bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for different kinases in thylakoid protein phosphorylation
Authors: Foyer C H
Published: August 2015

Thylakoid protein phosphorylation was facilitated in darkness by using the ferredoxin-NADPH system. CoCl2 and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) were potent inhibitors of LHCP (light-harvesting chlorophyll-binding protein) phosphorylation, but 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea and atrazine had no significant effect. Differential effects on phosphorylation of the 9 kDa polypeptide and LHCP were observed in darkness with DBMIB and certain other inhibitors specific for Photosystem-II electron transport. Similarly, during illumination of intact chloroplasts or of the reconstituted chloroplast system, a differential action of bicarbonate was observed on the relative phosphorylation of the two proteins. The degree of phosphorylation of the 9 kDa polypeptide was increased in the presence of bicarbonate compared with its absence, whereas that of LHCP was relatively unchanged. Changes in the degree of phosphorylation of the 32 kDa polypeptide in these experiments did not correlate consistently with changes in phosphorylation of either LHCP or the 9 kDa polypeptide, although changes in the 32 kDa polypeptide more often paralleled phosphorylation of the 9 kDa polypeptide rather than the phosphorylation of LHCP. These observations suggest that the protein kinase that phosphorylates LHCP is distinct from that which phosphorylates the 9 kDa polypeptide.
Threshold responses in production of reactive oxygen metabolites in individual neutrophils detected by flow cytometry and microfluorimetry
Authors: Patel A K , Hallett M B , Campbell A K
Published: August 2015

1. The fluorescent compound 2′,7′-dichlorofluorescein was used as an indicator of intracellular H2O2 production by neutrophils in order to compare the response of the cell population with that observed with individual cells determined by flow cytometry and quantitative fluorescence microscopy. 2. 2′,7′-Dichlorofluorescein diacetate was deacetylated by intracellular esterases to form reduced 2′,7′-dichlorofluorescein. The polar non-fluorescent intermediate remained trapped within both intracellular granules and the cytoplasm. Reduced dichlorofluorescein was oxidized by H2O2, a product of the oxidative burst, to yield the highly fluorescent product dichlorofluorescein. 3. A population of neutrophils stimulated by suboptimal concentrations of fMet-Leu-Phe (N-formylmethionyl-leucyl-phenylalanine) or phorbol ester (phorbol 12-myristate 13-acetate) resulted in an oxidation of 45-50% of the cellular dichlorofluorescein (non-fluorescent) to oxidized dichlorofluorescein within 30 min. Subcellular fractionation showed that, although dichlorofluorescein (non-fluorescent) occurred both in the cytoplasm and the granules, oxidation of dichlorofluorescein (non-fluorescent) occurred predominantly in the granules of stimulated neutrophils. 4. Flow cytometry showed that unstimulated cells consisted of a single population of cells with low cellular fluorescence. Activation of neutrophils (to produce reactive oxygen metabolites) resulted in the appearance of a second population of cells, with high fluorescence. The number of cells in this new population increased with time. fMet-Leu-Phe (0.1 microM) or phorbol ester (1 ng/ml) activated 45% of the cells within 8 min and 42% within 30 min respectively. 5. Analysis of individual cells by quantitative fluorescence microscopy demonstrated that, in the presence of a suboptimal concentration of stimulus, cells either failed to respond or were activated after different time delays, 4-120 s (39 +/- 18.4 s) by fMet-Leu-Phe or 12-200 s (59 +/- 17.4 s) by phorbol ester. Furthermore the oxidative bursts were of different magnitudes. 6. It is concluded that, in order for an individual cell to cross the activation threshold for the ‘end response’, a critical concentration of stimulus together with the necessary changes in intracellular signals are required.
Short-term incubation of cardiac myocytes with isoprenaline has no effect on heparin-releasable or cellular lipoprotein lipase activity
Authors: Severson D L , Carroll R , Kryski A , Ramírez I
Published: August 2015

Heparin (5 units/ml) produced a rapid (5-10 min) release of lipoprotein lipase (LPL) into the incubation medium of cardiac myocytes. Preincubation of myocytes for 30 min with 0.01-10 microM-isoprenaline, 100 microM-forskolin or 500 microM-8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate did not increase heparin-releasable LPL activity. Incubation with isoprenaline also did not change cellular LPL activity, even though the catecholamine did increase the phosphorylase a activity ratio.
Abundance of the Ca2+-pumping ATPase in pig erythrocyte membranes
Authors: Bewaji C O , Bababunmi E A
Published: August 2015

The Ca2+-pumping ATPase (Ca2+-ATPase) was purified from human and pig erythrocyte membranes by calmodulin affinity chromatography in the presence of phosphatidylcholine. The amount of enzyme present in pig erythrocytes is at least 7 times greater than that isolated from human erythrocyte ghosts. However, the properties of the enzyme from the two species are similar in many respects.
The effects of autolysis on the structure of chicken calpain II
Authors: Crawford C , Willis A C , Gagnon J
Published: August 2015

When chicken calpain II autolysed in the presence of Ca2+, it underwent limited proteolysis to give peptides of Mr 54,000 and 37,000, and several of Mr approx. 30,000 and 18,000. The autolytic peptides were purified and their N-terminal amino acid sequences determined. By comparison of these sequences with the known sequence of the complete calpain molecule, the autolytic cleavage sites were identified. The structural integrity of the molecule during autolysis was investigated by gel-permeation chromatography. Experiments were also done to test the reversibility of adding EDTA to calpain during autolysis, measured as recoverable enzyme activity assayed in the presence of Ca2+. The results are presented in terms of a model for the structural changes occurring in calpain during autolysis. It was concluded that the loss of enzymic activity, which is a consequence of autolysis, was due to dissociation of the autolytic peptides after cleavage of the calpain large subunit within the third domain.
A transient kinetic study of enthalpy changes during the reaction of myosin subfragment 1 with ATP.
Authors: Millar N C , Howarth J V , Gutfreund H
Published: August 2015

1. The enthalpy changes during individual reaction steps of the myosin subfragment 1 ATPase were studied with the use of a new stopped-flow calorimeter [Howarth, Millar & Gutfreund (1987) Biochem. J. 248, 677-682]. 2. At 5 degrees C and pH 7.0, the endothermic on-enzyme ATP-cleavage step was observed directly (delta H = +64 kJ.mol-1). 3. ADP binding is accompanied by a biphasic enthalpy change. 4. The release and uptake of protons was investigated by the use of two buffers with widely different heats of ionization. 5. Protons are involved in all four principal steps of the myosin subfragment 1 ATPase.
V
max. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A
Authors: Matten W T , Maness P F
Published: August 2015

A kinetic analysis of the tyrosine-specific protein kinase of pp60c-src from the C1300 mouse neuroblastoma cell line Neuro-2A and pp60c-src expressed in fibroblasts was carried out to determine the nature of the increased specific activity of the neuroblastoma enzyme. In immune-complex kinase assays with ATP-Mn2+ and the tyrosine-containing peptide angiotensin I as phosphoacceptor substrate, pp60c-src from the neuroblastoma cell line was characterized by a maximum velocity (Vmax.) that was 7-15-fold greater than the Vmax. of pp60c-src from fibroblasts. The neuroblastoma enzyme exhibited Km values for ATP (16 +/- 3 microM) and angiotensin I (6.8 +/- 2.6 mM) that were similar to Km values for ATP (25 +/- 3 microM) and angiotensin I (6.5 +/- 1.7 mM) of pp60c-src from fibroblasts. pp60v-src expressed in Rous-sarcoma-virus-transformed cells exhibited an ATP Km value (25 +/- 4 microM) and an angiotensin I Km value (6.6 +/- 0.5 mM) that approximated the values determined for pp60c-src in neuroblastoma cells and fibroblasts. These results indicate that the pp60c-src kinase from neuroblastoma cells has a higher turnover number than pp60c-src kinase from fibroblasts, and that the neural form of the enzyme would be expected to exhibit increased catalytic activity at the saturating concentrations of ATP that are found intracellularly.
Acyl-CoA-binding protein from cow. Binding characteristics and cellular and tissue distribution
Authors: Mikkelsen J , Knudsen J
Published: August 2015

Using the tyrosine fluorescence quenching as a criterion for acyl-CoA binding, we have shown that acyl-CoA-binding protein (ACBP) binds acyl-CoA esters with a chain length greater than C8 with equal affinity. The binding studies indicated a binding stoichiometry of 1 mol of acyl-CoA/2 mol of ACBP. The protein was found in liver, adipose tissue, intestinal mucosa, kidney, heart, brain, muscles and mammary gland. The highest concentration was found in liver cytosol and the lowest in muscles and mammary gland. ACBP could not be shown to bind non-esterified fatty acids.
Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements
Authors: Taylor S J , Exton J H
Published: August 2015

The effect of the GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5′-[beta gamma-imido]triphosphate, but not with adenosine 5′-[gamma-thio]triphosphate, and was inhibited by guanosine 5′-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.
Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase
Authors: Lewis R E , Czech M P
Published: August 2015

Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for 125I-insulin (Kd = 0.12 nM versus 0.76 nM). Inclusion of 8.3-16.7% phosphatidylserine into the reconstituted vesicles restores 40-50% of the insulin-sensitivity to the receptor kinase. An elevated apparent affinity for 125I-insulin of insulin receptors in vesicles containing phosphatidylcholine and phosphatidylethanolamine is also restored to the value observed in detergent solution by the inclusion of phosphatidylserine in the reconstituted system. The effect of phosphatidylserine on insulin receptor kinase appears specific, because cholesterol, phosphatidylinositol and phosphatidic acid are all unable to restore insulin-sensitivity to the receptor kinase. Autophosphorylation sites on the insulin receptor as analysed by h.p.l.c. of tryptic 32P-labelled receptor phosphopeptides are not different for insulin receptors autophosphorylated in detergent solution or for the reconstituted vesicles in the presence or absence of phosphatidylserine. These data indicate that the phospholipid environment of insulin receptors can modulate its binding and kinase activity, and phosphatidylserine acts to restore insulin-sensitivity to the receptor kinase incorporated into phosphatidylcholine/phosphatidylethanolamine vesicles.
Developmental forms of human skeletal-muscle AMP deaminase. The kinetic and regulatory properties of the enzyme
Authors: Kaletha K , Nowak G
Published: August 2015

AMP deaminase isoforms from human skeletal muscle can be separated chromatographically [Kaletha, Spychała & Nowak (1987) Experientia 43, 440-443]. In adult tissue nearly all the AMP deaminase activity was eluted from phosphocellulose with 0.75 M-KCl (‘adult’ isoform), and the remaining activity could be eluted with 2.0 M-KCl. Conversely, most of the AMP deaminase activity from 11-week-old fetal tissue was eluted from phosphocellulose with 2.0 M-KCl (‘fetal’ isoform). In the present paper the kinetic and regulatory properties of AMP deaminase extracted from 11- and 16-week-old fetal skeletal muscle are reported. The two isoforms from 11-week-old human fetus differed distinctly in these properties. The ‘fetal’ isoform had about 5-fold higher half-saturation constant (S0.5) value than the ‘adult’ form. It was also more sensitive to the influence of some important regulatory ligands (ADP, ATP and Pi), and exhibited a different pH/activity profile. The ‘adult’ isoform of AMP deaminase from fetal muscle and the enzyme from mature muscle possessed similar kinetic and regulatory properties. This isoform seems not to be subject to any major modifications during further ontogenesis. This is not true, however, for the ‘fetal’ isoform. In the muscle of 16-week-old human fetus, the ‘fetal’ isoform showed a peculiar, biphasic, type of substrate-saturation kinetics. This phenomenon may reflect appearance of the next, developmentally programmed, isoform of human skeletal-muscle AMP deaminase.
Glucocorticoids increase insulin binding and the amount of insulin-receptor mRNA in human cultured lymphocytes
Authors: Shibasaki Y , Sakura H , Odawara M , Shibuya M , Kanazawa Y , Akanuma Y , Takaku F , Kasuga M
Published: August 2015

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.
Partial purification of goat kidney β-mannosidase
Authors: Frei J I , Cavanagh K T , Fisher R A , Hausinger R P , Dupuis M , Rathke E J , Jones M Z
Published: August 2015

1. Goat kidney beta-mannosidase was purified 8500-fold to a specific activity of 65,000 nmol/h per mg of protein with a 6% yield by using multiple steps including cation-exchange and anion-exchange fast protein liquid chromatography. This is the first description of a highly purified preparation from goat tissue; however, it was not homogeneous, as judged by silver-stained SDS/polyacrylamide-gel electrophoresis. 2. The enzyme exhibited microheterogeneity when analysed by isoelectric focusing (pI 5.5-6.5). 3. Purified beta-mannosidase hydrolysed the terminal beta-(1→4)-linkage of oligosaccharides that accumulate in beta-mannosidosis.
Metal ion binding to d-xylose isomerase from Streptomyces violaceoruber
Authors: Callens M , Tomme P , Kersters-Hilderson H , Cornelis R , Vangrysperre W , De Bruyne C K
Published: August 2015

The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 × 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 × 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 × 10(5) M-1 and 5 × 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.
The vanadium-iron protein of vanadium nitrogenase from Azotobacter chroococcum contains an iron-vanadium cofactor
Authors: Smith B E , Eady R R , Lowe D J , Gormal C
Published: August 2015

N-Methylformamide extracts of acid-treated precipitated VFe protein of the V-nitrogenase of Azotobacter chroococcum are yellow-brown in colour and contain vanadium, iron and acid-labile sulphur in the approximate proportions 1:6:5. E.p.r. spectra of the extracts exhibit a weak signal with g values near 4.5, 3.6 and 2.0 characteristic of an S = 3/2 metal-containing centre. The N-methylformamide extracts activated the MoFe protein polypeptides from mutants of nitrogen-fixing bacteria unable to synthesize FeMoco, the active centre of Mo-nitrogenase. The active hybrid protein exhibited the characteristic substrate-reducing phenotype associated with the VFe protein except that it could not reduce N2 to NH3. The above data are interpreted as demonstrating the existence of an iron- and vanadium-containing cofactor, FeVaco, within the VFe protein. It is suggested that nitrogen fixation requires specific interactions between FeVaco or FeMoco and their respective polypeptides. The biosynthesis of these cofactors is discussed.
Hyaluronic acid in human articular cartilage. Age-related changes in content and size
Authors: Holmes M W A , Bayliss M T , Muir H
Published: August 2015

Total tissue content and molecular mass of hyaluronic acid was determined in papain digests of human articular cartilage using a sensitive radiosorbent assay [Laurent & Tengblad (1980) Anal. Biochem. 109, 386-394]. 1) Hyaluronic acid content increased from 0.5 microgram/mg wet wt. to 2.5 micrograms/mg wet wt. between the ages of 2.5 years and 86 years. 2) Hyaluronic acid chain size decreased from Mr 2.0 x 10(6) to 3.0 x 10(5) over the same age range. 3) There was no age-related change in the size of newly-synthesized hyaluronic acid, which was of very high molecular mass, in both immature and mature cartilage. The results are consistent with an age-related decrease in proteoglycan aggregate size and suggest that modification of the hyaluronic acid chain may take place in the extracellular matrix.
Two systems in vitro that show insulin-stimulated serine kinase activity towards the insulin receptor
Authors: Smith D M , King M J , Sale G J
Published: August 2015

Two systems in vitro are described that show insulin-stimulated phosphorylation of the insulin receptor on serine residues. In the first system, insulin receptor was purified partially from Fao rat hepatoma cells by direct solubilization of the cells in Triton X-100 and chromatography on wheat-germ-agglutinin-agarose. Phosphorylation of these preparations with [gamma-32P]ATP in the presence or absence of insulin resulted in 32P incorporation exclusively into phosphotyrosine residues. Serine kinase activity towards the insulin receptor was reconstituted by adding extracts of Fao cells. Prior exposure of the cells to insulin stimulated serine kinase activity towards the insulin receptor in extracts 7.2-fold. A receptor serine kinase activity enhanced by treatment of cells with cyclic AMP analogues was also retained in the reconstituted system. In the second system, insulin receptor and insulin-sensitive serine kinase activity towards the insulin receptor were co-purified from human placenta. The protocol involved preparation of membranes, before solubilization and chromatography on wheat-germ-agglutinin-agarose, by using gentle procedures designed not to disrupt a potentially labile association between the insulin receptor and the serine kinase. Serine kinase activity in these preparations towards the insulin receptor was stimulated up to 10-fold by insulin, and the stoicheiometry of serine phosphorylation was estimated to be approx 0.8 mol/mol of insulin receptor for phosphorylations performed in the presence of insulin. Thus a preparation of insulin receptor is described for the first time that is phosphorylated to high stoicheiometry on serine in an insulin-dependent manner. Conditions that facilitate recovery and assay of serine kinase activity are defined and discussed. These systems provide a basis for characterizing the nature of the insulin-sensitive serine kinase that phosphorylates the insulin receptor, and defining its role in insulin action and control of receptor function.
Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism
Authors: Danielson U H , Mannervik B
Published: August 2015

Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.
Complete amino acid sequence of p453-plasmid-mediated PIT-2 β-lactamase (SHV-1)
Authors: Barthélémy M , Peduzzi J , Labia R
Published: August 2015

The complete amino acid sequence of the p453-plasmid-mediated PIT-2 beta-lactamase (SHV-1) was determined. The protein contains 265 residues. Peptides resulting from digestions with trypsin, Staphylococcus aureus V8 proteinase, chymotrypsin and Lys-C proteinase and cleavage with CNBr were separated and purified by using reverse-phase h.p.l.c. The amino acid sequence of each peptide was manually determined with the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method. The primary structure of PIT-2 beta-lactamase was compared with those of two closely related enzymes, namely TEM-1 beta-lactamase and the beta-lactamase of Klebsiella pneumoniae strain LEN-1. The PIT-2 beta-lactamase amino acid sequence was strongly retained, with respectively 68% and 88% homology. Thus PIT-2 enzyme could represent an evolutionary step between a chromosomally encoded beta-lactamase and the plasmid-mediated TEM beta-lactamases.
Biosynthesis, assembly and secretion of fibrinogen in cultured rat hepatocytes
Authors: Hirose S , Oda K , Ikehara Y
Published: August 2015

The biosynthesis, assembly and secretion of fibrinogen were investigated in cultured rat hepatocytes which were incubated with [35S]methionine. When initial rates of the synthesis of three fibrinogen subunits were compared, the A alpha-subunit was found to be synthesized significantly slower than the B beta- and gamma-subunits. Pulse-chase experiments revealed that the secreted fibrinogen contained different proportions of the newly synthesized subunits, depending upon the chase times. Radioactivity in the A alpha subunit, which initially had the highest level of the three, was rapidly decreased in parallel with the chase time. The gamma-subunit had an increasing amount of the radioactivity in the secreted molecule during the chase periods, whereas that in the B beta-subunit was gradually decreased at the later stages of chase. Analysis of intracellular components of fibrinogen confirmed that the nascent A alpha-subunit was most rapidly exhausted, and the gamma-subunit occupied the largest proportion among the non-assembled subunits at later stages of chase. Taken together, these results suggest that the synthesis of A alpha-subunit, which has the lowest rate, could be the rate-limiting step in the production and secretion of fibrinogen in cultured rat hepatocytes, in contrast with what has been proposed for human and rabbit fibrinogen, namely that the synthesis of B beta-subunit is the rate-limiting step. The results also indicate that there is a large intracellular pool of gamma-subunit.
Electric shock-mediated transfection of cells. Characterization and optimization of electrical parameters
Authors: Winterbourne D J , Thomas S , Hermon-Taylor J , Hussain I , Johnstone A P
Published: August 2015

The effect of various parameters on the electric shock-mediated permeabilization and transfection of CHO cells has been investigated. Up to 70% of the cells can be maintained transiently permeable to erythrosin B for periods of at least 1 h at 20 degrees C. Electrical conditions optimal for transient permeabilization were also optimal for efficient DNA transfection by pSV2neo. However, the DNA must be present during exposure to the electric field for efficient transformation. The same requirement existed for voltage-induced DNA toxicity. The results suggest that DNA moves into the cells by electrophoresis, not by simple diffusion. Based on these observations a simple, rapid procedure for optimizing the conditions for electric shock-mediated DNA transfer into cells has been developed.
β-lactamase inhibitors. The inhibition of serine β-lactamases by specific boronic acids
Authors: Crompton I E , Cuthbert B K , Lowe G , Waley S G
Published: August 2015

Many beta-lactamases have active-site serine residues, and are competitively inhibited by boronic acids. Hitherto, the boronic acids used have lacked any structural resemblance to the substrates of beta-lactamases. Phenylacetamidomethaneboronic acid, trifluoroacetamidomethaneboronic acid and 2,6-dimethoxybenzamidomethaneboronic acid have now been synthesized. The first of these contains the side-chain moiety of penicillin G, and the last that of methicillin. The pH-dependence of binding of the first inhibitor to beta-lactamase I from Bacillus cereus revealed pK values of 4.7 and 8.2 for (presumably) active-site groups in the enzyme. The kinetics of inhibition were studied by cryoenzymology and by stopped-flow spectrophotometry. These techniques provided evidence for a two-step mechanism of binding of the first two boronic acids mentioned above to beta-lactamase I, and for benzeneboronic acid to a beta-lactamase from Pseudomonas aeruginosa. The slower step is probably associated with a change in enzyme conformation as well as the formation of an O-B bond between the active-site serine hydroxy group and the boronic acid.
Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides
Authors: Jackson A P , Bagshaw C R
Published: August 2015

The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.
Effect of glycerol on the molecular properties of cerebrosides, sulphatides and gangliosides in monolayers
Authors: Bianco I D , Fidelio G D , Maggio B
Published: August 2015

The presence of glycerol, free from surface-active impurities, modifies the molecular area, surface potential/molecule and thermodynamic parameters of compression of monolayers of galactosylceramide, sulphatide and gangliosides GM1, GD1a and GT1b. This may be due to changes of the composition and structural properties of the glycosphingolipid solvation shell with an influence on the intermolecular organization.
Investigation of endosomal compartments involved in endocytosis and transcytosis of polymeric immunoglobulin A by subcellular fractionation of perfused isolated rat liver
Authors: Perez J H , Branch W J , Smith L , Mullock B M , Luzio J P
Published: August 2015

1. A gamma camera was used to monitor continuously the uptake of radiolabelled polymeric immunoglobulin A (pIgA) into the rat body after intravenous injection. Uptake into liver was fast but, since the peak of liver labelling occurred only after 9-15 min, it was not sufficiently rapid to constitute a pulse dose. A perfused, isolated rat liver system was therefore established which could be given a single pass dose of pIgA; a variety of tests showed such livers remained viable for at least 3 h and could be subsequently fractionated on Ficoll and Nycodenz gradients with normal distributions of marker enzymes. 2. Subcellular fractionation at different times after a single pass dose of pIgA showed that whilst pIgA appeared sequentially in sinusoidal plasma membrane, light endosomes, dense endosomes, very dense endosomes and lysosomes as in vivo, the predominance of pIgA in the light endosome compartment disappeared much earlier than after injection in vivo of pIgA, presumably because this compartment was not being continuously loaded over the first 10-15 min. The time course of appearance of label in bile was unchanged. A large excess of unlabelled asialofetuin did not change these patterns, indicating that the asialoglycoprotein receptor was not involved. 3. Low doses of the microtubule agent colchicine reduced the proportion of pIgA reaching the bile, but subcellular fractionation of treated liver showed that distribution of label amongst liver fractions was little changed, although the overall liver pIgA content had increased. This would suggest that pIgA did not remain in the common compartment which could have supplied bile or lysosomes but rather flowed out of it as rapidly as in untreated liver but towards those compartments supplying the lysosomes. 4. Experiments with nocodazole, which reversibly disrupts microtubules, showed that very little of the pIgA taken into an inhibited liver appeared in the bile after nocodazole was removed 30 min later, even though a second dose of pIgA, given after nocodazole removal, appeared in bile with a normal time course. The first dose of pIgA must therefore have passed beyond the compartments competent to supply the bile before nocodazole was removed. Such compartments were undamaged since the second dose of pIgA appeared in bile normally. We therefore conclude that the bulk of pIgA must be supplied to the bile from light or dense endosomes rather than from very dense endosomes and lysosomes.
Parathyroid hormone and prostaglandin E2 stimulate both inositol phosphates and cyclic AMP accumulation in mouse osteoblast cultures
Authors: Farndale R W , Sandy J R , Atkinson S J , Pennington S R , Meghji S , Meikle M C
Published: August 2015

Parathyroid hormone (PTH) and prostaglandin E2 (PGE2) are physiological agonists which stimulate bone cells to resorb bone, a process by which the mineralized extracellular bone matrix is dissolved. Bone resorption has a key role in the maintenance of plasma calcium levels. It has been established that both PTH and PGE2 activate adenylate cyclase in osteoblasts, but it is apparent that (1) the two agents have qualitatively different effects on osteoblasts, and (2) the generation of cyclic AMP cannot account for all the effects of PTH on bone cell metabolism. Others have demonstrated that PTH and PGE2 may also elevate intracellular calcium levels, but the mechanism by which this is achieved has not been fully defined. Here we have investigated the effects of PTH on neonatal mouse osteoblasts in culture and shown that physiological concentrations of the hormone (50 nM) caused a small increase (22%) in total inositol phosphates accumulation, with a larger increase (40%) in inositol trisphosphate. We found that this activation occurred at lower concentration than was necessary to activate adenylate cyclase. PGE2 was a more effective activator of inositol phosphates accumulation than PTH, causing up to 300% increase in the total inositol phosphates after 30 min. Both PTH and PGE2 stimulated cyclic AMP accumulation, but the activation of adenylate cyclase by forskolin did not enhance inositol phosphates production. We conclude that both PTH and PGE2 stimulate phosphoinositide turnover in mouse osteoblasts and suggest that this mechanism may contribute to their elevation of intracellular calcium in bone cells.
Further studies of oligosaccharide recognition by the soluble 13 kDa lectin of bovine heart muscle. Ability to accommodate the blood-group-H and -B-related sequences
Authors: Abbott W M , Hounsell E F , Feizi T
Published: August 2015

Oligosaccharide recognition by the 13 kDa soluble lectin from bovine heart muscle has been investigated by inhibition of binding of the 125I-labelled lectin to trypsin-treated rabbit erythrocytes. The results indicate that the Type 1 (Gal beta 1-3GlcNAc) and the Type 2 (Gal beta 1-4GlcNAc) backbone structures are the basic recognition units, and that the blood-group-H structure, the blood-group-B structure, the ‘B-like’ structure [afucosyl-(blood group B)] and the alpha 2-3 sialylated analogues of the backbone structures can also be accommodated and hence are candidate receptor structures for the lectin. A comparison of available inhibition data on six other soluble beta-galactoside-binding lectins (three from human lung and three from rat lung) has shown some common features among these and the bovine lectin, e.g. in general a stronger reaction with N-acetyl-lactosamine than with lactose, and a lack of reaction with 3-fucosyl-lactose and 6-sialyl-lactose. However, there are distinctive features among the lectins, e.g. differences in relative reactions with the blood-group-A structure, and no two of the lectins appear to be identical in their fine specificities.
Conditioned media of Kupffer and endothelial liver cells influence protein phosphorylation in parenchymal liver cells. Involvement of prostaglandins
Authors: Casteleijn E , Kuiper J , Van Rooij H C , Koster J F , Van Berkel T J
Published: August 2015

The possible role of Kupffer and endothelial liver cells in the regulation of parenchymal-liver-cell function was assessed by studying the influence of conditioned media of isolated Kupffer and endothelial cells on protein phosphorylation in isolated parenchymal cells. The phosphorylation state of three proteins was selectively influenced by the conditioned media. The phosphorylation state of an Mr-63,000 protein was decreased and the phosphorylation state of an Mr-47,000 and an Mr-97,000 protein was enhanced by these media. These effects could be mimicked by adding either prostaglandin E1, E2 or D2. Both conditioned media and prostaglandins stimulated the phosphorylase activity in parenchymal liver cells, suggesting that the Mr-97,000 phosphoprotein might be phosphorylase. Parenchymal liver cells secrete a phosphoprotein of Mr-63,000 and pI 5.0-5.5. The phosphorylation of this protein is inhibited by Kupffer- and endothelial-liver-cell media, and prostaglandins E1, E2 and D2 had a similar effect. The data indicate that Kupffer and endothelial liver cells secrete factors which influence the protein phosphorylation in parenchymal liver cells. This forms further evidence that products from non-parenchymal liver cells, in particular prostaglandin D2, might regulate glucose homoeostasis and/or other specific metabolic processes inside parenchymal cells. This stresses the concept of cellular communication inside the liver as a way by which the liver can rapidly respond to extrahepatic signals.
A collagen-binding glycoprotein on the surface of mouse fibroblasts is identified as dipeptidyl peptidase IV
Authors: Bauvois B
Published: August 2015

A dipeptidyl aminopeptidase (DPP) was detected in plasma membranes from normal (3T3) and transformed (3T12) mouse fibroblasts. This enzyme was active in cleaving the prolyl bond in the synthetic dipeptide nitroanilide Gly-Pro-NH-Np, which is a specific substrate for DPP IV (Km 0.63 mM and Vmax 6.1 nmol/min per mg at pH 6.0 and 37 degrees C). However, it did not degrade Pro-NH-Np or other dipeptide nitroanilides such as Gly-Arg-NH-Np or Val-Ala-NH-Np. The enzyme was totally inhibited by di-isopropyl phosphorofluoridate (Pri2-P-F) and by phenylmethanesulphonyl fluoride, indicating a serine catalytic site for the proteinase. DPP IV is a glycoprotein that specifically recognized immobilized gelatin and type I collagen. Upon molecular exclusion chromatography, the proteinase exhibited an apparent Mr of 100,000. SDS/polyacrylamide-gel electrophoresis under non-reducing and reducing conditions revealed that the [3H]Pri2-P-protein was exclusively represented by a polypeptide of Mr 55,000. This suggested that DPP IV consists of two non-covalently linked 55,000-Mr subunits. Fibroblast adhesion to native or denatured collagen was significantly inhibited by the two dipeptide inhibitors of DPP IV, Gly-Pro-Ala and Ala-Pro-Gly, but not by the peptides Gly-Pro and Gly-Gly-Gly, which are not inhibitors of the proteinase. Moreover, preliminary fractionation of DPP IV by molecular exclusion chromatography and affinity chromatography indicated that this material was active in disrupting cell adhesion to collagens. Taken together, the above data suggest that a fibroblast membrane-associated collagen-binding glycoprotein, DPP IV, may play a role in cell attachment to collagen.
Biosynthesis of secreted β-hexosaminidase in Tetrahymena thermophila. A comparison of the wild type with a secretory mutant
Authors: Hünseler P , Tiedtke A , von Figura K
Published: August 2015

The synthesis and secretion of beta-hexosaminidase was studied in wild type and secretion-deficient Tetrahymena thermophila cells by metabolic labelling and immunoprecipitation. beta-Hexosaminidase is synthesized as a Mr 79,000 polypeptide which is within 10 min converted into a Mr 59,000 form. The Mr 59,000 polypeptide is further processed (within 20 min) into at least three major mature forms of Mr 58,000-54,000, which are almost quantitatively secreted into the culture medium within 1-2 h after their synthesis. Both precursor and mature forms contain asparagine-linked oligosaccharide chains which are cleavable by endoglucosaminidase F, but not by endoglucosaminidase H. Neither [32P]orthophosphate nor [35S]sulphate are incorporated into immunoprecipitable precursor and mature beta-hexosaminidases, suggesting the absence of a phosphorylated recognition marker. Biosynthesis and processing of beta-hexosaminidase is apparently unaltered in the secretory mutant MS-1; however the processed polypeptides remain cellular bound in the mutant, indicating that the mutation affects a late event in the secretion pathway of lysosomal enzymes.
Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy
Authors: Mörgelin M , Paulsson M , Hardingham T E , Heinegård D , Engel J
Published: August 2015

Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.
Post-translational processing of preprotachykinins. Isolation of protachykinin-(1-37)-peptide from human adrenal-medullary phaeochromocytoma tissue
Authors: Kage R , Thim L , Creutzfeldt W , Conlon J M
Published: August 2015

The biosynthetic precursors of the mammalian tachykinins, alpha-, beta-and gamma-preprotachykinins, contain a common N-terminal region of 74 amino acids. A polyclonal antiserum was raised against a synthetic peptide representing N-tyrosylated beta-preprotachykinin-(48-56)-peptide as predicted from the nucleotide sequence of cloned DNA complementary to human beta-preprotachykinin mRNA. By using this antiserum in radioimmunoassay, a single immunoreactive peptide was identified in an extract of a human pheochromocytoma that produced substance P and neurokinin A. Partial microsequencing and determination of the amino acid composition of the peptide indicated identity with preprotachykinin-(20-56)-peptide. Thus the data demonstrate that the Ala19-Glu20 bond in preprotachykinin is the site of cleavage of the signal peptide.
Activation of insect cell adenylate cyclase by Bacillus thuringiensis δ-endotoxins and melittin. Toxicity is independent of cyclic AMP
Authors: Knowles B H , Farndale R W
Published: August 2015

Insecticidal Bacillus thuringiensis (Bt) delta-endotoxins are cytolytic to a range of insect cell lines in vitro. Addition of Bt var. aizawai or var. israelensis toxins to Mamestra brassicae (cabbage moth) cells in vitro increased intracellular cyclic AMP, which was paralleled by activation of adenylate cyclase in isolated membranes. Var. kurstaki toxin, which does not lyse M. brassicae cells, had no effect on cyclic AMP concentrations in intact cells, but was able to stimulate adenylate cyclase in membrane preparations. In contrast, the bee-venom toxin melittin, which is also cytolytic, increased intracellular cyclic AMP in whole cells, but inhibited adenylate cyclase in isolated membranes. Octopamine and forskolin also increased cyclic AMP in cells, but were not cytolytic. When added to cells at concentrations exceeding their LC90 (concentration causing 90% cell death), melittin and var. israelensis toxins caused cell lysis without a concomitant increase in intracellular cyclic AMP. Taken together, these results suggest that activation of adenylate cyclase by cytolytic toxins is a secondary effect (related perhaps to interactions of these toxins with membrane lipids) and is neither necessary nor sufficient for cytolysis.
Inactivation of pyruvate dehydrogenase complex in heart muscle mitochondria of gold-thioglucose-induced obese mice is not due to a stable increase in activity of pyruvate dehydrogenase kinase
Authors: Caterson I D , Kerbey A L , Cooney G J , Frankland R , Denyer G S , Nicks J , Williams P F
Published: August 2015

The proportion of pyruvate dehydrogenase (PDH) complex in the active dephosphorylated form was decreased (compared with fed lean control mice) in heart muscle mitochondria after the induction of obesity with gold-thioglucose (by 54%) or starvation of lean mice for 48 h (by 81%). The effects of obesity to inactivate PDH complex were demonstrable 4 weeks after administration of gold-thioglucose, and occurred despite significant hyperinsulinaemia in obese animals. Phosphorylation and inactivation of PDH complex in mouse heart muscle in starvation was attributed to a stable increase (2.7-fold) in the activity of PDH kinase as measured in extracts of mitochondria mediated by increased specific activity of a protein activator of PDH kinase (KAP) [Denyer, Kerbey & Randle (1986) Biochem. J. 239, 347-354]. In obese mice no such increase in kinase activity was observed, and we conclude that phosphorylation and inactivation of PDH complex in heart muscle in obesity is not mediated by KAP, but rather is a consequence of increased lipid oxidation.