Showing papers in "Biochemical Society Transactions in 1980"

Active-site probes of flavoproteins

Abstract: Flavins are very versatile coenzymes, functioning with considerable efficiency in a wide variety of enzymic reactions involving either two-electron or one-electron transfers, with both functions often catalysed by the same enzyme. They are responsible for catalysing the dehydrogenation of many different types of compounds, including dithiols, reduced nicotinamide nuclwtides, alcohols and a-hydroxy acids, amines and a-amino acids, and even saturated C-C bonds, provided that a suitable activating group such as a carbonyl residue is situated a/% to the bond to be oxidized. In the process of catalysing these dehydrogenation reactions, the flavin is itself reduced, and, in order to function catalytically, the oxidized form must be regenerated at the expense of reduction of some acceptor. The acceptor may be in some cases the oxidized form of the same type of compound that serves as reducing substrate, e.g. it might be a disulphide, an oxidized nicotinamide nucleotide or an unsaturated compound such as fumarate or crotonylCoA. In such a case the enzyme might conveniently be classified as a transhydrogenase and subclassified as a C-C, C-S, C-N or N-N transhydrogenase, depending on the nature of the atoms acting as hydrogen donor and hydrogen acceptor (Hemmerich & Massey, 1979). In most cases, however, the acceptor molecule will be molecular 0, or another redox protein, such as an iron-sulphur protein or a cytochrome. In the latter cases the flavoprotein necessarily acts as a mediator between two-electron and one-electron transfers. Flavoproteins fill a unique spot in biochemistry with this capacity. An equal richness of possibilities exists in the reactions of different flavoproteins with molecular 0,. In some cases one-electron transfer is carried out, with superoxide (02-) and flavin semiquinone as the immediate products of the reaction. In other cases a direct two-electron reduction of 0, to H,O, appears to occur, and in another class of flavoproteins, the mono-oxygenases, one atom of the 0, molecule is incorporated into H20 and the other is incorporated into another substrate of the enzyme, to form an oxygenated product.

ADP-ribosylation of nuclear proteins.

Abstract: ADP-ribosylation can be defined as the postsynthetic modification of protein by the covalent attachment of the ADP-ribose moiety of NAD+. ADP-ribosylation of elongation-factor Tu is responsible for the inhibition of protein synthesis by both diptheria and Pseudomonas aeroginosa toxins (Hilz & Stone, 1976). The activation of membrane adenylate cyclase by cholera toxin is thought to occur by ADP-ribosylation of the adenylate cyclase-associated GTP binding protein (Gill & Meren, 1978). It has also been proposed as a mechanism by which Escherichia coli RNA polymerase activity is modified during bacteriophage T4 infection (Goff, 1974; Rohrer et al., 1975; Skorko et al., 1977). Coliphage N4 has also been reported to contain an intrinsic ADP-ribosyltransferase activity (Pesce et al., 1976). ADP-ribosyltransferase activity is also present in the mitochondria and nuclei of all eukaryotic organisms examined to date. The field of ADP-ribosylation has been comprehensively reviewed by Sugimura (1973), Hilz & Stone (1976) and Hayaishi & Ueda (1977). The present review is concerned exclusively with ADP-ribosylation carried out in eukaryotic nuclei and will concentrate on the large amount of data that has emerged subsequent to the publication of other reviews. The enzyme responsible for ADP-ribosylation in nuclei is termed poly(ADP-ribose) synthetase or polymerase; the name is derived from the fact that, unlike other ADP-ribosyltransferases, the enzyme is capable of synthesizing a protein-bound homopolymer of ADP-ribose, poly(ADP-ribose) (Chambon et al., 1966; Reeder et al., 1967; Fujimura et al., 1967). NAD is cleaved at the nicotinamide-ribose bond (bond energy 34 kJ/mol) and the ADP-ribose moiety transferred to either a nuclear protein or a protein-bound ADP-ribose molecule. The polymer thus formed is degraded by another nuclear enzyme, poly(ADP-ribose) glycohydrolase, producing the free monomeric form of ADP-ribose (Miwa & Sugimura, 1971) (Fig. 1 ) .

Monoclonal antibodies that bind to the human erythrocyte-membrane glycoproteins glycophorin A and Band 3

Abstract: Monoclonal antibodies from hybrid myelomas offer a powerful approach to the biochemistry of cell-surface components and the identification and marking of different types of cell (Milstein et al., 1979). Hybrid myelomas are made by hybridizing antibody-secreting spleen cells from an immunized mouse with a myeloma cell line that can be grown in culture or passaged in mice. The resulting hybrids make antibody molecules of a unique amino acid sequence specified by the spleen cell. Hybrid myelomas were made producing monoclonal antibodies to surface molecules of the human erythrocyte. Hybrids LICR LON/RlO, R20.16 and R18 were made by fusing spleen cells from mice immunized with human erythrocytes with the mouse myeloma NS 1, with poly(ethy1ene glycol) as described by Galfre et al. (1977), except that they were cloned from the outset in soft agar. The air-buffered medium L1S (Leibovitz, 1963) was used, and after dilution of the poly(ethy1ene glycol) and centrifugation, cells were resuspended with LO6 thymocytes/ml as a feeder (Andersson et al., 1977) in 0.25% (w/v) agar, in Ll5 medium + 20% h/v) foetal-calf serum and 0.5 ml portions were put in each of 48 2 cm2 agar-subbed wells. When the agar was set it was overlaid with 1.5 ml of Dulbecco’s medium (Smith et al., 1Y60) with serum and HAT (Littlefield, 1964). Visible hybrid colonies were picked from the agar 11 days after fusion, and tested for production of anti-erythrocyte antibody 4 days later.

Muscle alanine synthesis and hepatic gluconeogenesis.

Abstract: Gluconeogenesis is the metabolic process involving the synthesis de novo of glucose molecules from other carbon precursors during situations in which there is an enhanced oxidative utilization of body glucose or in which there is a dietary insufficiency of carbohydrate to supply the body requirements for oxidative glucose metabolism. Glucose synthesis is, for the most part, confined to the liver, although in prolonged starvation in man the contribution by the kidney becomes substantial (Owen et al., 1969). In certain situations the hepatic gluconeogenic pathway may be operative in the face of an adequate dietary supply of carbohydrate, when carbon precursors are diverted towards the repletion of liver glycogen rather than to glucose synthesis for hepatic release into the circulation. Such situations include re-feeding after starvation (Hems et al., 1972) and the analogous situation of weaning following the suckling stage of neonatal development (K. Snell, unpublished work). Hepatic gluconeogenesis is regulated within the liver by metabolic and hormonal factors and indirectly by the supply of gluconeogenic precursors from peripheral tissues (see Exton, 1972 for review). Not only are the various precursors utilized at different rates for glucose formation within the liver, but the physiological circulating concentrations of many precursors lie below the saturating levels for hepatic glucose synthesis, as demonstrated in the perfused liver in vitro (Exton & Park, 1967; Mallette et al., 1969a) and the whole animal in vivo (Aikawa et al., 1972). Thus both the rate of precursor supply and the nature of the precursors will influence the rate of glucose formation by the liver. The principal endogenous glucogenic precursors are lactate, pyruvate and amino acids derived from skeletal muscle and other tissues, and glycerol derived from the hydrolysis of triacylglycerol in adipose tissue. Cahill and his colleagues have quantified the relative contribution of these various precursors to glucose formation in man, particularly during starvation when gluconeogenesis is increased (Cahill, 1970). As starvation is extended beyond 24 h, the relative contribution of proteinderived amino acids to total hepatic glucose production increases. A similar increased contribution has been observed in diabetic subjects where gluconeogenesis is also elevated above normal (Wahren et al., 1972). Of these amino acids, the contribution by alanine to hepatic glucose output is quantitatively the most significant. Hepatic extraction of alanine exceeds that of all other amino acids in the rat (Ishikawa, 1977) and man (Felig et al., 1969a) in vivo, and, in addition, it has been shown in the perfused liver in vitro that alanine is the best amino acid substrate for gluconeogenesis in the adult rat (Ross et al., 1967; Exton & Park, 1967). A specific stimulation of alanine transport into the liver cell has been found in starvation and after glucagon stimulation of gluconeogenesis (Mallette et al., 19696; Fehlmann et al., 1979).

The role of calcium in the regulation of mitochondrial metabolism

Abstract: presence of rotenone and oligomycin (Scott & Nicholls, 1980). This combination of inhibitors should prevent any Ca’+ uptake into the matrices of the mitochondria. As is shown in Table 1, the pelleted Ca2+ was greatly diminished under these conditions, whereas the solubilized CaZ+ actually increases. This clearly indicates that the major proportion of the synaptosomal CaZ+ is located in a compartment that is responsive to the mitochondrial membrane potential, and can therefore be identified with the mitochondrial matrix. Abolition of the mitochondrial membrane potential of necessity inhibits oxidative phosphorylation. Thus it could be argued that the diminished pellet Ca2+ resulted from the inhibition of the ATP supply to a Ca2+dependent ATPase accumulating Ca2+ within digitonin-resistant vesicles. However glycolysis allows significant ATP concentrations to be maintained under these conditions (Scott & Nicholls, 1980), whereas the presence of oligomycin alone, which inhibits the mitochondrial ATP synthase without lowering the membrane potential of the internal mitochondria (Scott & Nicholls, 1980), does not diminish the Ca2+ in the pellet (Table 1). The conclusion is therefore that the mitochondrial matrix represents the major site of Ca2+ accumulation within isolated synaptosomes amounting to some 85nmol of Ca2+/mg of mitochondrial protein under these conditions. This is well within the capacity of brain mitochondria to accumulate Ca2+, but is sufficient to saturate the efRux pathway (Nicholls & Scott, 1980). It is therefore feasible that the mitochondria regulate cytosolic Ca2+ concentrations under these conditions. There is, however, one proviso to make: the total Ca2+ content of the synaptosome is still increasing at 16min (Fig. l), implying that the internal mitochondria are capable of decreasing the cytosolic free Ca2+ concentration sufficiently to impose a net inward Ca2+ flux across the plasma membrane. It remains to be established therefore whether this represents an approach towards a steady-state, or whether a proportion of synaptosomes with a defective plasma membrane Ca2+-eWux pathway contributes disproportionately to the observed Ca2+ uptake by the total synaptosomal population. Previous studies (Kendrick et al., 1977; Blaustein et al.. 1978; Rahamimoff & Abramovitz, 1978) emphasized the role of ATP-dependent Ca*+ accumulation by intra-synaptosomal reticular membranes. However, the present study demonstrates that the mitochondria, with their elaborate Ca2+-regulatory mechanism, play the predominant role within the cytosol of the isolated synaptosome.

A colorimetric assay for mucous glycoproteins using Alcian Blue

Abstract: The present paper describes a simple, sensitive technique for measurement of respiratory mucous glycoproteins that involves their precipitation with the cationic dye Alcian Blue. The assay was adapted from that described by Whiteman (1973) for measurement of urinary glycosaminoglycans. This method is useful for measurement of mucins secreted in response to stimulating agents in isolated airway preparations of animals, and of human airway secretions obtained by bronchoswpy. The procedure is also useful for measurement of mucin content of fractions obtained after column chromatography. The Alcian Blue assay was developed as an alternative to the usual methods of glycoprotein determination, which measure total hexose wntent (phenol/sulphuric acid, anthrone, orcinol), which are of limited sensitivity (Mantle & Allen, 1978) and are hazardous to operate, requiring the use of large amounts of sulphuric acid. Mucous glycoproteins are polyanionic, having both sialic acid and sulphate residues attached to their oligosaccharide structures. It is these groups that interact with Alcian Blue to form insoluble complexes. A 0.1% (w/v) solution of Alcian Blue in 0.1 M-sodium acetate/acetic acid buffer, pH 5.8, containing 25 mM-MgC1, was clarified by sedimentation at 1870ga, for 30min at 2OoC in an MSE Mistral 2L centrifuge. To 3.0ml portions of tracheal mucus samples in plastic test tubes was added 1 ml of the purified Alcian Blue solution. After at least 2 h equilibration at room temperature, mucus-Alcian Blue complexes were sedimented at 1870ga, for 30min at 2OOC. Supernatant solutions were discarded, and each pellet was washed twice by successive resuspension in 40% (v/v) ethanoV0.1 Msodium acetate buffer, pH 5.8, containing 25 mM-MgCI,, and sedimentation at 1870ga, for IOmin at 2OOC. A 40% (w/v) Manoxol 1 B (sodium dibutylsulphosuccinate) solution was clarified by filtration. Mucus-dye complexes were dissociated by addition of 1 ml of the Manoxol 1B solution and ultrasonication at SOW for 10s with a Dawes soniprobe. Samples were sedimented for 1 min as described above to eliminate the foam generated during sonication. Absorbance at 620nm was measured in a Cecil CE272 spectrophotometer. A calibration curve for Alcian Blue precipitation was determined for a cat tracheal mucus preparation (Fig. 1) obtained as described by Gallagher et al. (1975). The protein content of this preparation was determined by alkaline hydrolysis followed by ninhydrin reaction, and the carbohydrate content was determined by g.1.c. (Hall, 1978). The carbohydrates present were those typical of cat tracheal mucins (Gallagher et al., 1975) and contained no deoxyribose, ribose or uronic acids. This mucus preparation was not contaminated by nucleic acids or proteoglycans. The resultant absorbance at 620nm of the Alcian Blue 0.6 r

Protein and amino acid turnover during and after exercise.

Abstract: MICHAEL J. RENNIE,* RICHARD H. T. EDWARDS,* C. T. MERVYN DAVIES,? STEPHEN KRYWAWYCH,* DAVID HALLIDAYJ JOHN C. WATERLOW$ and DAVID J. MILLWARD$ *Department of Human Metabolism, University Colrege London Medical School, London W C l E SJJ, U.K., ?Medical Research Council Environmental Physiology Unit and $Department of Human Nutrition, London School of Hygiene and Tropical Medicine, London W C l E 7HT, U.K., and $Division of Human Nutrition, Clinical Research Centre, Northwick Park Hospital, Harrow HA1 JUJ, U.K.

Physical Chemical Aspects of Cell Surface Events in Cellular Regulation

Abstract: One of the manifest virtues of the Singer-Nicolson fluid-mosaic model of the cell membrane was that it provided tangible means whereby the membrane could act as a transducer of information. Signals received by membrane-spanning proteins and glycoproteins could result in significant biochemical events on the other side of the Davson-Danielli lipid-bilayer palisade. The fluid-mosaic model was, of course, erected in part to account for this known function of biological membranes and drew on evidence, both structural and functional, for transmembrane communication in its formulation. Nevertheless, its appearance was a considerable stimulus to work in the field of ligandreceptor interactions and the volume under review gathers together recent contributions in a number of areas where refinement of experimental technique has provided data sufficiently precise and extensive to permit analysis by the techniques of the theoretical physical chemist. It contains the formal papers together with some 60 pages of transcribed discussion of an international conference held at Bethesda in October 1978. The avowed aim of the conference was to catalyse interaction between specialists, both theoreticians and experimentalists, in an apparent diversity of disciplines and the Proceedings contain papers reflecting this spread of interests; he would be indeed well informed who could profess detailed acquaintance with the experimental systems from neurobiology, endocrinology, immunology, pharmacology and toxicology treated in the various sessions of the meeting. However, the role of the plasma membrane as the cell’s primary sense organ runs throughout the whole and should permit the reader to emerge Theseus-like having threaded the labyrinth. The kernel of the book is probably represented by a number of papers dealing with mathematical and physical models of membrane-ligand interaction, e.g. on receptor aggregation and its consequences (J. Schlessinger), a computer model of membrane fluidity (L. Finegold), reversible binding of bivalent antigen (A. s. Perelson) and a random-hit model by coupling (R. N. Bergman et al.). These are intermixed with experimentally based papers dealing with systems in which physiological function and molecular geometry have been highly correlated, such as the calcium pump of the sarcoplasmic reticulum (A. E. Shamoo), with ‘clean’ cell-agonist interacting systems, such as insulin with cultured cell lines (P. Demeyts), and with definitely ‘dirty’ systems such as the activation of B lymphocytes by antiimmunoglobulin G antibodies (D. G. Sieckmann et al.). The papers are generally self-contained and authoritative. Unusually full references for this type of publication will enable the reader ready access to the literature of any unfamiliar field, should he so wish. The reprinted discussion, jokes from the Chair and all, are in the main pertinent and highlight strengths and weaknesses in the formal papers. The constraints of rapid publication have left some marks on the book. Not all the cameraready scripts deserved that epithet and the worst is no pleasure at all to decipher; the index is a joke. Nevertheless, the contents of the book make a stimulating read, and it is to be hoped that, for example, the immunologist who may be attracted to the last section of the book will find time at least to dip in elsewhere so that the cross-fertilization of the original meeting may continue to yield fruit in the scientific community at large.

The methanogenic biodegradation of catechol by a microbial consortium: evidence for the production of phenol through cis-benzenediol.

Abstract: If the multiple forms of glutathione S-transferase arise solely as a result of non-enzymic deamidation (the process suggested to be operative in the case of human liver), then every rat tissue should contain the same forms in the same proportions. We have therefore analysed the forms in rat liver, kidney, spleen, brain and testis by CM-cellulose chromatography at pH6.7 after first removing acidic proteins by passage through DEAEcellulose at pH7.2. By using the liver chromatogram as a reference (with haemoglobin subforms as internal standards), we identified glutathione S-transferases B and AA in kidney (they co-chromatograph with the liver forms), C, A and AA in testis and only low activities of AA in spleen. These results are clearly not consistent with non-enzymic deamidation playing a major role in the origin of these multiple forms. Interestingly, brain contains virtually no glutathfone S-transferase activity. It remains to be seen if this absence of protective binding protein(s) relates to the known susceptibility of this tissue to bind toxic amounts of bilirubin. Prolonged dialysis after passing cytosol through DEAE-cellulose at pH7.2 did not increase activity in those tissues with low glutathione S-transferase activity (brain and spleen), neither did it effect the elution profile of liver or kidney cytosol on CM-cellulose chromatography, suggesting that differential binding of physiologically important ligands is not responsible for the observed multiple forms or the low activity seen in brain, spleen, muscle or adipose tissue. The tubes of material corresponding to the peaks of activity of the rat liver forms C, B, A and AA resolved by CM-cellulose chromatography were analysed by sodium dodecyl sulphate/ polyacrylamide-gel electrophoresis using the system of Maizel (1971) as modified by Thomas (1978). Bands corresponding to subunit mol.wts of 24000, 21500 and 19500 were tentatively identified as subunits c, b and a (using the terminology of Bass e t al., 1977), as shown in Fig. l(a). Further purification of forms C, B, A and AA on Sephadex G-100 (which exhibited elution volumes corresponding to mol.wts of 44000, 43000,40000 and 5 5 OOO respectively) resulted in highly purified preparations composed only of these three subunits (Fig. lb). There is some overlap of the forms during CM-cellulose chromatography; however, forms A and C are similar in that they appear to be dimers of b-type subunits. The posession of the same immunological determinants by these two forms (Habig et al., 1974a; Guthenberg & Mannervik, 1979) is consistent with this observation. The fact that they are separable by CM-cellulose chromatography indicates some difference between the subunits, and this aspect requires further study. Glutathione S-transferase B is a heterodimer composed of subunits a and c in accord with Hayes e t al. (1979), whereas form AA appears to be the cc-homodimer postulated by Hayes et al. (1 979). An attractive scheme to account for the origin of the multiple forms of the glutathione S-transferases in rat liver might be that there is only one gene product (c) and that two proteolytic cleavages, e.g. c + b +a, could then result in the dimeric forms, cc, cb, ca, bb, ba and aa. Unfortunately there is no evidence for bc or ba heterodimers in the present or in previous work (Bass et al., 1977; Hayes et al., 1979). In the absence of definitive data about the subunit compositions of forms D and E. the relationship between the glutathione S-transferases requires further consideration.

Interaction of vincristine with calf brain tubulin.

Abstract: The interaction of the antimitotic drug vincristine with tubulin has been investigated by the techniques of self-assembly, velocity sedimentation, fluorescence, circular dichroism, and differential spectroscopy. Vincristine has been shown to inhibit the self-assembly of tubulin into microtubules at substoichiometric concentrations. The sedimentation velocity patterns at low vincristine concentration (less than 1 X 10(-5) M to 7 X 10(-5) M) consist of a bimodal boundary with a 5.8 S peak and a fast moving peak, with a nominal S20,w value of 9 S. The data conform to the ligand-promoted self-association theory of Cann and Goad (Cann, J.R., and Goad, W.B. (1972) Arch. Biochem. Biophys. 153, 603-609). At higher vincristine concentrations (greater than 8 X 10(-5 M), most of the protein is polymerized and sediments as a hypersharp peak with a nominal S20,w value of approximately 20 S. The association constant for the binding of vincristine to tubulin, determined by spectrofluorometry, is 3.5 X 10(4) liters/mol at 25 degrees C. The binding of vincristine does not induce any significant conformational changes in tubulin; however, the difference spectral results indicate perturbation of both vincristine and protein chromophores.

Methanogenic fermentation of the naturally occurring aromatic amino acids by a microbial consortium.

Abstract: Medium 2 was similar to Medium 1 except for the replacement of sodium lactate by sodium benzoate (0.75 9). Sterile culture flasks (500ml) were filled to the brim with freshly prepared sterile Medium 1 and 2 separately, inoculated with a suspension of freeze-dried Desulfovibrio N.C.I.B. 8399, capped with sterile Suba-Seal rubber stoppers and incubated by total immersion in a water bath at 37°C. Growth was evident in Medium 1 within 2-3 days by the appearance of black iron pyrites; a drop of this culture on a microscope slide revealed motile bacteria of the expected size and form. The flask containing Medium 2 showed no evidence of growth for 3 months; shortly afterwards it was noticed that the precipitate in this culture had also turned black. Inoculating a few drops of this culture into fresh Medium 2 resulted in growth now starting within 3-4 days with production of ferrous sulphide and concomitant utilization of benzoate as estimated by spectrophotometry (Williams & Evans, 1975). Initially, it was thought that Desulfovibrio N.C.I.B. 8399 had become adapted for the metabolism of benzoate. Smears of the culture fluid however revealed two types of motile bacteria-the vibrio and a rod-present. The contaminant was separated in pure culture and identified as a strain of Pseudomonas aeruginosa (see the acknowledgements). Neither organism alone metabolized the benzoate in Medium 2; when put together, growth with utilization of this substrate ensued. The benzoate disappeared significantly faster when sodium lactate (1 g/litre) was also added to Medium 2. Thioglycollate, yeast extract and the preservation of strict anaerobiosis were all essential for the phenomenon to occur. At this stage it was decided to apply the methods developed by Balba & Evans (1977a,b) and Balba (1978) for the identification of benzoate metabolites in anaerobic cultures: thtse included t.1.c. and g.1.c. of neutral and aciddiethyl ether extracts of culture fluid after suitable treatments. The Pseudomonas aeruginosa strain grew aerobically in benzoate or p-hydroxybenzoate mineral-salts medium using the ortho (i.e. intradiol) pathway of metabolism. Nitrate would not serve as terminal electron acceptor for anaerobic benzoate dissimilation in the medium used by Williams & Evans (1975). When pyruvate or fumarate (2g/litre) were substituted for nitrate in the latter medium, the bacterium flourished anaerobically and benzoate was utilized; their replacement by succinate or lactate (2g/litre) gave poor growth and benzoate hardly disappeared. Examination of the mixed culture (i.e. Desulfovibrio vulgaris N.C.I.B. 8399 + Pseudomonas aeruginosa strain) growing in Medium 2, revealed the simultaneous disappearance of benzoate with the formation of cyclohexanol among the products in the neutral-ether extract and cyclohexane carboxylate in the acid-ether extract; these are known intermediates in the reductive pathway of benzoate metabolism. This syntrophic association of a facultative pseudomonad together with Desulfovibrio vulgaris N.C.I.B. 8399 accomplishes the utilization of benzoate through anaerobic sulphate respiration. It seems likely that the Desulfovibrio produces initially small quantities of organic acids capable of acting as electron acceptors for the facultative organism to attack the benzoate-the chain of events would then become autocatalytic. Whether this association is of wide occurrence among sulphate-reducers remains to be demonstrated.

Studies on the purification of glucose 6-phosphatase from rabbit liver microsomal fraction

Abstract: Pure glycoprotein was dissolved in CsCl at a starting density of 1.42g.ml-1; after centrifugation (1.5 x lO'g, 5\"C, 48h) the densities in fractions 1 and 8 were 1.38 and 1.56g.ml-I respectively. Four tubes contained 0.2 M-mercaptoethanol and CsCl (A, W) and four tubes contained CsCl only (4 0). Protein (A, A) was measured by the method of Lowry et a f . (I95 I ) and glycoprotein (H, 0) by the method of Mantle & Allen (1978).

The metabolism of ranitidine in animals and man

Abstract: Ranitidine hydrochloride (N-124 {[5-(dimethylaminomethyl)furan 2 yl]methyl}thio)ethyl] N’ methyl nitroethene 1,l diamine hydrochloride} is a new histamine H, receptor antagonist effective in decreasing hypersecretion of gastric acid (Bradshaw et al., 1979; Peden et al., 1979). This compound has a novel structure and does not contain the imidazole nucleus, which has been regarded as essential for H,-receptor blockade (Ganellin et al., 1976). The metabolism of [ 14Clranitidine hydrochloride (labelled as indicated in Scheme 1) has been studied in the rat, dog, mouse, rabbit and marmoset after oral administration of 10-100mg (base)/kg body wt. In the dog, up to 80% of the administered radioactivity was recovered in urine in 3 days, the bulk of this being excreted in 24 h. In the other animal species, between 30 and 60% of the administered radioactivity was rapidly excreted in urine, the balance being recovered in faeces. After oral and intravenous doses of 25 mg/kg to the mouse, 48 and 64% of the radioactive dose respectively was excreted in urine collected for . 3 days. The corresponding values for excretion of radioactivity in the faeces were 36 and 25%. Biliary-cannulation studies in the anaesthetized rot showed that, in this species, up to 17% of an intravenous dose was excreted in the bile during 4.5 h. Wholebody radioautography of the rat has confirmed the rapid excretion of radioactivity by the renal and biliary routes. Highest tissue concentrations of radioactivity after oral administration were found in the gut wall, liver and kidneys. Peak plasma total radioactivity of 2-17pg equivalents of ranitidine/ml and unchanged drug concentrations of 1-9pug/ml (determined by high-pressure liquid chromatography; Carey & Martin, 1979) were attained within 30min of oral administration of [“Clranitidine hydrochloride (50mg of base/kg) to the rat. Absorption was slower in the dog, the peak amounts of radioactivity and unchanged-drug concentrations were respectively 19-28 and 10-14pg/mI between 1 and 2h after administration of dose. In each species the drug and its metabolites were rapidly cleared from plasma. The compound was not bound to plasma protein to any significant extent in any of the species investigated. Urine collected from animals housed in metabolism cages can be contaminated with micro-organisms that could metabolize ranitidine. To prevent this, the urine used for chromatographic analysis was collected from rabbits and dogs by using an indwelling catheter and from rats in which the ureters were cannulated. Metabolism cages had to be used for marmosets and mice and therefore the urine was collected in containers cooled with solid CO,. The drug and its metabolites were isolated from urine by adsorption on Amberlite XAD-2 resin. The residue from the methanol eluate was examined by t.l.c., with Merck 0.25mm prelayered plates [system A: Kieselgel 60F2,,, solvent system methanol/NH, 49 : 1 (v/v); system B: aluminium oxide F,,, (type E), solvent system chloroform/methanol/water 75 : 15 : 1 (by vol.)]. Samples of pure ranitidim, ranitidine N-oxide (I) desmethyl ranitidine (11) and ranitidine S-oxide (111) were used as standards. 93

Structure and function of erabutoxins and related neurotoxins from sea snakes and cobras.

Abstract: This raises the possibility that the processing mechanisms may differ in different tissues. Since bovine /.-endorphin appears to have the same primary structure as rat /.-endorphin (Rubinstein et al., 1977; Li et al., 1977), it is anticipated that the four peptides isolated from ox pituitary, described here, will prove valuable as ‘marker’ peptides in studies on the distribution of /.-endorphin in regions of rat pituitary and brain. Bradbury, A. F., Smyth, D. G. & Snell, C. R. (1975) in Peptides: Structure and Biological Activity (Walter, R. & Meienhofer, J.. eds.), pp. 609-6 I5 Ann Arbor Sci., Ann Arbor, MI Bradbury, A. F., Smyth, D. G., Snell, C. R., Birdsall, N. J. M. & Hulme, E. C. (1 976a) Nature (London) 260,625-626 Bradbury, A. F., Feldberg, W. S., Smyth, D. G. & Snell, C. R. (19766) in Opiates and Endogenous Opioid Peptides (Kosterlitz, H. W., ed.). pp. 9-17, North-Holland, Amsterdam Deakin, J. F. W., Dostrovsky, J. 0. & Smyth, D. G. (1980) Biochem. J.

The interrelationship between glutamine and alanine in the intestine.

Abstract: protein purified from a control human liver. The observed very low but distinct activity of the H-protein from the patient could be accounted for by its modulating effect on P-protein, thus enabling the glycine-cleavage reaction to proceed to a certain degree even in the hyperglycinaemic condition. Serine is known to be a gluconeogenic amino acid. Two possible routes of gluconeogenesis have been suggested: one through the formation of pyruvate by serine dehydratase, and the other via hydroxypyruvate formed by the activity of serine-pyruvate aminotransferase (Lardy et al., 1970). The relative contributions of these two routes may differ considerably according t o animal species with different activities of the enzymes concerned. We have observed with liver slices that the amounts of radioactivity incorporated into glucose from either [3-I4C1or [l-'FI-serine (3.3mn4) in chicken liver were nearly equal, and the values were about twice the values obtained with rat liver slices under comparable experimental conditions, whereas the activity of serine dehydratase in chicken liver has been shown to be about one-hundredth of that in rat liver. Chicken liver contained considerable serine-pyruvate aminotransferase activity, which could account for the substantial gluconeogenesis from serine observed i~ this species Fujiwara, K., Okuda, K. & Motokawa, Y. (1979) Arch. Biochem.

The effect of tunicamycin on wall-polymer synthesis in Bacilli.

Abstract: Toluene-treated cells were incubated in the presence of UDPManUANAc, UDPGlcNAc and UDP-[14Clglucose and the incorporation of radioactivity into trichloroacetic acid-precipitated (0) or hot-SDS-insoluble (A) fractions was examined. tion of incorporation into the hot-SDS-insoluble fraction was 55% at 100pg of tunicamycin/ml, and higher concentrations had no greater effect. Again, the resistant 45% would appear to reflect the presence in the cells of partially synthesized teichuronic acid already inacessible to the action of tunicamycin. In the trichloroacetic acid-precipitated fraction, however, whereas the maximum inhibition was 60% at 5Opg of antibiotidml, higher drug concentrations steadily increased the observed incorporation until at 500pg/ml the residual inhibition was only 6% (Fig. 2). Since, as in wall-plus-membrane preparations, SDSsoluble polymer was simultaneously synthesized, it seems possible that, at high tunicamycin concentrations, a greater proportion of this soluble fraction consisted of molecules sufficiently large to be co-precipitated with the cells by trichloroacetic acid. How excess tunicamycin could achieve this effect is not clear. The synthesis of peptidoglycan by etherized Neisseria gonorrhoeae has been described (Brown & Perkins, 1979). In those experiments the incorporation of radioactivity from UDP[ “CIGlcNAc into the trichloroacetic acid-precipitated fraction was inhibited by 26% by 1 pg of tunicamycin/ml. In the present work, incorporation into hot-SDS-soluble and -insoluble fractions was examined (Fig. 3). Concentrations of tunicamycin as low as 0.005pg/ml caused a 20% fall in the synthesis of the hotSDS-soluble fraction and an initial small rise in the synthesis of the SDS-insoluble (i.e. wall-bound) fraction. Further effects were not observed until a concentration of 10pg/ml was exceeded, when considerable inhibition of incorporation into both fractions took place. The low-concentration effect of favouring the synthesis of wall-bound material at the expense of the soluble fraction could, perhaps, represent a tendency for existing partially cross-linked chains to be extended, rather than new chains to be initiated, in the presence of the drug. Alternatively, the limited effect may imply a separate population of polymer made at highly tunicamycin-sensitive synthetic centres. In Bacillus licheniformis preparations, Ward (1 977) also observed an increase in synthesis of wall-bound peptidoglycan at low concentrations of tunicamycin.