bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of protein synthesis in vitro by a lectin from Momordica charantia and by other haemagglutinins
Authors: Barbieri L , Lorenzoni E , Stirpe F
Published: August 2015

Protein synthesis by a rabbit reticulocyte lysate is inhibited by the haemagglutinating lectins from Momordica charantia and Crotalaria juncea seeds and from the roe of Rutilus rutilus, and by a commercial preparation of the mitogenic lectin from Phytolacca americana. The haemagglutinins from the seeds of Ricinus communis and of Vicia cracca acquired inhibitory activity after their reduction with 2-mercaptoethanol.
Calcium ion-dependent adenosine triphosphatase activity and plasma-membrane phosphorylation in the human neutrophil
Authors: Schneider C , Mottola C , Romeo D
Published: August 2015

A plasma-membrane fraction was isolated from a post-nuclear extract of human neutrophils by centrifugation through a linear sucrose density gradient. This fraction exhibited a Ca2+-dependent adenosine triphosphatase (ATPase) activity that could be differentiated from mitochondrial or myosin ATPase and from plasma-membrane Mg2+-dependent ATPase. When assayed in the presence of [gamma-32P]ATP, the Ca2+-dependent ATPase reaction resulted in the formation of an acid-resistant hydroxylamine-sensitive bond between the gamma-[32P] phosphate group and a membrane protein subunit with an apparent mol.wt. of 135000. Half-maximal activating effect of Ca2+ was found at 82nM and 0.18 microM for the ATPase and the formation of the 32P-membrane complex respectively. Generation of the phosphorylated product attained the steady state at 0 degrees C by about 30s, and was rapidly reversed by ADP. These results suggest that the Ca2+-activated ATPase reaction occurs through the formation of a phosphoprotein intermediate, similar to that described for some Ca2+-dependent ATPase enzymes associated with Ca2+ transport. The possibility thus exists that the neutrophil Ca2+-dependent ATPase catalyses a process of Ca2+ extrusion from the cell, thereby participating in the regulation of several Ca2+-dependent neutrophil functions.
Glycogen synthesis by hepatocytes from diabetic rats
Authors: Golden S , Wals P A , Okajima F , Katz J
Published: August 2015

Hepatocytes prepared from streptozotocin- and alloxan-diabetic rats starved for 24 h contain 0.5–2% wet wt. of glycogen. Glycogen synthesis in the hepatocytes from such rats, after prior depletion of the glycogen by glucagon injection, was studied. As distinct from cells from normal animals, there was no glycogen synthesis from glucose as sole substrate, even at concentrations of 60 mM. When supplied with glucose, a gluconeogenic precursor (lactate, dihydroxyacetone or fructose), and with glutamine there was concurrent synthesis of glucose and of glycogen. Without glutamine there was little or no glycogen synthesis. The rate of glycogen formation was in the same range as for cells from control rats. Glutamine addition markedly activated glycogen synthase in cells of starved diabetic rats, but there was no effect on phosphorylase. We obtained very little synthesis of glycogen with hepatocytes from fed diabetic rats, whereas with normal animals, synthesis by such cells equals or exceeds that obtained from starved rats. The conversion of synthase b (inactive) into the active form was studied in rat liver homogenates. The activation of the synthase in cells from starved diabetic rats is somewhat less than that from normal animals, but that from fed diabetic rats is markedly decreased compared with that in livers of fed control animals or that of starved diabetic animals.
Mössbauer spectra of photosystem-I-reaction centres from the blue-green alga Chlorogloea fritschii
Authors: Evans E H , Rush J D , Johnson C E , Evans M C W
Published: August 2015

Substantial amounts of iron have been shown by Mössbauer spectroscopy to be present in Photosystem-I preparations from the blue-green alga Chlorogloeta fritschii. Changes in the spectra on chemical reduction provide evidence that some of this iron is very similar to that found in the 4Fe-4S centres of ferredoxins. Such reduced samples also show e.p.r. signals consistent with maximum reduction of iron-sulphur centres A and B of Photosystem I. An unchanged component in the spectra indicates, assuming all centres A and B are reduced, the presence of another iron-containing species.
The subcellular distribution of alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase in dog liver
Authors: Okuno E , Minatogawa Y , Nakanishi J , Nakamura M , Kamoda N , Makino M , Kido R
Published: August 2015

The subcellular distributions of alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase in the particulate fraction of dog liver were examined by centrifugation in a sucrose density gradient. Most of both enzyme activities in the particulate fraction were localized in the mitochondria, but not in the peroxisomes.
Stimulation by secretin of bilirubin UDP-glycosyltransferase activities and of cytochrome P-450 concentration in rat liver
Authors: Ricci G L , Fevery J
Published: August 2015

The activity of bilirubin UDP-glucuronyltransferase in liver tissue was increased 1.5-fold after 90 min of secretion administration (4 i.u./h per kg body wt.) in anaesthetized Wistar rats biopsied half-hourly over a period of 2 h. In unanaesthetized R/A Wistar rats, activities of liver enzymes were assayed after administration secretin for 1 h. Bilirubin UDP-glycosyltransferase activities and cytochrome P-450 concentration were increased, but p-nitrophenol UDP-glucuronyltransferase and UDP-glucose dehydrogenase activities remained unchanged.
A reaction for the simple sensitive fluorimetric assay of heparin and 2-amino sugars
Authors: Scott John E.
Published: August 2015

1. 3,5-Diaminobenzoic acid reacted rapidly with the product from HNO2 deamination of heparin, heparan sulphate and 2-amino-2-deoxyhexoses under very mild conditions (pH3.0 and 37°C) to give stable fluorescent derivatives. 2. The fluorescence yield was rectilinearly related to the concentration of heparin etc. Less than 0.1μg of 2-amino-2-deoxyhexose was easily measurable in standard cuvettes. 3. The deamination products of glucosamine and (particularly) galactosamine were labile in the HNO2 reagent, with half-lives of 20–40min at room temperature. At 0°C they were much more stable. The analogous product from heparin was not so labile. 4. Under the standard conditions, and at room temperature, relative fluorescence yields (d-glucosamine=1.0) were: d-galactosamine, 0.75; d-gulosamine, 0.38; d-mannosamine, approx. 0.20. 5. Neutral sugars, chondroitin sulphates, DNA and N-acetylneuraminic acids did not react, nor did N-acetylamino sugars or non-deaminated hexosamines. 6. It is suggested that the Dische–Borenfreund [Dische & Borenfreund (1950) J. Biol. Chem.184, 517–522] indole method, the Kissane–Robins [Kissane & Robins (1962) J. Biol. Chem.233, 184–188] DNA assay and the proposed amino sugar method are all examples of simple aldehyde reactions. The specificity of the proposed method is considerably greater than that of the Dische–Borenfreund procedure, partly because of the much milder reaction conditions. 7. The proposed method is very reproducible, about 50–100 times as sensitive as the Elson–Morgan reaction, and 10–50 times as sensitive as the Dische–Borenfreund procedures. It is also convenient; acid hydrolysates of amino sugar-containing compounds can be directly neutralized with sodium acetate solution.
Structural characterization of ficaprenol-11 by 13C nuclear magnetic resonance
Authors: Tanaka Y , Takagi M
Published: August 2015

The location of the internal trans and cis isoprene units in ficaprenol-11 isolated from Ficus elastica was determined by 13C nuclear magnetic resonance. The alignment of the isoprene units was estimated to be in the order: omega-terminal unit, three trans units, six cis units and alpha-terminal cis alcohol unit.
Affinity labelling of the NADP+-binding site of glucose 6-phosphate dehydrogenase from Candida utilis
Authors: Bellini T , Signorini M , Dallocchio F , Rippa M
Published: August 2015

1. Periodate-oxidized NADP+ inhibits the catalytic activity of glucose 6-phosphate dehydrogenase from Candida utilis, competing with NADP+. 2. Incubation of the enzyme with the coenzyme analogue causes partial reversible inactivation of the enzyme as a result of affinity labelling of the coenzyme-binding site. 3. Some kinetic values of the reaction were calculated. 4. The inactivation can be made irreversible by treatment with NaBH4, which reduces a Schiff base formed between an aldehyde group on the coenzyme analogue and a lysine residue on the enzyme. 5. Complete inactivation can be correlated with the binding of only one inhibitor to each enzyme subunit. 6. The lysine residue involved in the binding of the inhibitor is present at the coenzyme-binding site.
Regulation of microsomal stearoyl-coenzyme A desaturase. Purification of a non-substrate-binding protein that stimulates activity
Authors: Jones D P , Gaylor J L
Published: August 2015

Crude cytosolic fraction from rat liver was examined for proteins that may be involved in regulation of microsomal stearoyl-CoA desaturase activity. Gel filtration revealed the presence of several components that either stimulate or inhibit this enzyme. In addition, other components bind the acyl-CoA substrate, thus affecting observed activities in vitro. A protein that stimulates stearoyl-CoA desaturase but does not bind substrate was purified approx. 1100-fold. The purified protein had no visible absorption spectrum and an approximate mol.wt. of 26500. Maximal stimulation of desaturase activity occurred with less than 2 micrometer purified protein. The protein was heat-labile and exhibited neither catalase nor glutathione peroxidase activity. Addition of the cytosolic protein produced no effect on the desaturase reaction stoicheiometry; the proportions O2 consumed/NADH oxidized/stearoyl-CoA desaturated remained 1:1:1. Because the Km' for stearoyl-CoA was also unchanged by addition of the cytosolic protein, no change in substrate affinity was suggested. Furthermore addition of the cytosolic protein also produced no effect on desaturase inhibition by oleoyl-CoA, which suggested that the protein does not simply relieve apparent product inhibition. These results indicate that, in analogy to other cytosolic proteins that stimulate microsomal oxidase activities, the protein may have a regulatory function, perhaps related to activity modulation via organization of the multienzymic desaturase in the membrane.
A new fluorogenic substrate for plasmin
Authors: Pierzchala P A , Dorn C P , Zimmerman M
Published: August 2015

A new fluorogenic peptide substrate for plasmin, 7-(N-succinoylalanylphenylalanyl-lysylamido)-4-methylcoumarin trifluoroacetate salt, was prepared that can be used in a simple and direct assay. The results obtained by the assay method are linear over a wide range of enzyme concentrations and sensitive enough to detect as little as 10(-5) CTA units of plasmin. By making use of the inhibitor Trasylol and the differences in kinetic constants, plasmin can be specifically assayed even in the presence of the plasminogen activator thrombin, as well as in culture fluids from HeLa cells.
Kinetic and spectroscopic evidence of cation-induced conformation changes in yeast K+-activated aldehyde dehydrogenase
Authors: Betts G F , Poole P L , Springham M G , Bostian K A
Published: August 2015

The activity, stability and spectroscopic properties of yeast K+ -activated aldehyde dehydrogenase were measured at various times after removal from, and after returning to a solution containing K+. Enzyme activity is rapidly lost on removal of most of the K+ and rapidly regained if K+ is replaced immediately. These activity changes are slower than likely rates of K+ dissociation and association. These rapid changes in concentration result in altered enzyme stability with enzyme in K+ the more stable. U.v. difference spectra are produced whenever enzyme in an activating environment (K+ or Tl+) is compared with enzyme in a non-activating environment (Tris+ or Li+). These spectral changes occur within 10s. The saturation characteristics with K+ are hyperbolic for all three phenomena of activation, stabilization and spectral change, with estimated apparent dissociation constants (Ks) for K+ of 7.5 mM, 5.5 mM and 6 mM respectively. Continued incubation of enzyme in the absence of K+ results in the accumulation of an enzyme form that re-activates only slowly on replacing K+. Stability characteristics in various concentrations of K+ over equivalent time scales are consistent with the existence of additional conformations. Spectroscopic evidence also indicates such additional slow conformation changes. Results have been interpreted in terms of two separate conformation transitions induced or stabilized by K+.
Acute effects of ethanol on the perfused rat liver. Studies on lipid and carbohydrate metabolism, substrate cycling and perfusate amino acids
Authors: Topping David L. , Clark Dallas G. , Storer Gerald B. , Trimble Rodney P. , Illman Richard J.
Published: August 2015

1. Livers from fed rats were perfused in situ with whole rat blood containing glucose labelled uniformly with 14C and specifically with 3H at positions 2, 3 or 6. 2. When ethanol was infused at a concentration of 24μmol/ml of blood the rate of utilization was 2.8μmol/min per g of liver. 3. Ethanol infusion raised perfusate glucose concentrations and caused a 2.5-fold increase in hepatic glucose output. 4. Final blood lactate concentrations were decreased in ethanol-infused livers, but the mean uptake of lactate from erythrocyte glycolysis was unaffected. 5. Production of ketone bodies (3-hydroxybutyrate+3-oxobutyrate) and the ratio [3-hydroxybutyrate]/[3-oxobutyrate] were raised by ethanol. 6. Formation of 3H2O from specifically 3H-labelled glucoses increased in the order [6-3H]<[3-3H]<[2-3H]. Production of 3H2O from [2-3H]glucose was significantly greater than that from [3-3H]glucose in both control and ethanol-infused livers. Ethanol significantly decreased 3H2O formation from all [3H]glucoses. 7. Liver glycogen content was unaffected by ethanol infusion. 8. Production of very-low-density lipoprotein triacylglycerols was inhibited by ethanol and there was a small increase in liver triacylglycerols. Very-low-density-lipoprotein secretion was negatively correlated with the ratio [3-hydroxybutyrate]/[3-oxobutyrate]. Perfusate fatty acid concentrations and molar composition were unaffected by perfusion with ethanol. 9. Ethanol decreased the incorporation of [U-14C]glucose into fatty acids and cholesterol. 10. The concentration of total plasma amino acids was unchanged by ethanol, but the concentrations of alanine and glycine were decreased and ([glutamate]+[glutamine]) was raised. 11. It is proposed that the observed effects of ethanol on carbohydrate metabolism are due to an increased conversion of lactate into glucose, possibly by inhibition of pyruvate dehydrogenase. The increase in gluconeogenesis is accompanied by diminished substrate cycling at glucose–glucose 6-phosphate and at fructose 6-phosphate–fructose 1,6-bisphosphate.
Activation of AMP aminohydrolase during skeletal-muscle contraction
Authors: Rahim Z H A , Lutaya G , Griffiths J R
Published: August 2015

AMP aminohydrolase activity is enhanced by 60% after 5 s tetanic stimulation of phosphorylase kinase-deficient mouse muscle and after 60 s tetanus in normal mice. During the recovery from tetanus the activity in the contralateral leg is similarly enhanced. The activation is stable to 1000-fold dilution and has a half-life of approx. 1 h.
Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Effect of adenosine 3′:5′-cyclic monophosphate
Authors: Roberts S , Morelos B S
Published: August 2015

Investigations were carried out on the phosphorylation of ribosomal proteins in vivo in cerebral cortices of immature rats. Two-dimensional electrophoresis revealed that the cerebral 40S subunit contained at least four ribosomal proteins which were phosphorylated in animals given [32P]orthophosphate intracisternally. These proteins exhibited electrophoretic properties similar to those of the constitutive basic proteins S2, S3a, S5 and S6. The cerebral 60S subunit contained several proteins that were phosphorylated in vivo, including three basic proteins with electrophoretic mobilities similar to those of ribosomal proteins L6, L14 and L19. Four other proteins associated with the 60S subunit that were more acidic were also phosphorylated. Phosphorylated congeners of 40S and 60S ribosomal proteins could often be detected in distinct protein-stained spots on two-dimensional electrophoretograms. The cerebral S6 protein consisted of at least five distinct species in different states of phosphorylation. Administration of N6O-2′ dibutyryl cyclic AMP increased the proportion of the more phosphorylated congeners of the S6 protein, but appeared to have little or no effect on phosphorylation of other cerebral ribosomal proteins. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated S6-protein phosphorylation; N2O2′-dibutyryl cyclic GMP had no effect on this process. These observations indicate that several ribosomal proteins of both subunits are normally phosphorylated in rat cerebral cortex in situ. The results also suggest that selective and specific alterations in the phosphorylation state of the S6 ribosomal protein of the cerebral 40S subunit may accompany the production of cyclic AMP during neural activation.
Metabolic oxidation of the ethynyl group in 4-ethynylbiphenyl
Authors: Wade A , Symons A M , Martin L , Parke D V
Published: August 2015

1. 4-Ethynylbiphenyl undergoes extensive metabolism in the rat and the rabbit, involving aromatic hydroxylation and oxidation of the ethynyl group. No metabolites containing the intact ethynyl group were detected. 2. In the rat unchanged 4-ethynylbiphenyl was concentrated initially in the adipose tissue. No other tissues accumulated significant amounts of radioactivity. 3. The major metabolites were the same in both the rat and the rabbit, namely 4′-hydroxybiphenyl-4-ylacetic acid (90-95% of dose) and biphenyl-4-ylacetic acid (2-10% of dose). 4. Excretion was slower in the rat than in the rabbit, probably because of greater biliary and faecal excretion in the rat. Biliary excretion and enterohepatic circulation of biphenyl-4-ylacetic acid and 4′-hydroxybiphenyl-4-ylacetic acid were demonstrated in the rat.
Synthesis of N-acetyl-l-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport
Authors: Patel Tarun B. , Clark John B.
Published: August 2015

1. The synthesis and efflux of N-acetyl-l-aspartate from brain mitochondria of rats of different ages has been studied. 2. Brain mitochondrial State 3 (+ADP) respiration rate, using 10mm-glutamate and 2.5mm-malate as substrates, increases during the suckling period and reaches approx. 50% of the adult value at 17 days after birth [adult State 3 respiration rate=160±7ng-atoms of O/min per mg of mitochondrial protein(mean±s.d.; n=3)]. 3. The influence of 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate on aspartate efflux from brain mitochondira from rats of different ages oxidizing glutamate and malate was studied. In all cases the aspartate efflux in State 3 was greater than in State 4, but, whereas the aspartate efflux in State 3 increased as the animals developed, that of State 4 showed only a small increase. However, the rate of aspartate efflux in the presence of pyruvate or 3-hydroxybutyrate as well as glutamate and malate was approx. 60–65% of that in the presence of glutamate and malate alone. 4. An inverse relationship between aspartate efflux and N-acetylaspartate efflux was observed with adult rat brain mitochondria oxidizing 10mm-glutamate and 2.5mm-malate in the presence of various pyruvate concentrations (0–5mm). 5. N-Acetylaspartate efflux by brain mitochondria of rats of different ages was studied in States 3 and 4, utilizing 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate as acetyl-CoA sources. A similar pattern of increase during development was seen in State 3 for N-acetylaspartate efflux as for aspartate efflux (see point 3 above). Also only very small increases in N-acetylaspartate efflux occurred during development in State 4.6. Rat brain mitochondria in the presence of iso-osmotic N-acetylaspartate showed some swelling which was markedly increased in the presence of malate. 7. It is concluded that N-acetylaspartate may be synthesized and exported from both neonatal and adult rat brain mitochondria. It is proposed that the N-acetylaspartate is transported by the dicarboxylic acid translocase and may be an additional mechanism for mitochondrial/cytosolic carbon transport to that of citrate.
The binding of the coenzyme pyridoxal 5'-phosphate and analogues of the substrate-coenzyme complex to tyrosine decarboxylase
Authors: Orlacchio A , Borri-Voltattorni C , Turano C
Published: August 2015

Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bound at the active site with great affinity. From a comparison of the interaction of a number of such compounds with the apoenzyme the delta G0 values for the binding of the substrate carboxy and phenyl groups and of the coenzyme aldehydic group were determined to be equal to (or more negative than) ‒3.8. ‒8.4 and ‒12.5kJ/mol (-0.9, ‒1.9 and ‒3kcal/mol) respectively; the delta G0 for the binding of the coenzyme phosphate group was shown to be more negative than ‒20.5kJ/mol (-4.9kcal/mol). Two features of the binding process of the coenzyme-substrate analogues to tyrosine decarboxylase have already been found in the case of tyrosine aminotransferase [Borri-Voltattorni, Orlacchio, Giartosio, Conti & Turano (1975) Eur. J. Biochem. 53, 151-160]: (1) in the binding of the substrate to the enzyme a significant fraction of the instrinsic delta G0 appears to be used for some associated endoergonic process; (2) the delta H0 and delta S0 of binding appear to be very sensitive indicators of the correct alignment of the substrate-coenzyme and analogues at the active site.
Identification of cyanogen bromide peptides involved in intermolecular cross-linking of bovine type III collagen
Authors: Nicholls A C , Bailey A J
Published: August 2015

Cyanogn bromide peptides derived from bovine type III collagen and containing reducible cross-links were isolated and identified. Two peptides, alpha 1 (III)CB7 and alpha 1 (III)CB9B, from within the helical portion of the molecule were shown to contain the ‘amino donor’ residues cross-linked to non-helical ‘aldehyde donor’ residues in the formation of cross-links. This information, in conjunction with previously published data for the order of the cyanogen bromide peptides [Fietzek, Allman, Rauterberg & Wachter (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 84-86], suggests that in type III collagen intermolecular cross-links are located in the end-overlap regions, so as to stabilize a quarter-stagger arrangement of molecules within the fibre in a similar manner to that proposed for type I and type II collagens.
New relations for activity analysis of associating enzymes
Authors: Thomes J C , Archambault De Vençay J , Julien R
Published: August 2015

New relations for catalytic-activity analysis are proposed for simple association/dissociation equilibria between the mono- and oligo-meric forms of enzymes in the case where these equilibria evolve very slowly in comparison with the binding of the substrates. This analysis of activity versus total enzyme concentration leads rapidly to information, complementary to that given by physico-chemical methods, on specific activities, the degree of polymerization of the enzyme forms and on their dissociation constants.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets
Authors: Mitropoulos Konstantinos A. , Knight Brian L. , Reeves Bernard E. A.
Published: August 2015

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg2+. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent Km of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent Km and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent Km for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high Km. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent Km for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent Km of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.
The site of phosphorylation of troponin I in the perfused rabbit heart. The effect of adrenaline
Authors: Moir A J G , Solaro R J , Perry S V
Published: August 2015

1. On treatment of the perfused rabbit heart with adrenaline, the total covalently bound phosphate of troponin I increased from 1.14 mol of phosphate/mol to 1.86 mol of phosphate/mol. 2. Covalently bound phosphate could be identified only in the region of the molecule of cardiac troponin I consisting of residues 1-48. 3. When 32P-labelled orthophosphate was present in the perfusion medium the phosphate at serine-20 became radioactively labelled. This residue was the only significant site of phosphorylation that could be identified. 4. The addition of adrenaline caused a 4-5-fold increase in covalently bound [32P]phosphate. Virtually all of the 32P was located at serine-20. 5. It was concluded from these studies that the extent of phosphorylation of serine-20 of cardiac troponin I increased from 30-40% in the control perfused heart to about 100% in the presence of adrenaline.
Identification of O-acetyl-5-methoxytryptophenol in the pineal gland by gas chromatography-mass spectrometry
Authors: Smith I , Francis P , Leone R M , Mullen P E
Published: August 2015

The presence of O-acetyl-5-methoxytryptophol in the pineal glands of rats kept in the dark for 8 h, but not in the light, has been shown by means of g.l.c.-mass spectrometry. It is suggested that this compound may be the biologically active precursor of circulating methoxytryptophol.
Isoenzymes of membrane-bound β-glucosidase of human spleen
Authors: Yaqoob M , Carroll M
Published: August 2015

Normal human spleen contains two forms of membrane-bound beta-glucosidase, distinguishable by their thermostability and kinetic properties. The spleen from a patient with adult Gaucher's disease was deficient in the major, thermolabile, form of the enzyme.
Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle
Authors: Walsh T P , Winzor D J , Clarke F M , Masters C J , Morton D J
Published: August 2015

The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 × 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 × 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant.
Interaction of aldolase with actin-containing filaments. Structural studies
Authors: Stewart M , Morton D J , Clarke F M
Published: August 2015

Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed.
Oestrogen-induced synthesis of thiamin-binding protein in immature chicks. Kinetics of induction, hormonal specificity and modulation
Authors: Muniyappa Kalappagowda , Adiga P. Radhakantha
Published: August 2015

A specific radioimmunoassay procedure was developed to monitor the plasma concentrations of thiamin-binding protein, a minor yolk constituent of the chicken egg. By using this sensitive assay, the kinetics of oestrogen-induced elaboration of this specific protein in immature chicks was investigated. After a single injection of the steroid hormone, with an initial lag period of 4–5h the thiamin-binding protein rapidly accumulated in the plasma, attaining peak concentrations around 75h and declining thereafter. A 4-fold amplification of the response was noticed during the secondary stimulation, and this increased to 9-fold during the tertiary stimulation with the steroid hormone. The magnitude of the response was dependent on the hormone dose, and the initial latent period and the duration of the ascending phase of induction were unchanged for the hormonal doses tested during both the primary and secondary stimulations. The circulatory half-life of the protein was 6h as calculated from the measurement of the rate of disappearance of the exogenously administered 125I-labelled protein. Simultaneous administration of progesterone, dihydrotestosterone or corticosterone did not alter the pattern of induction. On the other hand, hyperthyroidism markedly decreased the oestrogenic response, whereas propylthiouracil-induced hypothyroidism had the opposite effect. The anti-oestrogen E- and Z-clomiphene citrates, administered 30min before oestrogen, effectively blocked the hormonal induction. α-Amanitin and cycloheximide administered along with or shortly after the sex steroid severely curtailed the protein elaboration. A comparison of the kinetics of induction of thiamin- and riboflavin-binding proteins by oestrogen revealed that, beneath an apparent similarity, a clear-cut difference exists between the two vitamin-binding proteins, particularly with regard to hormonal dose-dependent sensitivity of induction and the half-life in circulation. The steroid-mediated elaboration of the two yolk proteins thus appears to be not strictly co-ordinated, despite several common regulatory features underlying their induction.
Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves
Authors: Barankiewicz J , Paszkowski J
Published: August 2015

The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed.
The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase
Authors: Methven Patricia , Lemon Marius , Bhoola Kanti
Published: August 2015

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (Km 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (Km 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg2+. Mn2+ (3mm) was as effective as Mg2+ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca2+ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg2+ was 3mm. At concentrations of Mg2+ between 0.1 and 1mm, 1mm-Ca2+ was activatory, and at concentrations of Mg2+ below 0.1mm, 1mm-Ca2+ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.
Metabolic control of the potassium permeability in pancreatic islet cells
Authors: Henquin Jean-Claude
Published: August 2015

The K+ permeability of pancreatic islet cells was studied by monitoring the efflux of 86Rb+ (used as tracer for K+) from perifused rat islets and measuring the uptake of 42K+. Glucose markedly and reversibly decreased 86Rb+ efflux from islet cells and this effect was antagonized by inhibitors of the metabolic degradation of the sugar, i.e. mannoheptulose, iodoacetate, glucosamine and 2-deoxyglucose. Among glucose metabolites, glyceraldehyde reduced the K+ permeability even more potently than did glucose itself; pyruvate and lactate alone exhibited only a small effect, but potentiated that of glucose. Other metabolized sugars, like mannose, glucosamine and N-acetylglucosamine, also decreased 86Rb+ efflux from islet cells. Fructose was effective only in the presence of glucose. Non-metabolized sugars like galactose, 2-deoxyglucose and 3-O-methylglucose had no effect. The changes in K+ permeability by agents known to modify the concentrations of nicotinamide nucleotides, glutathione or ATP in islet cells were also studied. Increasing NAD(P)H concentrations in islet cells by pentobarbital rapidly and reversibly reduced 86Rb+ efflux; exogenous reduced glutathione produced a similar though weaker effect. By contrast, oxidizing nicotinamide nucleotides with phenazine methosulphate or Methylene Blue, or oxidizing glutathione by t-butyl hydroperoxide increased the K+ permeability of islet cells. Uncoupling the oxidative phosphorylations with dicumarol also augmented 86Rb+ efflux markedly. In the absence of glucose, but not in its presence, methylxanthines reduced 86Rb+ efflux from the islets; such was not the case for cholera toxin or dibutyryl cyclic AMP. Glucose and glyceraldehyde had no effect on 42K+ uptake after a short incubation (10min), but augmented it after 60min; the effect of glucose was suppressed by mannoheptulose and not mimicked by 3-O-methylglucose. The results clearly establish the importance of the metabolic degradation of glucose and other substrates for the control of the K+ permeability in pancreatic islet cells and support the concept that a decrease in the K+ permeability represents a major step of the B-cell response to physiological stimulation.
Stabilization of rat liver tyrosine aminotransferase in vivo by pyridoxine administration
Authors: Snape B M , Badawy A A , Evans M
Published: August 2015

Administration of pyridoxine stabilizes rat liver tyrosine aminotransferase in vivo, whereas administration of cortisol, cyclic AMP, glucagon, insulin, tryptophan or tyrosine does not. The results of these and other experiments with pyridoxine are discussed in relation to the mechanisms of action of this vitamin on the activity of the enzyme.
Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches
Authors: Mollinedo F , Larraga V , Coll F J , Muñoz E
Published: August 2015

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.
Requirement for guanosine triphosphate for cholera-toxin-catalysed incorporation of adenosine diphosphate ribose into rat liver plasma membranes and for activation of adenylate cyclase
Authors: Doberska C A , MacPherson A J S , Martin B R
Published: August 2015

1. Cholera toxin was shown to require the presence of GTP to activate rat liver plasma-membrane adenylate cyclase. ATP did not affect the activation process. 2. Cholera toxin catalysed the incorporation of 32P from NAD labelled in the alpha-phosphate group of the ADP moiety into a rat liver plasma-membrane protein with a subunit mol.wt. of 42 500. This is taken to demonstrate ADP-ribosylation. The ADP-ribosylation of this protein also required GTP and was unaffected by ATP. 3. Nicotinamide inhibited both the activation of adenylate cyclase by cholera toxin and the ADP-ribosylation of the protein of 42 500 subunit mol wt. Neither the activation nor the ADP-ribosylation could be reversed by treatment with nicotinamide in the presence of cholera toxin.
Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue
Authors: Lucas John J. , Tepperman Helen , Tepperman Jay
Published: August 2015

Microsomal preparations from rat adipose tissue catalyse the transfer of [14C]mannose from GDP-[14C]mannose to an endogenous acceptor forming a [14C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [14C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate.

The kinetics of transfer of [14C]mannose from GDP-[14C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The Km and Vmax. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.

In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [14C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose.

Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.
Proteins of the kidney microvillar membrane. Asymmetric labelling of the membrane by lactoperoxidase-catalysed radioiodination and by photolysis of 3,5-di[125I]iodo-4-azidobenzenesulphonate
Authors: Booth A G , Kenny A J
Published: August 2015

Two methods were used to label pig kidney microvillar membrane proteins from the luminal and cytoplasmic surfaces of closed membrane vesicles. The first method was lactoperoxidase-catalysed radioiodination. The enzyme reagents, lactoperoxidase and glucose oxidase, were positioned inside the vesicles before sealing or externally after sealing, iodination being initiated by the subsequent addition of glucose and 125I-. After resolution of the labelled proteins by electrophoresis in the presence of dodecyl sulphate, asymmetric labelling patterns on radioautographs were observed. However, the major disadvantage of this method is the high degree of intramembrane labelling of the fatty acid chains of membrane lipids, a reaction that undermines any conclusions about the location of the label in that region of the protein supposedly exposed at the surface of the membrane. The second method overcame this disadvantage. A new hydrophilic photoreagent, 3,5-di[125I]iodo-4-azidobenzesulphonate, was synthesized via the intermediate, diazotized 3,5-di[125I]iodosulphanilic acid. It was transported by a Na+-dependent system into microvillar vesicles, thus permitting labelling from either side of the membrane when the vesicles were photolysed. The labelling of membrane lipids was less than with the first method and was essentially confined to the polar headgroups. The activity of several microvillar peptidases survived the labelling reaction and they could be identified in the immunoprecipitates after resolution of the detergent-solubilized membrane proteins by crossed-immunoelectrophoresis. Treatment with papain converted the detergent-solubilized form of susceptible enzymes into the proteinase-solubilized form, which lacked the intramembrane domain and any portion exposed at the cytoplasmic surface. Radioautography established that aminopeptidases M and A, dipeptidyl peptidase IV and neutral endopeptidase were transmembrane proteins. This novel approach to the investigation of membrane topology may be applicable to other complex membranes.
Purification and properties of lung lectin. Rat lung and human lung β-galactoside-binding proteins
Authors: Powell J T
Published: August 2015

Lung is one of the organs of the rat with a particular abundance of haemagglutinating activity that is inhibited by beta-galactosides. This lectin activity can be attributed to a single protein that has been purified from rat lung; a similar protein has been purified from human lung. The molecular weights and subunit structures were estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the human lung lectin appeared to be composed to two identical subunits, mol.wt. 14500, whereas rat lung lectin was observed as both a dimer and a tetramer of one subunit type, mol.wt. 13500. Both lectins bind to disaccharides or oligosaccharides with terminal beta-linked galactose residues. The carbohydrate moiety may be free [lactose or D-galactopyranosyl-beta-(1 leads to 4)-thiogalactopyranoside], protein-bound (asialofetuin) or lipid-bound (cerebrosides). The molecular properties of the beta-galactoside-binding proteins of rat lung and human lung are closely similar to those of embryonic chick muscle lectin [Nowak, Kobiler, Roel & Barondes (1977) Proc. Natl. Acad. Sci. U.S.A. 73, 1383–1387] and calf heart lectin [De Waard, Hickman & Kornfeld (1976) J. Biol. Chem. 251, 7581–7587].