Showing papers in "FEBS Journal in 1974"

Involvement of the Superoxide Anion Radical in the Autoxidation of Pyrogallol and a Convenient Assay for Superoxide Dismutase

Abstract: The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6. The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·−, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2·− -independent mechanisms rapidly become dominant. Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism. A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol. A plausible explanation is given for the non-competitive part of the inhibition of catechol O-methyltransferase brought about by pyrogallol.

A Film Detection Method for Tritium‐Labelled Proteins and Nucleic Acids in Polyacrylamide Gels

Abstract: A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X-Omat” film at -70 °C. Optimal conditions for each step are described. β-particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week. When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.

A Linear Steady‐State Treatment of Enzymatic Chains

Abstract: A theoretical analysis of linear enzymatic chains is presented By linear approximation simple analytical solutions can be obtained for the metabolite concentrations and the flux through the chain for steady-state conditions The equations are greatly simplified if the common kinetic constants are expressed as functions of two parameters, ie the thermodynamic equilibrium constant and the “characteristic time” Three cardinal terms are proposed for the quantitative description of enzyme systems The first two are the control strength and the control matrix; these indicate the dependence of the flux and the metabolite concentrations, respectively, on the kinetic properties of a given enzyme The third is the effector strength, which defines the dependence of the velocity of an enzyme on the concentration of an effector; it expresses the importance of an effector By linear approximation simple analytical expressions were derived for the control strength, the control matrix and the mass-action ratios The effector strength was calculated for two cases: for a competitive inhibitor and for allosteric effectors according to the Monod (1965) model The influence of an effector on the concentrations of the metabolites was considered

The Influence of Respiration and ATP Hydrolysis on the Proton‐Electrochemical Gradient across the Inner Membrane of Rat‐Liver Mitochondria as Determined by Ion Distribution

Abstract: 1 A technique is described, based on the distribution of rubidium, acetate and methylammonium ions, for the simultaneous estimation of membrane potential and pH gradient across the inner membrane of mitochondria The technique requires less than 05 mg mitochondrial protein and is independent of many factors which interfere with electrode determinations of protonmotive force (Δp) 2 With a limiting matrix volume of 04 μl/mg mitochondrial protein, the indicated value of Δp for rat liver mitochondria is 228 mV in state 4, 170 mV in state 3, and –06 mV in the presence of rotenone and uncoupler The relative contributions of the pH gradient and membrane potential are dependent on the availability of electrophoretically and electroneutrally translocatable species in the incubation medium 3 In a sucrose-based medium containing 05 mM KCl, rotenone and uncoupler, the technique indicated a membrane potential of + 85 mV and a pH gradient of + 146 (acidic in the matrix compartment) In state 4, under no conditions examined did the pH gradient contribute more than 50% of the total protonmotive force 4 The hydrolysis of ATP generates an optimal Δp of 220 mV 5 The proton conductance of the inner membrane is potential dependent, increasing when Δp is greater than 200 mV 6 The extra-mitochondrial phosphate potential sustainable by respiration was found to change in parallel to Δp, but to exceed the latter parameter when based upon a stoichiometry of two protons translocated per ATP synthesised

Proteinase K from Tritirachium album Limber

Abstract: The fungus Tritirachium album Limber was grown by submerged fermentation, investigating the conditions for maximal secretion of proteases into the medium. Enzyme secretion starts when the stationary phase of growth is reached, and when the culture medium is depleted of glucose and amino acids, suggesting a catabolite repression of the enzyme. The main proteolytic enzyme was named proteinase with respect to its keratin hydrolyzing activity. It was isolated from the culture medium and purified by crystallization and chromatography on DEAE-Sephadex and Sephadex G-75. Homogeneity was ascertained by disc gel electrophoresis and by isoelectric focusing. The molecular weight of proteinase K was determined by gel filtration to be 18500 ± 500, the isoelectrice point was pH 8.9. The enzyme was shown to be a serine protease by inactivation with diisopropyl phosphofluoridate and phenylmethylsulfonyl fluoride. It displayed strong proteolytic activity on denatured but also on native proteins as demonstrated by the rapid inactivation of bovine ribonuclease. The pH-optimum was in the range between pH 7.5–12.0. A specificity of the enzyme for peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids was observed.

Structure of the porcine vasoactive intestinal octacosapeptide. The amino-acid sequence. Use of kallikrein in its determination.

Abstract: The amino acid sequence of the porcine vasoactive intestinal octacosapeptide is His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-L ys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2. Its amino acid residues 1, 2, 6 and 7, counted from the N-terminus, are identical to those in the corresponding positions in both porcine glucagon and secretin. The residues in positions 3, 12, 13, 14 and 23 are identical to those in secretin, but not in glucagon, and position 10 is occupied by a tyrosyl and position 28 by an asparaginyl residue, like in glucagon but not in secretin. If in addition to identical amino acid residues also chemically similar residues, such as isoleucine in position 26 of the octacosapeptide, as compared to leucine in secretin and glucagon, are taken into consideration then the similarity between these three polypeptides is still more evident. At a more remote level there is some structural resemblance also between these peptides and the other four peptides from the intestinal wall, cholecystokinin-pancreozymin, motilin, the gastric inhibitory peptide and substance P, the structures of which are known. The elucidation of the structure of the octacosapeptide was facilitated by the finding that pancreatic kallikrein preferentially cleaved only one of the three bonds in its N-terminal cyanogen bromide heptadecapeptide that are susceptible to cleavage with trypsin.

The Neutral Proteases of Human Granulocytes

Abstract: Two electrophoretic variants of a collagenolytic enzyme from the cytoplasmic lysosome-like granules of leukaemic myeloid cells were purified by chromatography of the granule extract on Sephadex G-75, Bio Rex 70 and collagen–Sepharose, and electrophoresis on polyacrylamide gel. The granulocyte collagenases degraded native collagen in solution at about neutral pH with the production of the two specific fragments. They also acted on other substrates, such as fibrinogen and proteoglycans. The granulocyte collagenases were inhibited by α1-antitrypsin and by α2-macroglobulin. The two collagenases gave reactions of identity on Ouchterlony immunodifusion and crossed immunoelectrophoresis. Amino acid analyses suggested that the collagenases differed only in respect of a few residues. Ultracentrifugation indicated that both proteins were homogeneous and had an s020,w of 4.5 S. Thin-layer chromatography on Sephadex G-150 suggested that both collagenases had a molecular weight of 76000. Electrophoresis after treatment of the collagenases with sodium dodecylsulfate revealed two components with molecular weights of 42000 and 33000, respectively, suggesting that the native enzymes are built up of to two subunits.

The Amino-Acid Sequence of the Alkali Light Chains of Rabbit Skeletal-Muscle Myosin

Abstract: The amino-acid sequence of the alkali light chain 1 (Al) of rabbit skeletal muscle myosin has been determined and compared with the sequence of the smaller but related alkali light chain 2 (A2). The molecular weights of the two proteins calculated from these sequence determinations are 20700 and 16500, respectively. The results show that the two proteins have identical sequences over their C-terminal141 residues. There is an additional sequence of 41 residues at the N-terminal end of A1 which accounts for the extra 4000 molecular weight. Between this additional sequence and the sequence common to both proteins are eight amino-acid residues. Comparison of the sequences of these eight residues in A1 and A2 reveals five amino-acid substitutions. Thus in spite of the very extensive homology between the two proteins, these substitutions indicate that there must be two RNA coding sequences for these light chains in rabbit fast muscle myosin. Rabbit skeletal muscle myosin contains two heavy chains of molecular weight about 200000 [l 21 and four light chains of molecular weight about 20000 [3]. Two chemical classes of light chains have been characterised by their thiol peptides [4] and reaction of myosin with 5,5’-dithiobis(2-nitrobenzoic acid) (Nbs,) selectively dissociated a substantial proportion of one of these light chains without significant effect on the myosin ATPase activity. This light chain has been termed the “Nbs, light chain” l. The remaining light chains cannot be dissociated without total loss of ATPase activity and these were termed the “alkali light chains”, since they were first shown to be released under alkaline conditions [5]. Gel electrophoresis of myosin in the presence of sodium dodecylsulphate shows the presence of three light chain components with apparent molecular weights of 25000, 18000 and 16000 [3]. The 18000-M, component corresponds to the Nbs, light chain and the other two components are classified as alkali light chains since they both contains the single thiol sequence characteristic of this class of light chains in fast muscle myosin [6,7]. The extent of sequence homology between alkali light chain 1 (Al, Mr = 25000) and alkali light chain 2 (A2, Abbreviation. Nbs,, 5,5’-dithiobis(2-nitrobenzoic acid). Nomenclature. The term “alkali light chain” is based on the observation that these proteins are dissociated from myosin in alkali [5]. Other terminology used elsewhere includes LC1(= Al) and LC3 (= A2) [26] and OL and y chains [29]. 1 The “Nbs, light chain” was previously called the “DTNB light chain” [4]. Mr = 16000) was examined by peptide mapping of tryptic digests of the two proteins. Peptides from both proteins stained selectively for arginine, tyrosine and histidine had identical mobilities in the two-dimensional maps, and all the peptides present in A2 could be found in corresponding positions in Al. Such differences as there were could be accounted for by additional peptide material in Al, though the amino-acid compositions indicated minimum molecular weights of 21000 for A1 and 17000 for A2, a difference of 4000 instead of the 8000 estimated from apparent molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. From these results it was concluded that either A2 was a degraded fragment of A1 or the two proteins showed an exceptionally high degree of homology. The relative constancy in the amounts of A1 and A2 in rabbit myosin prepared under a variety of conditions together with observations that both components are found in myosin from chicken breast and leg muscles [S], and the fast skeletal muscle of pig, sheep and cat, make it unlikely that A2 arises as a result of adventitious proteolytic activity. Nevertheless we could not rule out a specific cleavage occurring in the polypeptide chains as a consequence of post-translational modification or during the myosin assembly process. For this reason detailed sequence analysis of both A1 and A2 has been carried out to investigate the relationship between them and show whether they represent products of different genes. Part of the results of this work have been published previously [9].

A spectroscopic study of the haemin--human-serum-albumin system.

Abstract: The interaction of haemin with human serum albumin has been reexamined. The absorption spectrum of the bound haem is identical with that of uncomplexed monomeric haemin in solution, and it is suggested, on the basis of an interaction of albumin with iron-free protoporphyrin IX, that the iron is not implicated in the interaction with the protein. A ferric cyanide derivative, and a ferrous haem derivative of methaemalbumin can be recognised, but not azide or fluoride derivatives. The bound haemin gives rise to extrinsic Cotton effects, which are different in detail in the derivatives, and in the complex with protoporphyrin IX Spectrophotometric titrations show that there is one strong binding site for haemin and several weaker sites. The latter are associated with optical activity opposite in sign to that of the strong complex. The binding profiles are little affected by pH over a wide range, by ionic strength or by the presence of 40% (v/v) dimethylsulphoxide/water, in which the free haemin is monomeric. The binding of haemin to albumin has been followed by spectrophotometry, circular dichroism and fluorescence. The binding of haemin quenches the protein fluorescence, which progressively changes in character from tryptophan to tyrosine type. The haem at the primary binding site thus strongly quenches the tryptophan specifically. From fluorescence titrations at a range of protein concentrations, binding isotherms were constructed, and gave an association constant for the strong site of 50 μM−1. From binding isotherms based on absorption measurements we can infer the existence of at least four sites with much lower binding constants. A long-chain fatty acid anion was found to compete with haemin only for the weaker binding sites, so that binding beyond one mole per mole of protein can be essentially eliminated. The open-chain tetrapyrrole, bilirubin, was found, in agreement with earlier work, not to compete with haemin, as regards the strongest binding sites of either ligand. Between the weaker sites, however, evidence of competition was obtained.

Active transport of maltose in Escherichia coli K12. Involvement of a "periplasmic" maltose binding protein.

Abstract: A “periplasmic” maltose binding protein was purified from Escherichia coli This protein is shown to be under the same positive control as the whole maltose system, and its synthesis is inducible by maltose This binding protein is coded by malE, one of the three cistrons of the malB region involved in the transport of maltose The binding characteristics of this protein, as well as the kinetics of release of bound maltose, suggest that the binding protein can exist in two states differing by their affinity towards maltose The significance of this result is discussed in view of the role that the protein is believed to play in maltose transport

Composition of the Photosynthetic Apparatus of Normal Barley Leaves and a Mutant Lacking Chlorophyll b

Abstract: Comparison of the composition of the energy-trapping apparatus of normal barley photo-synthetic membranes and those lacking chlorophyll b shows that not only is chlorophyll b absent from the mutant, but all constituents (chlorophyll a, chlorophyll b, carotenoids and the protein moiety) of the major chlorophyll-protein complex of normal higher plant chloroplast membranes are also missing; other chlorophyll-containing components, as far as can be ascertained, are essentially unaffected by the mutation. The nuclear gene which codes for the protein moiety of this complex is suggested as a possible site of the mutation in this barley mutant. This chlorophyll α/β-protein complex which accounts for such a large proportion of the chlorophyll and protein of the photosynthetic apparatus of higher plants is thus not essential for a plant to grow photosynthetically, and therefore a new name, light-harvesting chlorophyll-protein, is proposed for the complex, which was formerly termed the photosystem II chlorophyll-protein. The biosynthetic machinery in the mutant is unable to compensate for the loss of the light-harvesting chlorophyll-protein by adding extra chlorophylls into alternative sites in the membrane, thus the chlorophyll/P700 ratio in the mutant is two-thirds that of the normal plant. The light-harvesting chlorophyll-protein is the major location for chlorophyll b in higher plant membranes. The function of the complex is thought to be analogous to that of the algal biliproteins, i.e. light energy absorbed by the complex is fed preferentially, but not exclusively, to the photosystem II reaction-center or trap. It is reasoned that the complex exerts a strong influence on the organization of photosynthetic lamellae in higher plant chloroplasts by maintaining lamellae in contact with each other.

Heterogeneity of erythrocyte catalase II. Isolation and characterization of normal and variant erythrocyte catalase and their subunits

Abstract: 1 Dissociation of the erythrocyte catalase tetramer into its dimer form proceeds readily in urea: the rate depends on urea concentration, time of inactivation and temperature, but is not influenced by enzyme concentration. This process is accompanied by loss of catalase activity, generation of peroxidase activity and an alteration of antigenic properties. 2 Recombination of dimer subunits to the tetramer molecule can be accomplished by removing urea by dialysis. Under optimal conditions up to 80% of the original catalase activity can be recovered. The reconstituted material is identical with the native catalase regarding enzymatic and antigenic properties, but differs with respect to electrophoretic mobility and heat stability. 3 Normal human erythrocyte catalase and the enzyme species present in blood of individuals heterozygous for Swiss-type acatalasemia differ with regard to electrophoretic mobility and heat stability. Moreover, dissociation of the heterozygote catalase tetramer into dimer subunits occurs at lower urea concentrations when compared to the normal enzyme. Since heterozygote erythrocyte catalase exerts properties intermediate to those of the normal enzyme and of the variant found in homozygotes for Acatalasemia, this enzyme species is considered to represent a molecular hybrid. 4 Hybridization of catalases can be accomplished in vitro by dissociating a mixture of normal and heterozygote catalase preparations in 8 M urea and subsequent dialysis. The resulting enzyme species reveals intermediate electrophoretic mobility and heat stability when compared with its native constituents.

Purification and Properties of the Cholinergic Receptor Protein from Electrophorus electricus Electric Tissue

Abstract: The acetylcholine (nicotinic) receptor from the electric organ of Electrophorus electricus has been purified approx. 300-fold from Triton X-100 extracts of electroplax membrane fragments. The receptor protein was first adsorbed from crude extracts on an affinity column containing 1,3-bis(tri-ethylammonium ethoxy)-4-iodoacetamidobenzene diiodide, an analogue of flaxedil, linked to Sepharose 2B, then eluted with a solution of flaxedil and concentrated. The receptor protein was further purified by centrifugation on sucrose gradients containing 1 Triton X-100, 1% Emulphogene, or 0.5% sodium cholate. If desired, detergent could be removed after centrifugation in the presence of sodium cholate by gel filtration on Sephadex G-75. At all stages the receptor was assayed by incubation with α-[3H]toxin from Naja nigricollis followed by isolation of the toxin · receptor complex by filtration on Millipore filters at low detergent concentration. The best preparations bound 1 mol toxin per 150000 g protein. In the presence of non-denaturing detergents the preparations gave a single major band for both protein and activity upon polyacrylamide disc gel electrophoresis or sucrose gradient centrifugation. The receptor protein appears essentially homogeneous in the electron microscope after negative staining with uranyl acetate. Upon gel electrophoresis in the presence of dodecylsulphate, there were two major bands corresponding to molecular weights of 43000 and 48000. The ultraviolet-absorption spectrum and amino-acid composition of the purified receptor protein are typical of those of globular, water-soluble proteins in general and do not reflect the hydrophobic character of the macromolecule. Furthermore, the purified protein interacts with concanavalin A and other plant agglutinins indicating that it contains a carbohydrate moiety. The purified receptor protein carries a single class of binding sites for [3H]decamethonium (Kd= 0.02 μM) at ligand concentrations up to 1 μM and for acetylcholine (Kd= 0.06 μM) at up to 1 μM. No evidence for cooperativity in binding of cholinergic effectors to the receptor protein, either purified or in crude extracts, has been detected and all effectors tested inhibit the binding of [3H]decamethonium in a competitive manner. The affinity of agonists for the solubilized receptor protein is approx. 20-fold greater than for the membrane-bound protein.

A linear steady-state treatment of enzymatic chains. Critique of the crossover theorem and a general procedure to identify interaction sites with an effector.

Abstract: A theoretical analysis of the crossover theorem is presented based on a linear approximation. Cases are considered in which the simple crossover theorem may lead to erroneus conclusions. Among them are the following : more than one interaction site of an effector with the enzymatic chain; influx and efflux of metabolites regulated by outer metabolic processes ; existence of inner effectors ; conservation equations for metabolite concentrations ; and changes of the state of complexes with the metabolites. It is shown that the action of an effector does not always produce a crossover at the affected enzyme. On the other hand, examples are given where “pseudo-crossovers’’ occur at unaffected enzymes. It is concluded that for real systems the identification of the interaction sites of an effector with an enzymatic chain cannot be achieved by the simple crossover theorem. Furthermore, even the identification of “rate controlling” or “regulatory important” enzymes by means of crossovers must be done with great caution. A simple and general procedure for the identification of interaction sites of an outer effector with an enzymatic chain is proposed. It requires the determination of the flux through the chain, the concentrations of the substrates and products of the enzymatic step under consideration and the rate law by which an inner effector, if present, influences the reaction rate of this step. The crossover theorem was first formulated by Chance et al. [l-41. It deals with the influence of outer effectors on the levels of the metabolites in an enzymatic chain. In its simplest form, the “classical” crossover theorem can be stated in the following way: the variations of the concentrations of the metabolites before and beyond an enzyme which is influenced by an effector have different signs. It has been widely used to identify the interaction sites of effectors within the chain. Chance et al. [l--51 have applied the crossover theorem for the investigation of the changes in the steady-state oxidation-reduction levels of the components of the respiratory chain. With the help of this theorem they were able to identify the sites of phosphorylation. An inspection of the literature on metabolic regulation shows that subsequently the crossover theorem has been applied to a variety of systems including very complicated ones. It was tacitly assumed that in all these cases the crossover theorem is valid in its simple form (e.g. [6-8)). Only few theoretical considerations of the crossover theorem have been published so far. Chance et al. [7] have studied the crossover behaviour of the respiratory chain by means of analogue computers. In a more general manner, Holmes [9] considered several types of sequences of chemical reactions and proved the theorem for some simple cases. For the characterization of a crossover he used pairs of neighbouring metabolites where the signs (-, +) and (+, -) indicate the direction of the variation of the concentrations produced by the effector. For the condition that the effector increases the flux the pairs were called “forward)’ and “backward” crossovers, respectively, by Williamson [lo]. The present paper deals with the limitations of the simple crossover theorem in its application to real systema. We shall consider five situations where the crossover theorem is not valid. It will be shown that the uncritical application of the crossover theorem may lead to serious misinterpretations. On the other hand, the procedure proposed in this paper for the identification of the interaction sites of an effector with an enzymatic chain will give correct results if the linear approximation holds. Parts of the results have been presented at the FEBS Advanced Course on “Mathematical Models of Metabolic Regulation”, Oberhof, November 1972.

Crystal and molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI.

Abstract: The structure of the variable part of a χ-type Bence-Jones protein RE1 has been determined at a resolution of 0.28 nm. It forms a dimer in the crystal related by a local diad, held together by hydrogen bonding interactions of residues Tyr-36, Gln-38, Ala-43, Pro-44, Tyr-87, Gln-89 and Phe-98, which are largely conserved in light chains. The structure consists of two hydrogen-bonded sheets covering a hydrophobic interior made up of mostly conserved amino-acid side chains. Approximately 50% of the residues form β-pleated sheets. Several of the pleated sheet strands are connected by hair-pin bends, which contain glycine residues conserved in most light chains. The arrangement of the hypervariable segments especially in comparison with the Fab structure suggests that the V dimers form a primitive antibody. The folding of the poly-peptide chain and the spatial relationship of the two monomers appear to be the same as in the λ-type Bence-Jones protein dimer and the Fab fragment.

Hamster Brown-Adipose-Tissue Mitochondria

Abstract: The rate of controlled respiration of hamster brown-adipose-tissue mitochondria is dependent on the pH of the incubation medium, and on the presence of albumin and exogenous purine nucleotide. The steady-state protonmotive force (Δp) across the inner membrane is dependent on the same parameters. The freshly prepared mitochondria fail to maintain a proton electrochemical gradient during respiration. The maximal Δp which may be attained is 220 mV, in the presence of both albumin and purine nucleotide. The concentration of purine nucleotide required to raise ΔP is dependent on the pH of the medium and on the presence of albumin. The rate of controlled respiration is an inverse linear function of Δp over the range 220 mV to 166 mV. Albumin, purine nucleodites and medium pH control Δp by modulating the effective proton conductance of the inner membrane. At pH 7.1, the effective proton conductance of the freshly prepared mitochondria is greater than 35 nequiv. H+× min−1× mg−1× mV−1 at 23°C, a figure which is reduced to 7–10 in the presence of albumin, to 2 in the presence of 1 mM GDP, and to 1 in the presence of both inhibitors of proton conductance. In the presence of albumin, the concentration of GDP required for 50% inhibition of the proton conductance is 10 μM at pH 7.1. The additional proton conductance induced by the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone is a linear function of the uncoupler added. The significance of these findings is discussed in terms of theories of energy transduction, and the possible physiological mechanisms controlling respiration in brown adipose tissue in vivo.

Human Glutathione Reductase: Purification of the Crystalline Enzyme from Erythorocytes

Abstract: Glutathione reductase from human erythrocytes has been purified 40 000-fold in 10 steps with an overall yield of 36%. The procedure included bulk separations on DEAE-Sephadex and CM-Sephadex, gel-filtration on Sephadex G-200, and salt fractionation with ammonium sulphate. The purified enzyme was crystallised from aqueous solution. The final preparation had a specific activity of 240 units/mg. The purification procedure was completed in 4 weeks, and gave 40 mg enzyme from 35 1 blood. The schedule for glutathione reductase allowed the concurrent isolation of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. In solutions of thiols, the glutathione reductase preparation was homogeneous, as indicated by gel-filtration, ultracentrifugation and electrophoresis. In the absence of thiols, the enzyme showed a tendency to form aggregates. Some of the physical properties of the enzyme are discussed in relation to previous difficulties encountered in its isolation.

Transfer of Heme from Ferrihemoglobin and Ferrihemoglobin Isolated Chains to Hemopexin

Abstract: Incubation of ferrihemoglobin as well as of hemoglobin isolated chains in ferri-form with hemopexin at pH 7.0 leads to the gradual transfer of heme to the hemopexin molecules until the equilibrium is attained. The reaction of ferrihemoglobin with hemopexin can be described as a four-stage process involving successive transfer of the four heme groups from hemoglobin tetramer to the four molecules of hemopexin. The heme binding sites of the hemoglobin molecule are nonequivalent and cooperative. The transfer of heme from the isolated hemoglobin chains in ferri-form occurs as the simple competitive reaction which can be characterized by a single equilibrium constant. The affinity constant value for the binding of heme to the single binding center of hemopexin molecule was estimated as 1.9 × 1014× M−1. The direct transfer of heme from hemoglobin to hemopexin may occur in vivo as a part of hemoglobin degradation process.

Control of the Rate of Hepatic Uptake and Catabolism of Liposome-Entrapped Proteins Injected into Rats. Possible Therapeutic Applications

Abstract: 131I- or 125I-labelled albumin and β-fructofuranosidase were entrapped in liposomes composed of phosphatidylcholine, cholesterol and a charged lipid (molar ratio 7:2:1). Investigations on the fate of such liposomes injected into rats revealed the following. 1. Initially there is an increased rate of removal from plasma (rapid phase) followed by a decreased one (slow phase). The rate of removal during the rapid phase was highest for negatively charged liposomes and lowest for positively charged liposomes with neutral liposomes in between. Uniform rates of removal, regardless of charge, were obtained during the slow phase. 2. The rate of hepatic uptake of such liposomes paralleled that observed for their removal from plasma during the rapid phase. 3. Concurrent administration of a large quantity of positively charged liposomes and of a smaller quantity of negatively charged liposomes led to a diminution of the rapid phase of the latter suggesting a common site of uptake for liposomes of either charge. 4. Blockade of the reticuloendothelial system with carbon led to an increased hepatic localization of liposomes during the rapid phase suggesting a bimodal uptake of liposomes with both the Kupffer and the liver parenchymal cells involved. 5. Cross-linking of liposome-entrapped albumin and β-fructofuranosidase increased the stability of the latter and following injection into rats rendered both proteins less vulnerable to degradation by the liver lysosomal enzymes. 6. The possibility of controlling both the rate of removal from plasma and catabolism of liposome-entrapped proteins adds more scope to the use of liposomes in therapy.

Renaturation of Escherichia coli Tryptophanase after Exposure to 8 M Urea

Abstract: The renaturation of Escherichia coli K 12 tryptophanase after treatment with 8 M urea has been studied. It is shown that, during the renaturation process, an inactive as well as an active form of the protein can be obtained. The influence on the relative amounts of these two forms of factors such as temperature, the presence of the coenzyme, the protein concentration and the conditions for removal of urea has been investigated. The results obtained indicate that the inactive form is characterized by incorrect quaternary interactions occurring specifically between tryptophanase protomers. These findings are discussed in the general frame of the mechanism of protein folding, and are taken as an evidence for the existence of nucleation centers in the folding of tryptophanase.

The Complete Amino‐Acid Sequence of Non‐Immunolobulin Amyloid Fibril Protein AS in Rheumatoid Arthritis

Abstract: The primary structure of a non-immunoglobulin amyloid protein AS has been determined. The protein was found to consist of 76 amino acid residues corresponding to a molecular weight of 9145. The sequence analysis showed clearly that the protein was homogeneous. A characteristic distribution of hydrophobic amino acids was observed and suggested as being of importance for the ability of this protein to form fibrils. A comparison of the protein with other amyloid protein AS showed a high degree of variability, particularly in the carboxyl-terminal region.

The sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase in liver after the administration of glucose to mice and rats. The mechanism of the hepatic threshold to glucose.

Abstract: The use of anesthetized animals and of quick-freezing techniques has allowed to obtain a reliable estimation of the level of phosphorylase a in the liver. A striking inverse relationship was observed between the levels of phosphorylase a and of synthetase a in the liver of fed mice; this observation is in agreement with the hypothesis that the inactivation of phosphorylase is a prerequisite to the activation of glycogen synthetase. Accordingly, the administration of glucose to fed rats caused an extensive inactivation of liver phosphorylase which was usually terminated within two minutes; glycogen synthetase became activated only when and if the level of phosphorylase a had been taken down below a threshold value equal to approximately 10% of the total phosphorylase. This threshold is of the same magnitude in mice. Glucose also caused a slight decrease in the activity of liver phosphorylase when the animals had been previously treated with glucagon. These observations are adequately explained by the previously described stimulation of the phosphorylase phosphatase reaction by glucose and inhibition of synthetase phosphatase by phosphorylase a. In some experiments, insulin caused a partial inactivation of phosphorylase in the liver of rats but more often was without effect, whereas a subsequent load of glucose caused the usual precipitous response, demonstrating that this effect of glucose was not mediated by insulin.

Some kinetic properties of human-milk galactosyl transferase.

Abstract: Reactions catalyzed by human milk N-acetyl lactosamine synthetase in the presence and absence of a-lactalbumins have been investigated by steady-state kinetics. N-Acetyl lactosamine synthesis and lactose synthesis in the absence of a-lactalbumin appear to proceed by an ordered sequential reaction, with substrates attaching in the order : Mn2+, UDP-galactose and monosaccharide. Under the conditions used (pH 7.4, 37 “C) the attachment of Mn2+ is not at thermodynamic equilibrium and it appears that the enzyme can accept either free UDP-galactose or its Mn2+ complex as substrate. Evidence is presented which suggests that the Mn2+ complex of UDP may be the final product released from the enzyme. Reactions in the presence of a-lactalbumin proceed by a similar ordered mechanism. Kinetic effects observed in the presence of human a-lactalbumin with three different monosaccharide acceptors, and in the presence of bovine a-lactalbumin with glucose, can be reasonably explained only by assuming that a-lactalbumin attaches to the enzyme immediately before monosaccharides, contrary to suggestions by other workers. It is proposed that a-lactalbumin attaches to an enzyme . Mn2+ . UDPgalactose complex at thermodynamic equilibrium, producing a new enzyme form with increased affinity for monosaccharides. The inhibitory effects of a-lactalbumin on N-acetyl lactosamine synthesis are attributed to inhibition resulting from attachment of the protein to a central complex in an alternative pathway in the reaction scheme. The kinetic effects of four a-lactalbumins with human galactosyl transferase are characterized and intrinsic differences shown to be independent of the source of galactosyl transferase. The function deduced for a-lactalbumin in the lactose synthetase system is discussed in relation to its structure and a procedure is indicated for quantitatively measuring activity differences in a-lactalbumins.

Relations between Structure and Function in Cytoplasmic Membrane Vesicles Isolated from an Escherichia coli Fatty-Acid Auxotroph

Abstract: The results presented in this paper establish relationships between structural, morphological and functional properties of cytoplasmic membrane vesicles isolated from an Escherichia coli unsaturated fatty acid auxotroph. The membranes were isolated from cells grown in the presence of either oleic, linoleic, linolenic or elaidic acids. High-angle X-ray diffraction studies show that the order-disorder transitions induced by temperature variations and associated with the paraffin chains of the lipids are a function of the fatty acid composition of the membranes. In some cases “cocrystallization” of various lipid species takes place within a single type of ordered domains. In other cases there is segregation of various lipid species into more than one distinct type of ordered domain. The various order-disorder transitions observed induce morphological changes in the hydrophobic core of the membranes which can be detected by freeze-etch electron microscopy. A random distribution of particles on the fracture faces is observed when the paraffin chains of the lipids are disordered. Upon ordering of the paraffin chains, particles are excluded from the ordered domains and as a consequence, smooth areas and areas with densely packed particles are observed. The ratio of the smooth surface to particulated surface is proportional to the amount of ordered paraffin chains present. Moreover, the size of the smooth domains is a function of the fatty acid composition of the membranes. Discontinuities in the rate of d-lactate-dependent proline uptake as a function of temperature correlate with the order-disorder transitions observed. The high energies of activation at low temperatures are attributed to decreased mobility of the carrier proteins upon aggregation. In contrast, phosphoenolpyruvate-dependent vectorial phosphorylation does not respond to the ordering of the paraffin chains.

UDP-glucuronyltransferase in perfused rat liver and in microsomes: influence of phenobarbital and 3-methylcholanthrene.

Abstract: Liver microsomal UDP-glucuronyltransferase (1-naphthol as substrate) is activated about 10-fold by treatment of native microsomes in vitro with Triton X-100. Following pretreatment of rats with 3-methylcholanthrene or phenobarbital the specific activities of detergent-activated microsomes were increased by about 300% and 40%, respectively, while the corresponding increases in native microsomes were only about 70% and 10%, respectively. In isolated liver perfused with 0.5 mM 1-naphthol, the rate of glururonidation of naphthol, as determined by the appearance of naphthol glucuronide in the perfusate, bile and liver tissue, was 0.1 μmol × min−1× g liver−1. This rate of glucuronidation was increased by 70% in rats pretreated with 3-methylcholanthrene and by 140% following phenobarbital treatment, in contrast to the changes observed in microsomes. A UDP-glucuronic acid level of 0.3 μmol/g liver was determined in perfused liver from control rats. This level remained unchanged during perfusion with 0.5 mM 1-naphthol. In the perfused liver from phenobarbital or 3-methylcholanthrene-treated rats, the UDP-glucuronate level increased to 0.5 μmol/g liver. In control, 3-methylcholanthrene or phenobarbital-treated rats, rates of glucuronidation of 1-naphthol in the perfused liver system correspond best with the UDP-glucuronyltransferase activities determined in native microsomes when the different UDP-glucuronate levels, microsomal protein contents and liver weights are taken into account. The results suggest that the enzyme is mostly latent in the microsomal membrane and that this latency is increased after pretreatment of rats with 3-methylcholanthrene despite the marked enzyme induction.

Pyruvate Formate‐Lyase of Escherichia coli: the Acetyl‐Enzyme Intermediate

Abstract: The reaction pyruvate + CoA ⇌ acetyl-CoA + formate, catalyzed by pyruvate formate-lyase of Escherichia coli, occurs by the succeeding half-reactions (a) E + pyruvate ⇌ E-acetyl + formate; (b) E-acetyl + CoA ⇌ E + acetyl-CoA. Making use of coupled optical assays, a ‘ping-pong mechanism’ was derived from the complete kinetic investigation of the forward and reverse reactions. The thermodynamic equilibrium constant of the overall reaction was calculated from the kinetic constants to be K= 750 (30°C, pH 8.1), which agrees with chemically determined values. The intermediate acetyl-enzyme, which had been previously proposed from the [14C]formate-pyruvate exchange, was detected by product-pulse experiments with [2-14C]pyruvate and trapped by acid precipitation. The acetyl group is linked to a sulfhydryl group of the protein. The value of the equilibrium constant of the first half-reaction is about 50, as directly measured and calculated from the kinetic data. It was concluded that the standard free energy of hydrolysis of acetyl-enzyme is about 1.7 kcal (7.1 kJ) more negative than that of acetyl-CoA. The intermediate was found to react with dithiothreitol with a second-order rate constant at 30°C and pH 7.6 of 1160 M−1× min−1. It resulted in a half-life of 4 s (or 20 s at 0°C) in the particular buffer which was required for enzyme stabilization. The enzyme (about 60 U/mg) was prepared by carrying its purified inactive form through the enzyme-II-dependent activation reaction, employing photoreduced flavodoxin along with the effector compounds S-adenosylmethionine and oxamate (as a pyruvate analogue).

The Amino‐Acid Sequence of Porcine Adenylate Kinase from Skeletal Muscle

Abstract: 1 Adenylate kinase (ATP:AMP phosphotransferase) has been purified 490-fold from porcine muscle with a final yield of 60 mg/kg muscle. 2 The amino-acid composition is Asp11, Asn2, Thr14, Ser11, Glu19, Gln6, Pro6, Gly19, Ala8, Cys2, Val17, Met6, Ile9, Leu18. Tyr7, Phe5, Lys21, His2, Arg11. 3 The protein molecule is a single polypeptide chain of 194 amino-acid residues with an acetyl-methionine at the N-terminus and a lysine residue at the C-terminus. 4 Cyanogen bromide cleavage of carboxymethylated adenylate kinase yielded six fragments which were further degraded by using trypsin, chymotrypsin, thermolysin, subtilisin or α-protease. Sequence data on the resulting peptides are summarized in the present report, full details are given in a supplementary paper which has been deposited at CNRS from where copies can be obtained. 5 The primary structure of porcine adenylate kinase is: Ac-Met-Glu-Glu-Lys-Leu-Lys-Lys-Ser-Lys-Ile10-Ile-Phe-Val-Val-Gly-Gly-Pro-Gly - Ser-Gly20-Lys-Gly-Thr-Gln-Cys-Glu-Lys-Ile-Val-Gln30-Lys-Tyr-Gly-Tyr-Thr-His-Leu-Ser-Thr-Gly40-Asp-Leu-Leu-Arg-Ala-Glu-Val-Ser-Ser-Gly50-Ser-Ala-Arg-Gly-Lys-Met-Leu-Ser-Glu-Ile60-Met-Glu-Lys-Gly-Gln-Leu-Val-Pro-Leu-Glu70-Thr-Val-Leu-Asp-Met-Leu-Arg-Asp-Ala-Met80-Val-Ala-Lys-Val-Asp-Thr-Ser-Lys-Gly-Phe90-Leu-Ile-Asp-Gly-Tyr-Pro-Arg-Glu-Val-Lys100-Gln-Gly-Glu-Glu-Phe-Glu-Arg-Lys-Ile-Gly110-Gln-Pro-Thr-Leu-Leu-Leu-Tyr-Val-Asp120-Ala-Gly-Pro-Glu-Thr-Met-Thr-Lys-Arg-Leu-Leu130-Lys-Arg-Gly-Glu-Thr-Ser-Gly-Arg-Val-Asp140-Asp-Asn-Glu-Glu-Thr-Ile-Lys-Lys-Arg-Leu150-Glu-Thr-Tyr-Tyr-Lys-Ala-Thr-Glu-Pro-Val160-Ile-Ala-Phe-Tyr-Glu-Lys-Arg-Gly-Ile-Val170-Arg-Lys-Val-Asn-Ala-Glu-Gly-Ser-Val-Asp180-Asp-Val-Phe-Ser-Gln-Val-Cys-Thr-His-Leu190-Asp-Thr-Leu-Lys.

Regulatory behavior of monomeric enzymes. 1. The mnemonical enzyme concept.

Abstract: A new concept, that of a mnemonical transition, is proposed to explain the departure from Michaelian behavior of monomeric enzymes following ordered reaction mechanisms. The concept integrates three simple ideas: the free enzyme occurs under two conformational states in equilibrium; the collision of any of these forms with the first substrate induces the same third new configuration required for proper substrate binding; the collision of only one of these enzyme forms with the last product stabilizes that form without any new conformational change. This whole set of definitions is equivalent to assuming that the free enzyme which is released after catalysis, is in a conformation different from the initial one. The enzyme can be said to “recall” for a while the configuration stabilized by the last product before relapsing to the initial conformation. The non-hyperbolic behavior is thus the consequence of the cooperation of two different conformations of the free enzyme to the overall reaction process. The reciprocal steady-state rate equations have been established and thoroughly discussed both for one-substrate, one-product and two-substrate, two-product mnemonical enzymes. The departure from Michaelian behavior does not appear as a consequence of a slow conformational transition, but is defined in a simpler way by the relative values of the activation free energies of conformation changes required for substrate binding on the two enzyme forms. A two-substrate, two-product enzyme following an ordered reaction mechanism and exhibiting the mnemonical transition has a very distinctive kinetic behavior. The curvature of the primary plots is observed with regard to the first substrate only, and is independant of the concentration of the second substrate as well as that of the first product. The enzyme is not inhibited by an excess of the substrate and the primary plots are either concave up or down. The slopes and the intercepts of the straight lines obtained in double reciprocal plots with the second substrate should give, when these are replotted against the reciprocal of the first substrate concentration, a straight line and a curve, respectively. The cooperation of the enzyme conformations to the overall reaction process can be either positive or negative. Since the reciprocal plots cannot exhibit an extreme, the extent of that cooperation can be measured by the numerical value of the second derivative of the reciprocal rate equation. The extent of cooperation between the free enzyme forms is highly controlled by the concentration of the last product. If the cooperation was already negative, the product strengthens that cooperation. If, on the other hand, the cooperation was positive, the product decreases or even reverses that cooperation. A very general property of one-sited mnemonical enzymes is that cooperation between enzyme forms is only kinetic and does not appear in the substrate binding isotherms.