity of this isoenzyme towards 1-chloro-2,4-dinitrobenzene; the degree of inhibition of conjugating activity correlated with the extent of methylation of GST 11-11. GST-MT activity toward GST subunits 3, 4 and 11 was present in kidney and liver, detectable in lung and heart, but absent from brain and testis. Anion-exchange chromatography of GST-MTs from liver and kidney suggested the presence of four different forms of GST-MT (I-IV) and indicated that GST-MT isoenzymes III and IV were present at significantly lower concentrations in kidney than liver. The present paper shows that methylation is an enzyme-catalysed reaction that differs in substrate-specificity with respect to different GST isoenzymes, that expression of GST-MT is tissue-dependent and multiple forms of the enzyme are present in liver and kidney, and that methylation inhibits GST activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Iron-induced ascorbate oxidation in plasma as monitored by ascorbate free radical formation. No spin-trapping evidence for the hydroxyl radical in iron-overloaded plasma
Authors: Minetti M , Forte T , Soriani M , Quaresima V , Menditto A , Ferrari M
Published: August 2015

A study was made of the interaction of plasma ascorbate and ascorbate free radical (AFR) with exogenously added iron. The quantitative determination of AFR has the advantage that transient increases in ascorbate oxidation can be directly monitored by e.p.r. spectroscopy. An AFR signal was found in the plasma of all donors and was unaffected by superoxide dismutase, catalase and the strong iron chelator deferoxamine. These findings and the rapid decrease in AFR under a nitrogen atmosphere suggest that plasma AFR is probably a result of air auto-oxidation. Iron loading of plasma did not affect the intensity of the AFR signal until the iron concentration approached or exceeded the plasma latent iron-binding capacity. In iron-overloaded plasma, the intensity of the AFR signal increased to about 10 times the normal level before decreasing rapidly to undetectable levels after 15-20 min. Determination of plasma ascorbate showed that the disappearance of AFR was due to a complete loss of the vitamin. When 50 microM-ascorbate was loaded with iron in iso-osmotic phosphate buffer there was an increase in the AFR signal, independent of the iron concentration, which was stable at least for 15 min. Thus the rate of ascorbate loss in the iso-osmotic phosphate buffer was considerably lower than in iron-overloaded plasma. The addition of different iron chelators produced comparable effects on the intensity of the AFR signal in both iron-overloaded plasma and ascorbate solution. These results suggest that the characteristic behaviour of plasma AFR after iron loading is due to its specific iron-binding capacity and to plasma ferroxidase activity. The ferroxidase activity of plasma is important to promote the transfer of Fe2+ into transferrin without a transient ascorbate oxidation. Spin-trapping studies with 5,5-dimethyl-1-pyrroline N-oxide and N-t-butyl-alpha-phenylnitrone revealed that iron-overloaded plasma was unable to produce spin-trap adducts even in the presence of 50-300 microM-hydrogen peroxide or 100 microM-azide. Evidence of OH. radical formation was obtained only after the addition of EDTA. Therefore, iron-overloaded plasma itself does not produce a Fenton reaction and, if ascorbate does indeed have a free-radical-mediated pro-oxidant role, it is not detectable in plasma by spin-trapping experiments.
Effect of selenium deficiency on hepatic type I 5-iodothyronine deiodinase activity and hepatic thyroid hormone levels in the rat
Authors: Beckett G J , Russell A , Nicol F , Sahu P , Wolf C R , Arthur J R
Published: August 2015

Selenium deficiency in rats for a period of up to 6 weeks inhibited both the production of 3,3′, 5-tri-iodothyronine (T3) from thyroxine (T4) (5′-deiodination) and also the catabolism of T3 to 3,3′-di-iodothyronine (5-deiodination) in liver homogenates. The hepatic stores of T3 were decreased by only 8% in selenium deficiency, despite the T3 production rate from T4 being only 7% of the rate found in selenium-supplemented rats. Hepatic glutathione S-transferase (GST) activity was increased in both hypothyroidism and selenium deficiency, but apparently by different mechanisms, since mRNA expression for this family of enzymes was lowered by hypothyroidism and increased in selenium deficiency. It is concluded that, since both T3 production and catabolism are inhibited by selenium deficiency, there is little change in hepatic T3 stores, and therefore the changes in the activity of certain hepatic enzymes, such as GST, that are found in selenium deficiency are not the result of tissue hypothyroidism.
Proteolytic dissection of the isolated platelet fibrinogen receptor, integrin GPIIb/IIIa. Localization of GPIIb and GPIIIa sequences putatively involved in the subunit interface and in intrasubunit and intrachain contacts
Authors: Calvete J J , Mann K , Alvarez M V , López M M , González-Rodríguez J
Published: August 2015

Human platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the subunits of the Ca(2+)-dependent heterodimer GPIIb/IIIa, which belongs to the integrin family of phylogenetically related receptors mediating a wide variety of cell-cell and cell-substratum interactions. GPIIb/IIIa plays a central role in haemostasis as a receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. The covalent structure of the subunits is largely known; however, the tertiary and quaternary structures of the heterodimer remain to be determined. To this end, our approach consisted of limited proteolysis of the isolated heterodimer with proteinases of different specificities, followed by protein-chemical and immunochemical analyses of the peptide fragments within each isolated proteolytic product. From the information obtained, we have drawn a rudimentary map which outlines the demarcation of compact domains and the subunit peptide stretches carrying the sequences putatively involved in intrachain, intrasubunit and intersubunit non-covalent connectivity in the heterodimer. Three compact domains have been well defined: one in the heavy (H) chain of GPIIb [GPIIbH-(600-700)], and two in GPIIIa, the N-terminal [GPIIIa-(1-52)] and the core [GPIIIa-(423-622)] domains. Between the latter two domains there is a proteolysis-susceptible region, which is partly involved in ligand binding [GPIIIa-(100-220)] and partly implicated as being in teh subunit interface of the heterodimer. Contrary to GPIIIa, GPIIbH is highly susceptible to proteolysis all along its sequence. Equally susceptible are the extracellular end of the transmembrane segment of both GPIIIa and the light (L) chain of GPIIb (GPIIbL), and the N-terminal end of GPIIbL. Three sequence stretches along the C-terminal half of GPIIbH, one sequence stretch in GPIIbL and three sequence stretches within the GPIIIa-(217-421) region were putatively involved in the subunit interface of the heterodimer. Most likely, the N-terminal end of GPIIbL is folded over the N- and C-terminal regions of GPIIbH, and the N-terminal end of GPIIbH is folded against the GPIIbH-(600-700) domain. This map of GPIIb/IIIa does not fit the current accommodation of the amino acid sequence of GPIIb and GPIIIa in the head/two-tails image of the heterodimer obtained by metal-rotary-shadowing electron microscopy.
Spectrofluorimetric assessment of the surface hydrophobicity of proteins
Authors: Cardamone M , Puri N K
Published: August 2015

The equilibrium binding of the apolar fluorescent dye 1-anilinonaphthalene-8-sulphonate (ANS) to bacteriorhodopsin, BSA, chicken egg lysozyme, ovalbumin, porcine somatotrophin (PST) and bovine pancreatic ribonuclease (RNAase) was quantitatively evaluated using Scatchard- and Klotz-plot analyses. On the basis of the average association constant for ANS binding sites (Ka), the proteins could be ranked in order of surface hydrophobicity as: Bacteriorhodopsin greater than BSA greater than ovalbumin greater than PST greater than lysozyme greater than RNAase. The number of protein-ANS binding sites was determined as 54, 10, 3, 1, 2 and 1 respectively. The ANS-based assessment of the surface hydrophobicity of these proteins was generally in agreement with the average hydrophobicity based on amino acid sequence [Bigelow (1967) J. Theor. Biol. 16, 187-211], except for results with PST and ovalbumin. The proteins were also analysed by reversed-phase h.p.l.c. using C1 and C8 columns. There was no significant correlation between ANS and reversed-phase-h.p.l.c. assessment of hydrophobicity, with the results obtained by h.p.l.c. being dependent upon the column used. ANS-based measurement of surface hydrophobicity appears to be the most appropriate means for assessing proteins such as to reflect their overall three-dimensional structure in solution.
Mechanisms of caeruloplasmin biosynthesis in normal and copper-deficient rats
Authors: Gitlin J D , Schroeder J J , Lee-Ambrose L M , Cousins R J
Published: August 2015

To examine the mechanisms of holo-caeruloplasmin biosynthesis, we measured the serum caeruloplasmin concentration and oxidase activity, hepatic caeruloplasmin mRNA content and hepatocyte caeruloplasmin biosynthesis and secretion in normal and copper-deficient rats. Copper deficiency resulted in a near-complete loss of serum caeruloplasmin oxidase activity, yet only a 60% reduction in serum caeruloplasmin concentration and no change in the abundance of hepatic caeruloplasmin mRNA or the rate of caeruloplasmin biosynthesis. Both interleukin-1 alpha and lipopolysaccharide increased hepatic caeruloplasmin mRNA content and caeruloplasmin biosynthesis in normal and copper-deficient animals, but neither mediator increased caeruloplasmin oxidase activity in the copper-deficient group. Pulse-chase studies in primary hepatocytes from normal and copper-deficient rats revealed that the secretory rates for newly synthesized caeruloplasmin were identical, despite little or no holo-caeruloplasmin synthesis in hepatocytes of copper-deficient rats. We conclude that hepatocyte copper content has no effect on hepatic caeruloplasmin-gene expression or caeruloplasmin biosynthesis and that the incorporation of copper into newly synthesized caeruloplasmin is not a rate-limiting step in the biosynthesis or secretion of the apoprotein from rat hepatocytes.
Phosphorylation of elongation factor 2 during Ca2+-mediated secretion from rat parotid acini
Authors: Hincke M T , Nairn A C
Published: August 2015

In this paper we report the rapid phosphorylation of a cytosolic 100 kDa protein during stimulation of secretion from dispersed aggregates of parotid acinar cells with Ca(2+)-mobilizing secretagogues (carbachol, Substance P, ATP and the Ca2+ ionophore A23187). Phosphorylation was inhibited by removal of extracellular Ca2+ but was not observed during stimulation with phorbol esters, suggesting that this protein is not a substrate for protein kinase C. Two-dimensional PAGE and immunoprecipitation with a specific antiserum indicated that this protein is elongation factor 2, whose Ca2+ calmodulin-dependent phosphorylation has been shown to inhibit protein synthesis [Nairn & Palfrey (1987) J. Biol. Chem. 262, 17299-17303]. These results suggest that phosphorylation of elongation factor 2 is the molecular mechanism for the inhibition of protein synthesis which has been previously observed in rat parotid cells during stimulation with Ca(2+)-mobilizing secretagogues.
Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1
Authors: Kolm R H , Sroga G E , Mannervik B
Published: August 2015

The coding region of cDNA corresponding to human class Pi glutathione transferase P1-1 was amplified by the PCR, subcloned into an expression vector, pKHP1, expressed in Escherichia coli, and characterized. The physicochemical and catalytic properties of recombinant glutathione transferase P1-1 were indistinguishable from those of the enzyme previously isolated from human placenta. The active-site residue Tyr-8 of the wild-type enzyme was converted into Phe by means of oligonucleotide-directed mutagenesis. The mutant enzyme Y8F displayed a 300-fold decrease in specific activity, ascribable mainly to a lowered k(cat.) (or V) value. Kinetic parameters reflecting binding affinity, S0.5 (substrate concn. giving 1/2V) and I50 (concn. of inhibitor giving 50% remaining activity), were only moderately elevated in the mutant enzyme. These results indicate that Tyr-8 contributes primarily to catalysis as such, rather than to binding of the substrates. The dependence of k(cat.)/Km on pH shows an optimum at pH 7.0, defined by acidic and basic ionic dissociation constants with pKa1 = 6.7 and pKa2 = 7.3 respectively. The mutant enzyme Y8F does not display the basic limb of the k(cat.)/Km versus pH profile, but shows a monotonic increase of k(cat.)/Km with an apparent pKa1 of 7.1. The results indicate that the phenolic hydroxyl group of Tyr-8 in un-ionized form, but not the phenolate of Tyr-8, contributes to catalysis by glutathione transferase P1-1.
Regulation of carboxypeptidase E. Effect of pH, temperature and Co2+ on kinetic parameters of substrate hydrolysis
Authors: Greene D , Das B , Fricker L D
Published: August 2015

Carboxypeptidase E is a member of the carboxypeptidase A and B gene family, with many of the putative active-site and substrate-binding residues conserved between these enzymes. However, the pH optimum of carboxypeptidase E is substantially lower than that of carboxypeptidases A and B. To evaluate whether the difference in the pH optima of these carboxypeptidases reflects fundamental differences in the ionization behaviour of active-site residues, the influence of pH on carboxypeptidase E activity was examined. The V(max) for hydrolysis of dansyl-Phe-Ala-Arg is pH-independent between 5 and 7, but decreases at pH values below 5. The pKa for the group the protonation of which leads to the loss of activity is approximately 4.8, and the slope of the V(max.)/pH profile suggests that only a single ionizable group is involved. In contrast, Km and V(max.)/Km are dramatically influenced by pH over the range 5-7, with multiple ionizable groups detected in this pH range. The pKa of the group the protonation of which decreases the V(max.) of substrate hydrolysis is lower (4.5) for carboxypeptidase E which had been reconstituted with Co2+. The enthalpy of ionization of the group observed in the V(max.) profile for carboxypeptidase E is approx. 28.9 kJ/mol. These results are compatible with the active-site model of the homologous carboxypeptidase A: in this model the ionization of a metal-bound water molecule is responsible for the observed decrease in V(max.).
C-terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity
Authors: Elder R H , Tumelty J , Douglas K T , Margison G P , Rafferty J A
Published: August 2015

A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; methylated-DNA: protein-cysteine methyltransferase) has been manipulated to generate a C-terminally deleted protein which retains full methyl-transfer activity. The elimination of 22 amino-acid residues from the C-terminus was achieved by endonuclease-SacI digestion of the 623 bp cDNA coding sequence and ligation of a SacI/HindIII linker containing an in-frame stop codon. The truncated protein was characterized by its reduced molecular mass in immunoblots probed with an antiserum against the full-length protein and by fluorography after incubation with [3H]methylated calf thymus DNA. The rate of methyl transfer was virtually identical for the full-length and truncated ATases. The construction of such a truncated, yet still functional, ATase, with a molecular mass of 19.7 kDa should facilitate a detailed n.m.r. structural study and help to determine the functional significance of the C-terminal domain of mammalian ATases.
Human placental β-galactosidase. Characterization of the dimer and complex forms of the enzyme
Authors: Hubbes M , D'Agrosa R M , Callahan J W
Published: August 2015

GM1 ganglioside beta-galactosidase (beta-Gal) is deficient in the autosomal recessive disorder GM1 gangliosidosis. A portion of the enzyme occurs in a complex with neuraminidase and an additional glycoprotein, protective protein, but the nature of the interactions conferring the stability of the complex is unknown. Affinity chromatography of beta-Gal on p-aminophenylthiogalactose-Sepharose (PATG-Sepharose) at pH 4.3, the pH optimum of beta-Gal, resulted in a 260-fold enrichment of beta-Gal, but the major protein in the fraction had an M(r) value of 74,000. Affinity chromatography on PATG-Sepharose at pH 5.2 showed substantial enrichment (4000-fold) of beta-Gal, and the mature form of the enzyme (M(r) 64,000) was the major protein in the preparation. Using h.p.l.c. molecular-sieve chromatography, we found that about 15% of the total beta-Gal occurred in a high-M(r) form (greater than 600,000), the presumptive complex, with 85% eluting at M(r) 150,000, suggestive of a dimer. This distribution was independent of both high (60 mg/ml) and low (5 mg/ml) protein concentration and the pH (pH 4.3 or 5.2) of the sample applied to the column. Furthermore, incubation for 90 min at 37 degrees C, conditions which had previously been suggested as optimal for formation of the complex, had no effect on this distribution. Further fractionation by anion-exchange chromatography and a second affinity column step yielded a beta-Gal preparation that contained a single polypeptide chain (M(r) 64,000), was devoid of neuraminidase and protective protein (absent carboxypeptidase activity), and when injected into rabbits gave rise to monospecific rabbit antisera. We conclude that the protein composition of the complex is variable (i.e. it is different when isolated at pH 4.3 and 5.2) and that the amount of beta-Gal tightly associated with the complex constitutes a small fraction of the total beta-Gal activity. The more prevalent form of the enzyme is a beta-Gal homodimer that is stable and devoid of either neuraminidase activity or protective protein.
Solubilization of growth hormone and other recombinant proteins from Escherichia coli inclusion bodies by using a cationic surfactant
Authors: Puri N K , Crivelli E , Cardamone M , Fiddes R , Bertolini J , Ninham B , Brandon M R
Published: August 2015

Recombinant pig growth hormone (rPGH) was solubilized from inclusion bodies by using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The solubilizing action of CTAC appeared to be dependent on the presence of a positively charged head group, as a non-charged variant was inactive. Relatively low concentrations of CTAC were required for rapid solubilization, and protein-bound CTAC was easily removed by ion-exchange chromatography. Compared with solubilization and recovery of rPGH from inclusion bodies with 7.5 M-urea and 6 M-guanidinium chloride, the relative efficiency of solubilization was lower with CTAC. However, superior refolding efficiency resulted in final yields of purified rPGH being in the order of CTAC greater than urea greater than or equal to guanidinium chloride. Detailed comparison of the different rPGH preparations as well as pituitary-derived growth hormone by h.p.l.c., native PAGE, c.d. spectral analysis and radioreceptor-binding assay showed that the CTAC-derived rPGH was essentially indistinguishable from the urea and guanidinium chloride preparations. The CTAC-derived rPGH was of greater biopotency than pituitary-derived growth hormone. The advantages of CTAC over urea and guanidinium chloride for increasing recovery of monomeric rPGH by minimizing aggregation during refolding in vitro were also found with recombinant sheep interleukin-I beta and a sheep insulin-like growth factor II fusion protein. In addition, the bioactivity of the CTAC-derived recombinant interleukin-1 beta was approximately ten-fold greater than that of an equivalent amount obtained from urea and guanidinium chloride preparations. It is concluded that CTAC represents, in general, an excellent additional approach or a superior alternative to urea and in particular guanidinium chloride for solubilization and recovery of bioactive recombinant proteins from inclusion bodies.
Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase
Authors: Byrne P C , Sanders P G , Snell K
Published: August 2015

A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5′ end and 653 bp at the 3′ end. The full-length cDNA was cloned into an expression vector and transfected into COS-1 cells. Serine hydroxymethyltransferase activity was increased by 33% in the transfected cells and a new protein band of the appropriate size was seen by SDS/PAGE analysis of proteins extracted from transfected cells. The protein sequence for rabbit cytosolic serine hydroxymethyltransferase derived from the cDNA nucleotide sequence was compared with three other derived or known prokaryotic and eukaryotic sequences. An overall sequence similarity of 34% was noted between all four sequences, whereas the similarity between the rabbit cytosolic and mitochondrial isoforms was 62%.
Eicosapentaenoic acid inhibits cell growth and triacylglycerol secretion in McA-RH7777 rat hepatoma cultures
Authors: Fox J C , Hay R V
Published: August 2015

The plasma triacylglycerol-decreasing effect of fish-oil fatty acids was studied in vitro by using the rapidly growing cultured rat hepatoma cell line McA-RH7777. Cells were exposed to albumin-complexed eicosapentaenoic acid (C20:5n-3; EPA), to oleic acid (C18:1n-9; OA), or to albumin alone. Cell growth was similar in albumin- and OA-supplemented cultures, but EPA treatment inhibited growth. As estimated by [14C]glycerol incorporation, OA stimulated both net triacylglycerol synthesis and secretion over control levels in a dose-dependent manner. EPA stimulated triacylglycerol synthesis in similar fashion to OA, but paradoxically decreased net triacylglycerol secretion and led to exaggerated intracellular accumulation of radiolabelled triacylglycerol. The EPA and OA effects were additive at low concentrations of total fatty acid, but at higher fatty acid concentrations OA appeared to negate some effects of EPA. Chemical analysis of albumin- and OA-treated cultures revealed OA-dominant profiles for both cellular and medium triacylglycerol-associated fatty acids. In contrast, EPA was the principal fatty acid in cellular triacylglycerol of EPA-supplemented cultures, whereas medium triacylglycerol from these cultures contained very little EPA. We conclude that McA-RH7777 hepatoma cells readily synthesize EPA-containing triacylglycerol molecules, but they have variable capacity for secreting them. We consider potential mechanisms to account for the effects of EPA in this system.
The effect of insulin-like growth factor II on glucose uptake and metabolism in rat skeletal muscle in vitro
Authors: Bevan S J , Parry-Billings M , Opara E , Liu C T , Dunger D B , Newsholme E A
Published: August 2015

The effect of insulin-like growth factor II (IGF II) on the rates of lactate formation, glycogen synthesis and glucose transport in the presence of a range of concentrations of insulin were investigated using an isolated preparation of rat skeletal muscle. IGF II, at a concentration of 65 ng/ml, caused a small but significant increase in the rates of these processes at a basal physiological insulin concentration (10 muunits/ml), but was without effect in the presence of 1, 100, 1000 or 10,000 muunits of insulin/ml. Hence IGF II increased the insulin sensitivity of this tissue. This effect was removed if the incubation medium was supplemented with an equimolar concentration of IGF binding protein 1 (BP1). It is suggested that changes in the concentration of IGF II and/or BP1 may regulate glucose uptake and metabolism in skeletal muscle and have physiological significance in the control of blood glucose level.
Definition of surface-exposed and trans-membranous regions of the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum using anti-peptide antibodies
Authors: Mata A M , Matthews I , Tunwell R E A , Sharma R P , Lee A G , East J M
Published: August 2015

Peptides have been synthesized representing parts of the transduction, phosphorylation, nucleotide-binding and hinge domains of the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR), and corresponding to segments of all of the postulated short inter-membranous loops of the (Ca(2+)-Mg2+)-ATPase (residues 77-88, 277-287, 780-791, 808-818, 915-924 and 949-958). A number of antibodies raised to these peptides have been shown to bind to the ATPase, defining surface-exposed regions. Many of these are concentrated in the phosphorylation and nucleotide-binding domains, suggesting that these domains could be exposed on the top surface of the ATPase. The cytoplasmic location of the loop containing residues 808-818 was confirmed by the finding that proteinase K treatment of intact SR vesicles enhanced the binding of antibodies against this segment. These findings support the 10-alpha-helix model of the ATPase. These results also suggest that only inter-membranous loops larger than about 20 residues are likely to be detected by immunological methods in transmembranous proteins. Binding of anti-peptide antibodies to proteolytic fragments of the ATPase has been used to define the domain structure of the enzyme. Some of the anti-peptide antibodies have been characterized by studying their binding to sets of hexameric peptides synthesized on plastic pegs. A wide pattern of responses is observed, with a restricted range of epitopes being recognized by each anti-peptide antibody.
Increased intestinal protein synthesis during sepsis and following the administration of tumour necrosis factor α or interleukin-1 α
Authors: von Allmen D , Hasselgren P O , Higashiguchi T , Frederick J , Zamir O , Fischer J E
Published: August 2015

The influence of sepsis on intestinal protein synthesis was studied in rats. Sepsis was induced by caecal ligation and puncture (CLP); control rats were sham-operated. Protein synthesis was measured in vivo in the jejunum and ileum following a flooding dose of [14C]leucine. At 8 h after CLP the protein synthesis rate was increased by approx. 15% in jejunal mucosa, and at 16 h after CLP, the protein synthesis rate was increased by 50-60% in the mucosa and seromuscular layer of both jejunum and ileum. In a second series of experiments, rats were treated with recombinant tumour necrosis factor alpha (rTNF alpha) or recombinant interleukin-1 alpha (rIL-1 alpha) administered at a total dose of 300 micrograms/kg body weight over 16 h. Control rats received corresponding volumes of solvent. Treatment with rTNF alpha resulted in an approx. 25% increase in mucosal protein synthesis in jejunum. Following treatment with rIL-1 alpha, protein synthesis increased by 25% in jejunal mucosa and almost doubled in ileal mucosa. The results suggest that sepsis stimulates intestinal protein synthesis and that this response may, at least in part, be mediated by TNF and/or IL-1.
Changes in the adenine nucleotide and inorganic phosphate content of Escherichia coli F1-ATPase during ATP synthesis in dimethyl sulphoxide
Authors: Beharry S , Bragg P D
Published: August 2015

Escherichia coli F1-ATPase contained 2.9 +/- 0.1 mol of adenine nucleotide and 3.1 +/- 0.3 mol of Pi/mol of enzyme. After preincubation with ATP, the nucleotide and phosphate contents were 5.6 and 6.0 +/- 0.5 mol/mol of enzyme respectively. The F1-ATPase was induced to synthesize ATP in the presence of 30% (v/v) dimethyl sulphoxide (Me2SO). The ATP originated from endogenous bound ADP. The bound adenine nucleotide and Pi contents of the enzyme during the time course of ATP synthesis were investigated by using F1-ATPase which had been preincubated with ATP. We show that the process of ATP synthesis in Me2SO involves (i) an initial rapid loss of nucleotide from the enzyme, the process being facilitated by exogenous Pi, (ii) a rapid loss of Pi from the enzyme, at least in the absence of exogenous Pi, (iii) re-binding of a portion of the lost nucleotide, and (iv) synthesis of ATP from bound ADP and exogenous Pi. It is proposed that transfer of the F1-ATPase to the Me2SO medium induces a change in the conformation of the enzyme to a form favouring ATP synthesis.
Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions
Authors: Smith C , Mitchinson M J , Aruoma O I , Halliwell B
Published: August 2015

Lipid peroxidation within human arterial lesions is thought to play an important role in the development of atherosclerosis. Peroxidation can be accelerated by the presence of ‘catalytic’ iron or copper ions. Gruel samples from advanced atherosclerotic lesions in the abdominal aortae of human cadavers were tested for pro-oxidant properties. All samples contained bleomycin-detectable iron and phenanthroline-detectable copper. Almost all gruel samples stimulated peroxidation of rat liver microsomes, and this was usually inhibited by the iron-ion chelator desferrioxamine. Some samples stimulated formation of hydroxyl radicals from H2O2 in the presence of ascorbate, a reaction again inhibited by desferrioxamine. We conclude that the interior of human advanced atherosclerotic lesions is a highly pro-oxidant environment, and that the use of copper or iron ions to promote peroxidation of low-density lipoproteins in vitro may be a valid model for events in the arterial wall.
Identification and characterization of aminopeptidases from Aplysia californica
Authors: Bawab W , Querido E , Crine P , DesGroseillers L
Published: August 2015

Aminopeptidase activities were identified in extracts of kidney, ovotestis, head ganglia, heart and haemolymph of Aplysia californica. These enzyme preparations hydrolysed [3H][Leu]enkephalin at the Try-1-Gly-2 bond as determined by h.p.l.c. analysis of cleavage products. In all these tissues, enkephalin-degrading aminopeptidase activities were present both in membrane-bound and cytosolic fractions. The bivalent-cation-chelating agent, 1,10-phenanthroline, inhibited kidney membrane aminopeptidase activity with an IC50 of 30 microM, suggesting that this enzyme is a metalloproteinase. The aminopeptidase inhibitor amastatin was the most potent inhibitor of [Leu]enkephalin degradation (IC50 25 nM) by membrane-bound aminopeptidase, and bacitracin, bestatin and puromycin were about 100-1000 times less potent. In contrast with membrane-bound aminopeptidase, the cytosolic form is sensitive to puromycin. Angiotensin-converting enzyme inhibitor had no effect on [Leu]enkephalin degradation by kidney membranes, while the neutral endopeptidase inhibitors were poor inhibitors of the enzymes in this preparation. The Km values of the aminopeptidase in the kidney membranes and cytosolic fractions for the [Leu]enkephalin substrate were 2.4 and 7.4 microM respectively. The aminopeptidase present in the kidney membranes also hydrolysed endogenous Phe-Met-Arg-Phe-amide peptide at the Phe-1-Met-2 bond as well as synthetic alanine p-nitroanilide and leucine p-nitroanilide. When used in a competition assay, these substrates inhibited hydrolysis of [3H][Leu]enkephalin, suggesting that the same enzyme degraded all these substrates. Taken together, these results suggest that Aplysia tissues contain both a membrane-bound aminopeptidase related to the mammalian aminopeptidase N and a cytosolic puromycin-sensitive aminopeptidase.
Small-angle X-ray-scattering studies of the C hordeins of barley (Hordeum vulgare)
Authors: I'Anson K J , Morris V J , Shewry P R , Tatham A S
Published: August 2015

Small angle X-ray scattering was used to study the solution conformation of the C hordeins of barley (Hordeum vulgare), a group of proteins whose primary structure consists predominantly of an octapeptide repeat motif. Measurements on the protein in 0.1 M-acetic acid at 25 degrees C are consistent with a model for the protein conformation of a stiff coil, the so-called ‘worm-like’ chain. The characteristic parameters (the Kuhn statistical segment length and the contour length) of the protein were calculated as 5.11 and 71.5 nm respectively.