Showing papers in "Biochemical Journal in 1974"

The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters

Abstract: A new plot is described for analysing the results of kinetic experiments in which the Michaelis–Menten equation is obeyed. Observations are plotted as lines in parameter space, instead of points in observation space. With appropriate modifications the plot is applicable to most problems of interest to the enzyme kineticist. It has the following advantages over traditional methods of plotting kinetic results: it is very simple to construct, because it is composed entirely of straight lines and requires no calculation or mathematical tables; the kinetic constants are read off the plot directly, again without calculation; it may be used during the course of an experiment to judge the success of the experiment, and to modify the experimental design; it provides clear and accurate information about the quality of the observations, and identifies aberrant observations; it provides a clear indication of the precision of the kinetic constants; constructed with care, it provides unbiased estimates of the kinetic constants, the same as those provided by a computer program; it may be used to simulate results for illustrative purposes very rapidly and simply.

A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors (Short Communication)

Abstract: A new method is described for plotting kinetic results for inhibited enzyme-catalysed reactions. It provides a simple way of determining the inhibition constant, K′i, of an uncompetitive, mixed or non-competitive inhibitor.

A rapid method for the preparation of microvilli from rabbit kidney

Abstract: A simple method for the isolation of microvilli from kidney brush border is described. The method depends on the preferential aggregation of other subcellular structures by bivalent metal ions. MgCl(2) is added to a homogenate of cortical tissue prepared from frozen rabbit kidneys. Aggregated material is removed by a low-speed centrifugation and the supernatant centrifuged at 15000g to yield a pellet enriched in microvilli. This is resuspended and given a second treatment with Mg(2+). The purified preparation is obtained after four short differential centrifugations. The six brush-border enzymes that were monitored were enriched 11-17-fold compared with the original homogenate and were obtained in about 10% yield. Marker enzymes for other subcellular components showed the preparation to be essentially free of mitochondria and to be less contaminated with endoplasmic reticulum and baso-lateral plasma membranes than are conventional brush-border preparations. The main contamination was of lysosomal origin, about half of which was attributable to adsorbed acid hydrolases rather than to intact lysosomes. The aggregated components in the low-speed pellet bound less Mg(2+) than did the microvillus fraction. A possible mechanism for the role of Mg(2+) is discussed.

Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids.

Abstract: 1. Monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, 2-chloropropionate, 2,2'-dichloropropionate and 3-chloropropionate were inhibitors of pig heart pyruvate dehydrogenase kinase. Dichloroacetate was also shown to inhibit rat heart pyruvate dehydrogenase kinase. The inhibition was mainly non-competitive with respect to ATP. The concentration required for 50% inhibition was approx. 100mum for the three chloroacetates, difluoroacetate and 2-chloropropionate and 2,2'-dichloropropionate. Dichloroacetamide was not inhibitory. 2. Dichloroacetate had no significant effect on the activity of pyruvate dehydrogenase phosphate phosphatase when this was maximally activated by Ca(2+) and Mg(2+). 3. Dichloroacetate did not increase the catalytic activity of purified pig heart pyruvate dehydrogenase. 4. Dichloroacetate, difluoroacetate, 2-chloropropionate and 2,2'-dichloropropionate increased the proportion of the active (dephosphorylated) form of pyruvate dehydrogenase in rat heart mitochondria with 2-oxoglutarate and malate as respiratory substrates. Similar effects of dichloroacetate were shown with kidney and fat-cell mitochondria. Glyoxylate, monochloroacetate and dichloroacetamide were inactive. 5. Dichloroacetate increased the proportion of active pyruvate dehydrogenase in the perfused rat heart, isolated rat diaphragm and rat epididymal fat-pads. Difluoroacetate and dichloroacetamide were also active in the perfused heart, but glyoxylate, monochloroacetate and trichloroacetate were inactive. 6. Injection of dichloroacetate into rats starved overnight led within 60 min to activation of pyruvate dehydrogenase in extracts from heart, psoas muscle, adipose tissue, kidney and liver. The blood concentration of lactate fell within 15 min to reach a minimum after 60 min. The blood concentration of glucose fell after 90 min and reached a minimum after 120 min. There was no significant change in plasma glycerol concentration. 7. In epididymal fatpads dichloroacetate inhibited incorporation of (14)C from [U-(14)C]glucose, [U-(14)C]fructose and from [U-(14)C]lactate into CO(2) and glyceride fatty acid. 8. It is concluded that the inhibition of pyruvate dehydrogenase kinase by dichloroacetate may account for the activation of pyruvate dehydrogenase and pyruvate oxidation which it induces in isolated rat heart and diaphragm muscles, subject to certain assumptions as to the distribution of dichloroacetate across the plasma membrane and the mitochondrial membrane. 9. It is suggested that activation of pyruvate dehydrogenase by dichloroacetate could contribute to its hypoglycaemic effect by interruption of the Cori and alanine cycles. 10. It is suggested that the inhibitory effect of dichloroacetate on fatty acid synthesis in adipose tissue may involve an additional effect or effects of the compound.

The purification and specificity of a neutral endopeptidase from rabbit kidney brush border.

Abstract: 1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [(125)I]iodoglucagon at a rate comparable with that for [(125)I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the alpha-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.

Regulation of the pentose phosphate cycle

Abstract: 1. A search was made for mechanisms which may exert a ;fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP(+)) show that at physiological concentrations of free NADP(+) and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn(2+) did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg(2+) and NADP(+) before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.

Cathepsin B1. A lysosomal enzyme that degrades native collagen

Abstract: 1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released 14C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human α2-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5–5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an α-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.

Reduction and inactivation of superoxide dismutase by hydrogen peroxide

Abstract: Reactions of H2O2 with superoxide dismutase were studied by e.p.r. (electron paramagnetic resonance) spectroscopy and other methods. In agreement with earlier work, the Cu2+ of the enzyme is reduced by H2O2, although the reaction does not go to completion and its kinetics are not simple. With dilute enzyme the time for half-reduction with 9mm-H2O2 is about 150ms. It is suggested that the reaction is a one-electron reduction, involving liberation of O2−. On somewhat more prolonged exposure to H2O2, the enzyme is inactivated. For enzyme in dilute solution and over a limited range of H2O2 concentrations, inactivation is first-order with respect to enzyme and reagent, with k=3.1m−1·s−1 at 20–25°C. Inactivation is accompanied by marked changes in the e.p.r. and visible spectra and appears to be associated with destruction of one histidine residue per subunit. It is suggested that this histidine is close to the metal in the native enzyme and essential for its enzymic activity.

Statistical considerations in the estimation of enzyme kinetic parameters by the direct linear plot and other methods

Abstract: The statistical implications of the direct linear plot for enzyme kinetic data, described in the preceding paper (Eisenthal & Cornish-Bowden, 1974), are discussed for the case of the Michaelis-Menten equation. The plot is shown to lead directly to non-parametric confidence limits for the kinetic parameters, V and K(m), which depend on far less sweeping assumptions about the nature of experimental error than those implicit in the method of least squares. Median estimates of V and K(m) can also be defined, which are shown to be more robust than the least-squares estimates in a wide variety of experimental situations.

The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction.

Abstract: Evidence is presented that the myosin subfragment-1-ADP complex, generated by the addition of Mg(2+) and ADP to subfragment 1, is an intermediate within the myosin Mg(2+)-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5 degrees C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21 degrees C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s(-1) at 21 degrees C, 0.07s(-1) at 5 degrees C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1-product complex (rate=0.055s(-1) at 21 degrees C, 0.036s(-1) at 5 degrees C). By means of studies on the P(i) inhibition of nucleotide-association rates, a myosin subfragment-1-P(i) complex was characterized with a dissociation equilibrium constant of 1.5mm. P(i) appears to bind more weakly to the myosin subfragment-1-ADP complex. The studies indicate that P(i) dissociates from subfragment 1 at a rate greater than 40s(-1), and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg(2+)-dependent ATPase. In this ATPase mechanism Mg(2+) associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H(+) is released/mol of subfragment 1 concomitant with the myosin-product isomerization or P(i) dissociation, and 0.23 mol of H(+) is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H(+) is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H(+) is released into the medium in the step involving ATP cleavage.

The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red.

Abstract: An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of (45)Ca(2+) transport by rat liver mitochondria. This method was used in conjunction with studies of Ca(2+)-stimulated respiration to examine the mechanisms of inhibition of Ca(2+) transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca(2+) transport non-competitively with K(i) 3x10(-8)m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La(3+) is competitive (K(i)=2x10(-8)m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the ;low-affinity' Ca(2+)-binding sites). La(3+) competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La(3+). The results are discussed briefly in relation to possible models for a Ca(2+) carrier.

Production and utilization of acetate in mammals

Abstract: 1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.

Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by α-cyano-4-hydroxycinnamate (Short Communication)

Abstract: α-Cyano-4-hydroxycinnamate greatly inhibits the transport of pyruvate but not that of acetate or butyrate in liver mitochondria and erythrocytes. In the latter, lactate uptake is also inhibited. It is concluded that a specific carrier is involved in membrane transport of pyruvate and that the plasma-membrane carrier may also be involved in lactate transport.

Characterization of plasma lipoproteins separated and purified by agarose-column chromatography.

Abstract: 1. A simple method for isolation of individual human plasma lipoprotein classes is presented. In this technique, lipoproteins are removed from plasma at d1.225 by ultracentrifugation, after which they are separated and purified by agarose-column chromatography. 2. Three major classes are obtained after agarose-column chromatography. Separation between classes is excellent; more than 95% of the lipoproteins eluted from the column are recovered in the form of a purified lipoprotein class. 3. Each lipoprotein class was characterized immunologically, chemically, electrophoretically and by electron microscopy. A comparison of the properties of the column-isolated lipoproteins was made with very-low-density lipoproteins, low-density lipoproteins, and high-density lipoproteins separated by sequential ultracentrifugation at densities of 1.006, 1.063 and 1.21 respectively. 4. By each criterion, peak-I lipoproteins from the agarose column are the same as very-low-density lipoproteins, peak-II lipoproteins are the same as low-density lipoproteins, and peak-III lipoproteins are the same as high-density lipoproteins. Thus the lipoprotein classes isolated by both methods are similar if not identical. 5. The agarose-column separation technique offers the advantage of a two- to three-fold saving in time. In addition, the column-elution pattern serves as a recording of the size distribution of lipoproteins in plasma. 6. The most complete characterization is reported for human plasma lipoproteins. The results with rhesus-monkey and rabbit lipoproteins were identical.

Hyaluronic acid in cartilage and proteoglycan aggregation

Abstract: 1 Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969) 2 This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid 3 The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH45 4 The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH45, whereas the amount of hyaluronic acid extracted appeared constant from pH30 to 85 5 Sequential extraction of cartilage with 015m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH45 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively 6 Most of the hyaluronic acid in cartilage, representing about 07% of the total uronic acid, was associated with proteoglycan aggregates 7 The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores

The mechanism of action of superoxide dismutase from pulse radiolysis and electron paramagnetic resonance. Evidence that only half the active sites function in catalysis

Abstract: 1. Detailed studies on the mechanism of the enzymic reaction of bovine superoxide dismutase were carried out by using pulse radiolysis and electron paramagnetic resonance (e.p.r.). 2. The second-order rate constant for reaction between superoxide dismutase and the superoxide ion was redetermined as (2.37±0.18)×109m−1·s−1 at 25°C. This reaction governs the turnover, and any first-order steps must have rate constants higher than about 106s−1. Turnover has a low activation energy and is slowed substantially when the viscosity is increased with glycerol, confirming that the reaction rate is near the limit for diffusion control. In water a reversible conformation change to a less active form appears to take place above about 40°C. 3. Pre-steady-state rates of reduction and reoxidation of copper in the enzyme are consistent with these processes being rate-limiting in enzyme turnover. 4. Examination, with the help of computer simulation, of the e.p.r. spectra at 9 and 35GHz of native superoxide dismutase indicated that, apart from 10–20% of impurities, only one species of Cu2+ is distinguishable. Further, the specific activity of our enzyme preparations, measured by pulse radiolysis, is at least as high as that obtained by other workers. 5. Nevertheless, measurement of the proportion of copper present as Cu2+ (determined both optically and by e.p.r. spectroscopy) in the steady states approached from both the oxidized and the reduced forms of the enzyme, indicates (after allowing for the impurities) that only half of the copper atoms participate in turnover. E.p.r. spectroscopy provided no evidence for differences between functioning and non-functioning Cu2+ atoms. 6. It is suggested that the results may be best interpreted in terms of an allosteric type of mechanism, with two initially indistinguishable copper atoms in the enzyme. Reaction of one of these with a superoxide ion then renders the other, at least transiently, unreactive.

Polyphenol-protein interactions.

Abstract: The association of some natural and synthetic polyphenols with β-glucosidase was examined and some observations on the chemical nature of the complex were made.

The influence of substrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust in vitro

Abstract: The rate at which isolated corpora allata of adult female Schistocerca gregaria incorporate [3H]farnesenic acid and [14C]methionine into C16juvenile hormone in vitro was examined at different concentrations of farnesenic acid, methionine, O2 and H+ ions. Maximum juvenile hormone biosynthesis is obtained at a farnesenic acid concentration of 20μm. The range of optimum l-methionine concentrations (0.1–0.4mm) encompasses the physiological concentration of this substrate in the haemolymph. Hormone biosynthesis is dependent on O2, but is not stimulated by hyperbaric oxygen tension. The glands had a maximum synthetic activity at pH8.0, but their activity was more reproducible in the the physiological range pH7.0–7.5. At pH6.5 and less, the synthetic ability was considerably decreased. The relative incorporations of the labelled substrates into methyl farnesoate and C16 juvenile hormone indicate that [3H]farnesenic acid comes into isotopic equilibrium within the gland more rapidly than [14C]methionine. The incorporations into methyl farnesoate become stoicheiometric after 20min incubation and into C16 juvenile hormone after a further 10min. Labelled juvenile hormone is detectable after 10min incubation and the rate of incorporation is constant for up to 4h. It is proposed that the described method may be usefully employed to assess the physiological changes in the enzymic competence of the glands to effect the last two stages in C16 juvenile hormone biosynthesis.

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles.

Abstract: 1. Synaptic vesicles separated by density-gradient centrifugation from extracts of the cholinergic nerve terminals of the electric organ of Torpedo marmorata were found to contain appreciable amounts of ATP as well as acetylcholine. 2. Vesicular ATP was stable in the presence of concentrations of apyrase and myokinase that rapidly destroyed equivalent amounts of endogenous or added free ATP; pre-treatment of cytoplasmic extracts of electric tissue with these enzymes destroyed endogenous free ATP, but did not affect the vesicular ATP. 3. When [U-14C]ATP was added to electric tissue at the time of comminution and extraction of the vesicles, all the radioactivity was associated with soluble components in the subsequent fractionation: none was associated with vesicles or membrane fragments; thus it is unlikely that vesicular ATP can be accounted for by the sequestration of endogenous free ATP within any vesicles formed during comminution and extraction of the tissue. 4. When synaptic vesicles were passed through iso-osmotic columns of Bio-Gel A-5m, which separates vesicles from soluble proteins and small molecules, all the recovered ATP and acetylcholine passed through together in the void volume. 5. Regression analysis showed that vesicular ATP content was highly correlated with vesicular acetylcholine content in different experiments, the molar ratio acetylcholine/ATP being 5.32±(s.e.m.) 0.45 (21 expts.) for the peak density-gradient fraction. The ratio varied, however, somewhat across the density-gradient peak suggesting some degree of chemical heterogeneity in the vesicle population.

Proton/sodium ion antiport in Escherichia coli

Abstract: In anaerobic suspensions of Escherichia coli, after H(+) ions have been translocated outwards across the plasma membrane by a respiratory pulse, re-equilibration is catalysed by Na(+). The sudden addition of a Na(+) salt causes the effective outward translocation of H(+) by an electroneutral process. We conclude that the plasma membrane of E. coli contains a Na(+)/H(+) antiport system that normally translocates Na(+) outwards under the influence of an inwardly directed H(+)-activity gradient.

A spectrophotometric assay of beta-lactamase action on penicillins.

Abstract: A new method for measuring the enzymic hydrolysis of the beta-lactam ring in penicillins is described. The change in extinction in the u.v. region is determined. The method is sensitive (50mum-benzylpenicillin can be used) and convenient.

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation.

Abstract: Mounted on the Internet with the permission of the Biochemical Society (1974). Also available from http://www.biochemj.org/bj/141/0351/1410351.pdf.

Structures for polyinosinic acid and polyguanylic acid

Abstract: X-ray-diffraction analysis of oriented, partially crystalline fibres of polyinosinic acid has resulted in a new molecular model. This model consists of four identical polynucleotide chains related to one another by a fourfold rotation axis. The coaxial helices are righthanded (screw symmetry 23(2)) and have an axial translation per residue h=0.341nm and a rotation per residue t=31.3 degrees . Incorporated in the model are standard bond lengths, bond angles and C-2-endo furanose rings. The nucleotide conformation angles, determined by linked-atom least-squares methods, are orthodox and the fit with the X-ray intensities is good. Each hypoxanthine base is linked to two others by hydrogen bonds involving O-6 and N-1. Further stability may arise from intrachain hydrogen bonds between each ribose hydroxyl group and the phosphate oxygen O-3. If guanine were to be substituted for hypoxanthine in an isogeometrical molecular structure, additional hydrogen bonds could be made between every N-2 and N-7.

Regulation of glucose and ketone-body metabolism in brain of anaesthetized rats

Abstract: 1. The effects of starvation and diabetes on brain fuel metabolism were examined by measuring arteriovenous differences for glucose, lactate, acetoacetate and 3-hydroxybutyrate across the brains of anaesthetized fed, starved and diabetic rats. 2. In fed animals glucose represented the sole oxidative fuel of the brain. 3. After 48h of starvation, ketone-body concentrations were about 2mm and ketone-body uptake accounted for 25% of the calculated O2 consumption: the arteriovenous difference for glucose was not diminished, but lactate release was increased, suggesting inhibition of pyruvate oxidation. 4. In severe diabetic ketosis, induced by either streptozotocin or phlorrhizin (total blood ketone bodies >7mm), the uptake of ketone bodies was further increased and accounted for 45% of the brain's oxidative metabolism, and the arteriovenous difference for glucose was decreased by one-third. The arteriovenous difference for lactate was increased significantly in the phlorrhizin-treated rats. 5. Infusion of 3-hydroxybutyrate into starved rats caused marked increases in the arteriovenous differences for lactate and both ketone bodies. 6. To study the mechanisms of these changes, steady-state concentrations of intermediates and co-factors of the glycolytic pathway were determined in freeze-blown brain. 7. Starved rats had increased concentrations of acetyl-CoA. 8. Rats with diabetic ketosis had increased concentrations of fructose 6-phosphate and decreased concentrations of fructose 1,6-diphosphate, indicating an inhibition of phosphofructokinase. 9. The concentrations of acetyl-CoA, glycogen and citrate, a potent inhibitor of phosphofructokinase, were increased in the streptozotocin-treated rats. 10. The data suggest that cerebral glucose uptake is decreased in diabetic ketoacidosis owing to inhibition of phosphofructokinase as a result of the increase in brain citrate. 11. The inhibition of brain pyruvate oxidation in starvation and diabetes can be related to the accelerated rate of ketone-body metabolism; however, we found no correlation between the decrease in glucose uptake in the diabetic state and the arteriovenous difference for ketone bodies. 12. The data also suggest that the rates of acetoacetate and 3-hydroxybutyrate utilization by brain are governed by their concentrations in plasma. 13. The finding of very low concentrations of acetoacetate and 3-hydroxybutyrate in brain compared with plasma suggests that diffusion across the blood–brain barrier may be the rate-limiting step in their metabolism.

Properties of a prolactin receptor from the rabbit mammary gland

Abstract: Receptors for human, simian, ovine, bovine and murine prolactin, human growth hormone and human placental lactogen have been identified in plasma-membrane-containing subcellular particles isolated from rabbit mammary glands. The association and dissociation of 125I-labelled prolactin are time- and temperature-dependent processes, both being maximal at 37°C. 125I-labelled prolactin prepared by the enzymic iodination procedure with lactoperoxidase binds better to receptors than does the preparation obtained by using chloramine-t as the oxidizing agent. The binding of 125I-labelled prolactin to receptors is strongly influenced by pH and ionic composition but not by many low-molecular-weight compounds tested, e.g. steroids, nucleotides and several drugs. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipid moieties are essential for the binding of 125I-labelled prolactin. The binding of 125I-labelled prolactin to receptors is a saturable and reversible process. Scatchard and Lineweaver–Burk analyses suggest that 125I-labelled prolactin has a high affinity for its receptor. Binding of 125I-labelled prolactin to receptors does not result in the destruction of the hormone. Considerable prolactin-binding activity is also observed in subcellular fractions isolated from the adrenal gland, liver, ovary and kidney of the pregnant rabbit, a finding that is consistent with other reported actions of prolactin in these organs.

Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle.

Abstract: 1. On electrophoresis in dissociating conditions the tropomyosins isolated from skeletal muscles of mammalian, avian and amphibian species migrated as two components. These were comparable with the α and β subunits of tropomyosin present in rabbit skeletal muscle. 2. The α and β components of all skeletal-muscle tropomyosins contained 1 and 2 residues of cysteine per 34000g respectively. 3. The ratio of the amounts of α and β subunit present in skeletal muscle tropomyosins was characteristic for the muscle type. Muscle consisting of slow red fibres contained a greater proportion of β-tropomyosin than muscles consisting predominantly of white fast fibres. 4. Mammalian and avian cardiac muscle tropomyosins consisted of α-tropomyosin only. 5. Mammalian and avian smooth-muscle tropomyosins differed both chemically and immunologically from striated-muscle tropomyosins. 6. Antibody raised against rabbit skeletal α-tropomyosin was species non-specific, reacting with all other striated muscle α-tropomyosin subunits tested. 7. Antibody raised against rabbit skeletal β-tropomyosin subunit was species-specific.

Equilibrium relations between the oxidation–reduction reactions and the adenosine triphosphate synthesis in suspensions of isolated liver cells

Abstract: 1. The redox state of cytochrome c, cytochrome a and the mitochondrial NAD couple, and the phosphorylation state of the adenine nucleotides, were measured in suspensions of isolated rat liver cells. 2. The DeltaG for the transfer of two electrons from the mitochondrial NAD to the cytochrome c couple is calculated to be 104kJ (24.8kcal). 3. The DeltaG associated with the synthesis of ATP at the measured phosphorylation state is calculated to be 95kJ (22.7kcal)/2mol of ATP. 4. The near equality of DeltaG of the electron-transport process and DeltaG required for ATP synthesis indicates near-equilibrium between the mitochondrial respiratory chain and the extramitochondrial phosphorylation state. 5. The existence of near-equilibrium in the coupled reactions implies that the respiratory activity depends on the ratio [ATP]/[ADP][P(i)] and not on the concentrations of the individual reactants. 6. If the overall system of oxidative phosphorylation is at near-equilibrium, all intermediary reactions must also be at equilibrium. Hence if the intramitochondrial and extramitochondrial phosphorylation states are indeed different, it follows that any differences in the activities of ATP, ADP and P(i) must be coupled to ion gradients and/or potentials across the inner mitochondrial membrane in such a way that translocation occurs without loss of free energy. 7. The metabolic state of the mitochondria in the cell can be defined by the turnover number of the cytochromes, the cytoplasmic phosphorylation state, and the oxidation-reduction potential of the NAD couple, rather than by the availability of ADP, substrate and O(2).

The interaction of the calcium-binding protein (troponin C) with bivalent cations and the inhibitory protein (troponin I)

Abstract: 1. The molecular weight of the calcium-binding protein of rabbit white skeletal muscle was estimated to be 18500 by sedimentation equilibrium and electrophoresis in sodium dodecyl sulphate. 2. Addition of 2 Ca2+ ions per molecule produced reversible changes in the u.v.-absorption spectrum that are interpreted as arising from conformational changes in the structure of the protein. 3. Cd2+ was almost as effective as Ca2+ in producing the spectral changes. Other bivalent metal ions, particularly Mg2+, were less effective. 4. Binding of Ca2+ by the calcium-binding protein produced an increase in mobility to the anode on electrophoresis in 6m-urea at pH8.6. The Ca2+-saturated form of the protein was more retarded on gel filtration than the Ca2+-free form. 5. In the presence of Ca2+ the calcium-binding protein formed an equimolar complex with the inhibitory protein. This complex was stable in 8m-urea and in the pH range 7.0–8.6. 6. An isotope-dilution method for the measurement of the content of calcium-binding protein in whole muscle is described. In rabbit psoas muscle the ratio of actin monomers to molecules of calcium-binding protein was approx. 7:1. Similar values were obtained for red skeletal and cardiac muscle. 7. Evidence is presented indicating that in the rabbit the inhibitory protein of the troponin complex of red skeletal and cardiac muscles is different from the inhibitory protein of white skeletal muscle.

Phosphorylation of troponin and the effects of interactions between the components of the complex

Abstract: 1. The troponin complex from skeletal muscle contains approximately 1 mol of phosphate/80000g of complex, covalently bound to the troponin T component. 2. On prolonged incubation of the troponin complex or troponin T with phosphorylase kinase the phosphate content of troponin T was increased to approx. 3mol/mol. 3. On prolonged incubation of troponin I with phosphorylase kinase up to 1.6mol of phosphate/mol were incorporated. 4. Phosphorylation of troponin I was greatly inhibited by troponin C owing to the strong interaction between these proteins. Thus in the troponin complex troponin T was the main substrate for phosphorylase kinase. The phosphorylation of isolated troponin T was also inhibited by troponin C. 5. Troponin I was phosphorylated when the troponin complex was incubated with a bovine cardiac 3′:5′-cyclic AMP-dependent protein kinase. Troponin T either in its isolated form or in the troponin complex was not phosphorylated by bovine protein kinase to any significant extent under the conditions used. 6. If the troponin complex was dephosphorylated to 0.2mol/mol, or phosphorylated up to 2.5mol/mol there was no significant effect on the ability of normal concentrations to confer Ca2+ sensitivity on the adenosine triphosphatase of densensitized actomyosin.

Compartmentation of free amino acids for protein synthesis in rat liver.

Abstract: The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [3H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2–3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.