Showing papers in "FEBS Journal in 1973"

Isolation of high-molecular-weight DNA from mammalian cells.

Abstract: A preparative method for isolating high-molecular-weight DNA from animal cells is described. This method is based on the use of proteinase K, a powerful proteolytic enzyme with a broad action spectrum, which is very active in the presence of sodium dodecylsulfate and ethylene-diamine tetraacetate. The DNA preparation is free of RNA, protein and degrading enzymes. The number-average molecular weight of the native DNA is 190 × 106, whereas it is 90 × 106 for single-stranded DNA, indicating that the DNA molecules do not contain single-stranded nicks. The native DNA molecules range in molecular weight from 40 × 106 to more than 500 × 106.

Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae.

Abstract: A simple scheme has been devised for the purification of Aspergillus nuclease S1 from commercially available crude amylase powder. Five purification steps yield 90% pure nuclease with an overall yield of 27%. The enzyme is shown to be absolutely specific for single-stranded nucleic acids. It degrades both DNA and RNA. The nuclease exhibits a sharp pH optimum at pH 4.2 and is active in low concentrations of dodecylsulfate. Aspergillus nuclease S1 is a metalloprotein of molecular weight 32000. The utility of this nuclease for measuring annealing of labeled DNA is discussed.

A New Group of Chromatin-Associated Proteins with a High Content of Acidic and Basic Amino Acids

Abstract: A new group of chromatin-associated proteins having a high content of acidic and basic amino acids have been isolated from the 0.35 M NaCl-extractable proteins from calf thymus. This new group, designated “high-mobility group” proteins have been partially separated and some interesting new proteins identified. One such protein contains over 55% acidic and basic amino acids.

The Subunit Structure of Rabbit‐Skeletal‐Muscle Phosphorylase Kinase, and the Molecular Basis of Its Activation Reactions

Abstract: Phosphorylase kinase was isolated from rabbit skeletal muscle in a state approaching homogeneity as judged by the criteria of ultracentrifugal analysis, ion-exchange chromatography, and antigen-antibody precipitation in agar. The purified enzyme showed four protein-staining components upon acrylamide gel electrophoresis in the presence of sodium dodecylsulphate, termed α, α′, β and γ. A variety of precautions designed to eliminate the possible action of proteases and ensure complete reduction and denaturation of the protein had no influence on the gel pattern. The molecular weights of the four components determined by gel electrophoresis, and supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride, following partial separation of the chains by gel filtration on Sephadex G-200 in the presence of sodium dodecylsulphate, were:–α= 145000, α′= 140000, β= 128000 and γ= 45000. The α' component was present only in trace amounts and the evidence suggests it may be derived from the α component. The three subunits α, β, and γ, were found to exist in equimolar quantities by densitometric analysis of acrylamide gels, by gel filtration on Sephadex G-200 in the presence of sodium dodecylsulphate, and by carboxymethylation with iodo[14C]acetate, suggesting a minimal binding molecular weight, αβγ, of 318000. The molecular weight of the native enzyme was determined as 1.28 × 106, demonstrating that phosphorylase kinase is composed of 4.0 αβγ units. Activation of the enzyme by incubation with adenosine-3′:5′-phosphate-dependent protein kinase, adenosine 3′:5′-phosphate and Mg-ATP, was accompanied by the phosphorylation of one site on the β subunit, although a second site on the α subunit was phosphorylated at a slower rate. Activation of the enzyme by proteolysis resulted from a limited cleavage of the α subunit. The products of proteolytic attack suggest that the α and β subunits may be structurally related. The γ subunit was not phosphorylated, was resistant to proteolysis and distinct from the (α+β) subunits in its amino acid composition. The possible functions of the chains, and the implications of the activation reactions to the nervous and hormonal control of glycogenolysis in vivo are discussed.

Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane.

Abstract: A new structure of a lipid and its covalent linkage to a protein (murein-lipoprotein of the Escherichia coli outer membrane) is described Glycerylcysteine (S-(propane-2′,3′-diol)-3-thio-2-aminopropanic acid) at the N-terminal end of the polypeptide chain is the attachment site of two ester-bound fatty acids An additional fatty acid is bound as amide to the N-terminal group The diglyceride residue on the cysteine could be derived from the phospholipid pathway since the fatty acid composition is very similar to that of the phospholipids from the same cells In contrast, 65% of the amide-linked fatty acid is palmitate The main fatty acids are palmitic acid (53%), cis-vaccenic acid (207%), 9,10-methylene-hexadecanoic acid (106%) and palmitoleic acid (94%) The structure of glycerylcysteine was established by chemical degradation of the compound containing the fatty acids, by incorporation studies of [35S]sulfate, [35S]cysteine, [14C]cystine, [methyl-3H]methionine and [2-3H]glycerol and by chemical synthesis Glycerylcysteine was isolated as a constituent of the peptide (Ser)-Ser-Am-Ala-Lys The C-terminal part of the lipopeptide sequence overlaps with the N-terminal end of the known sequence of the polypeptide chain Its N-terminal position was confirmed by isolation of the N-terminal polypeptide fragment (position 1–31) after cleavage with cyanogen bromide This contained the lipid

Reversible photolysis of the purple complex in the purple membrane of Halobacterium halobium.

Abstract: 1 A suspension of the purple membrane from Halobacterium halobium in concentrated salt solutions saturated with diethylether is bleached by light. During this process, the purple complex is converted in to a compound absorbing maximally at 412 nm (412-nm complex). The purple complex reappears in the dark with a rate constant of 0.04 s−1 at 20°C. 2 The initial rate of formation of the 412-nm complex is proportional to the intensity of the bleaching light and a quantum yield of 0.79 is measured. The action spectrum of the photolytic reaction is identical to the absorption spectrum of the purple complex. 3 The light reaction is temperature independent. The regeneration of the purple complex requires an activation energy of 11.4 kcal × mol−1. 4 Illumination causes a decrease in the tryptophan fluorescence of the membrane. Regeneration of the purple complex restores the original fluorescence level. The half times of both processes correlate with the half times observed by absorption spectroscopy. 5 The photochemical reaction is accompanied by release of protons during bleaching and uptake of the same amount of protons during regeneration of the purple complex. The half times of the measured pH changes again correspond to the one observed by absorption spectroscopy. The rapid release of protons upon illumination is temperature independent. For the dark uptake of protons an activation energy of 14 kcal × mol−1 has been determined. The significance of this isolated photochemical system is discussed with respect to a possible function of the purple membrane in vivo.

The kinetics of the redox reactions of ubiquinone related to the electron-transport activity in the respiratory chain.

Abstract: The role of ubiquinone (Q) in the respiratory chain is quantitatively analyzed by correlating both the steady-state redox level of Q and the kinetics of oxidation and reduction with the flux of the overall electron transport in uncoupled submitochondrial particles. This is achieved by experimentally defining donor and acceptor activities for Q. 1 The degree of reduction of Q in the steady state is proportional to the respiratory activity with NADH and succinate, if the respiratory activity is varied by titrating the donor side for Q with rotenone and malonate, respectively. The proportionality constant (acceptor activity, Vox) is independent of the substrate used. 2 The degree of oxidation of Q in the steady state is proportional to the respiratory activity as varied by titration of the acceptor side for Q with antimycin. The proportionality constant (donor activity, Vred) is independent of the acceptor activity and depends on the dehydrogenase activity for NADH and succinate. 3 From these experimental relations, the redox state of Q in the steady state and the respiratory activity can be described as functions of the donor and the acceptor activity only. The equations are valid with both NADH and succinate as the substrates. 4 The kinetics of oxidation of Q on the addition of oxygen as measured by the quench-flow method are in agreement with that measured by direct absorption recording in a mixing chamber. The reaction is first order with a rate constant equal to the acceptor activity divided by the amount of redox-active Q (Vox/Qa). The final steady-state level (in the present case 15% reduction) is a result also of the reduction reaction with the first-order rate constant, VredQa. 5 The acceptor activity can also be measured as maximum respiratory activity with duro-hydroquinone. This activity is independent of the presence of Q but sensitive to antimycin. Thus, the acceptor activity for ubiquinone can be measured by three independent methods. 6 It is concluded, that also in the steady state the reduction and oxidation of the active Q-pool follow pseudo-first-order reactions, the rates of which are equal to the respiratory rate. The total amount of redox-active Q is kinetically and functionally homogeneous and is not divided into substrate-specific compartments.

Structure primaire de la caséine β bovine

Abstract: In a previous report [1], we have given the complete primary structure of ϰB1-caseinomacro-peptide which is the soluble COOH-terminal fragment split from bovine ϰB1-casein by rennin. We also reported on the COOH-terminal sequence of the NH2-terminal fragment, the so-called para-ϰ-casein. The present paper deals with the complete amino acid sequence of bovine ϰB-casein, which has now been achieved by establishing the primary structure of para-ϰB-casein of which we discuss the salient features. This work has been reported briefly in a short communication [2]. SCM-para-ϰB-casein and maleyl ϰBCN1, the NH2-terminal cyanogen bromide fragment of ϰB-casein [1], were used as starting material. The tryptic and peptic peptides of SCM-para-ϰB-casein and the chymotryptic peptides of ϰBCN1 were isolated on Dowex 50 and Sephadex G-50 or G-25, and their sequences were determined either partially or completely by using classical methods and in some cases mass spectrometry. All these peptides and a NBS fragment of SCM-para-ϰB-casein have provided all the overlaps needed for the completion of the amino acid sequence of para-ϰB-casein. Para-ϰB-casein is a single polypeptide chain containing 105 amino acid residues: Asp3, Asn4, Thr3, Ser7, PyroGlu1, Glu4, Gln12, Pro12, Gly1, Ala9, Val5, 1/2 Cys2, Met1, Ile6, Leu7, Tyr9, Phe4, Lys6, His3, Trp1, Arg5, and its molecular weight has been calculated to be 12269. The average hydrophobicity, calculated according to Bigelow [3], is 5.48 kJ (1.310 kcal) per residue, and para-ϰB-casein can be therefore considered to be a very hydrophobic molecule. Its net positive charge at pH of native milk (about 6.8) is very close to 4.5. The high content (11.5%) and rather uniform distribution of prolyl residues are incompatible with much α-helical organization of the molecule, as previously shown for ϰ-casein [4]. Both hydrophobic and charged amino acid residues are distributed non-uniformly along the chain. Two regions, 1–24 and 80–105, are hydrophilic: the very hydrophilic former, with NH2-terminal pyroglutamic acid, contains a cysteinyl residue located inside a cluster of eleven ionizable residues including six out of the seven total dicarboxylic amino acids; the 80–105 region, which contains the second cysteinyl residue in position 88, is rather hydrophilic, except at the COOH-terminal end which is hydrophobic in spite of the presence of a cluster of four basic residues. These two hydrophilic regions are very likely to be on the outisde of the molecule and this may favor the formation of intermolecular S-S bonds. The very hydro-phobic central part of the chain, viz., 25–79, where most of the aromatic residues are located, has a para-ϰ-casein-like behaviour in aqueous solution, and it may be responsible for the aggregation ability of para-ϰ-casein. According to the sequence data of both ϰB1-caseinomacropeptide [1] and para-ϰ-casein, it is concluded that bovine ϰB-casein is made up of a single polypeptide chain containing 169 amino acid residues: Asp4, Asn7, Thr14, Ser12, SerP1, PyroGlu1, Glu12, Gln14, Pro20, Gly2, Ala15, Val11, 1/2 Cys2, Met2, Ile13, Leu8, Tyr9, Phe4, Lys9, His3, Trp1, Arg5, with NH2-terminal pyroglutamic acid. However, in accordance with the well-known lability of glutamine residues in NH2-terminal position [5], such a pyroglutamic acid residue may arise from the subsequent cyclisation of a NH2-terminal glutamine residue present originally in native ϰ-casein. The content of proline residues is high (11.8%). The molecular weight of the carbohydrate-free monomer of ϰB-casein is 19023. Its net negative charge at pH of native milk is close to 3.5 and its average hydrophobicity is about 5.37 kJ (1.285 kcal) per residue. We have already reported [6] the location of the two amino acid substitutions differentiating the two known genetic variants : the B variant differs from the A variant by the respective amino acid substitutions Ile 136/Thr and Ala 148/Asp. Tryptic and chymotryptic peptides isolated by Jolles et al. [7] from ϰA-casein correspond obviously to the fragments 11–16, 17–25,31–34, 63–67, 1–10 and 86–105 of ϰ-casein strand, but there is only partial agreement concerning the sequences of amino acid proposed for the last three fragments. More recently, Fiat et al. [8] have studied a short glycopeptide isolated from bovine ϰ-casein, but neither the amino acid sequence nor the location of the phosphate group agree with our results.

The Mechanism of Enzymatic Cellulose Degradation

Abstract: 1 A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cx enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. 2 The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-Sepharose 6B) and isoelectric focusing. 3 The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. 4 Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. 5 The molecular weights of the enzymes were 12500 and 50000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. 6 The low-molecular-weight enzyme was isoelectric at pH 4.60 (10 °C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. 7 Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard.

Interaction of Mixed-Function Oxidation with Biosynthetic Processes

Abstract: Gluconeogenesis from lactate was studied in perfused livers from phenobarbital-pretreated rats fasted for 24 h Maximal rates of glucose production obtained with lactate concentrations of more than 2 mM were suppressed to about 50% following the addition of aminopyrine, a substrate for NADPH-utilizing mixed-function oxidations Submaximal rates (ie with 05–2 mM lactate) or gluconeogenesis from dihydroxyacetone were only slightly affected by aminopyrine, whereas no inhibition was observed at low gluconeogenic rates The data are consistent with the following hypothesis In the presence of active NADPH-utilizing processes (such as mixed-function oxidation of aminopyrine) a futile cycle involving malic enzyme occurs in the pathway of gluconeogenesis which is compensated for by an increased flux through the pyruvate carboxylase reaction Thus, gluconeogenesis is suppressed when maximal activity of pyruvate carboxylase is reached It is assumed that the observed inhibition of gluconeogenesis is not specific for mixed-function oxidation, but may be an example for a more general mechanism of metabolic interdependences involving the extramitochondrial NADPH pool

The Amino-Acid Sequence of the αA2 Chain of Bovine α-Crystallin

Abstract: Isolated cyanogen bromide fragments of the αB2 chain of α-crystallin contained 67 and 107 residues, respectively. Part of the sequence of the C-terminal fragment was elucidated by Edman and Begg sequenator analysis. Sequences of the tryptic peptides were determined by a combination of various Edman degradation techniques. Overlaps of tryptic peptides were established by selective modification of lysine residues and subsequent tryptic cleavage and by isolation of peptides after hydrolysis with chymotrypsin, thermolysin and a staphylococcal protease. The sequence of the αB2 chain comprises 175 residues corresponding to a molecular weight of 20070. This value is somewhat lower than that assumed hitherto. A high degree of homology between the αB2, and the αA2 chain, the major polypeptides, of bovine α-crystallin was observed.

Reversible dissociation of the purple complex in bacteriorhodopsin and identification of 13-cis and all-trans-retinal as its chromophores.

Abstract: 1 Suspensions of the purple membrane from Halobacterium halobium in water-dimethyl-sulfoxide mixtures change their absorption maximum dependent on Me2SO concentration. Between 30 and 60% Me2SO the purple complex equilibrates with a second complex form called the 460-nm complex. The apparent thermodynamic parameters of this reaction have been determined. 2 The equilibrium is influenced by pH and the nature of the buffering ion species in such a way that low pH and triethanolamine or histidine favour the purple complex. 3 The kinetics of the reaction indicate the participation of more than two species and thin-layer chromatography reveals two retinal isomers to be present in both complexes, i.e. 13-cis and all-trans-retinal. Light causes isomerization of the 13-cis to the all-trans form, in the dark a mixture of both isomers is found. 4 Only the 460-nm complex reacts with hydroxylamine to form retinaloxime. Retinaloximes derived from authentic retinal isomers show two spots on thin-layer plates, presumably due to syn and anti forms of the oxime. The reaction product from detergent-treated purple membrane shows the four spots corresponding to the 13-cis and all-trans-retinaloximes. The product from the 460-nm complex forms mainly two spots corresponding to 13-cis and all-trans-retinaloxime in only one form, either the syn or the anti form. 5 The reaction with hydroxylamine has a fast second-order and a slow zero-order component. The products formed at any time are the same. After the end of the fast initial phase only the purple complex is present in the sample and has first to be isomerized before it is dissociated by Me2SO and can react with hydroxylamine. This rate-limiting step does not involve C°C double bond isomerization but interconversion of two unknown complex configurations. 6 Light accelerates the slow part of the reaction with hydroxylamine. The product then consists mainly of syn and anti all-trans-retinaloxime but also syn and anti 13-cis-retinaloxime. This is interpreted as intermediate formation of the hydroxylamine sensitive 412-nm complex by reversible photolysis of the purple complex as described in [16]. In this case apparently no preference for the formation of the syn or the anti form exists.

Studies on Soybean Trypsin Inhibitors

Abstract: For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel nitration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy-peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and nine minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor.

Pancreatic lipase and co-lipase. Interactions and effects of bile salts and other detergents.

Abstract: The effect of conjugated bile salts on the activity of pancreatic lipase depends on their concentration in relation to their critical micellar concentration. Below the critical micellar concentration, conjugated bile salts slightly stimulate the initial rate of hydrolysis of tributyrine, an effect that may be caused by a protection of the enzyme from inactivation at the substrate-water interface. Above the critical micellar concentration, conjugated bile salts almost completely inhibit lipase. The inhibition is more marked in alkaline reactions resulting in a pH optimum shift with increasing bile salt concentration. Bile salt inhibition of lipase is related both to the concentration of bile salt and to the substrate concentration or rather to substrate surface area, and most probably is complete when the interface is saturated with detergent. Co-lipase, in the absence of bile salts, stimulates the activity of lipase, 1.3 to 1.4-fold in the whole pH range of its activity. Co-lipase overcomes the inhibition of lipase caused by bile salts with a shift in the pH optimum to 6–7 compared to 8–9 for lipase alone. The different conjugated bile salts have similar effects, consideration being taken to differences in their critical micellar concentration; free bile salts have a less inhibitory effect on lipase and the stimulation by co-lipase shows no pH optimum shift. Detergents of the acyltaurine type such as decanoyltaurine and dodecanoylsarcosyltaurine inhibit lipase in a similar manner to the conjugated bile salts and this inhibition is also released by co-lipase. Detergents such as dodecylsulphate above the micellar concentration, irreversibly inhibit lipase. The simultaneous presence of bile salts protects the enzyme from being irreversibly inactivated. Lipase and co-lipase interact in a stoichiometrical relationship and it appears justified to classify co-lipase as a co-enzyme for lipase.

Phenol hydroxylase from yeast

Abstract: An enzyme, able to carry out an NADPH-dependent hydroxylation of monocyclic phenols, was purified 20–30-fold from Trichosporon cutaneum grown on phenol or resorcinol as a major carbon source The purified enzyme was homogeneous upon analytical disc electrophoresis The enzyme is a bright-yellow protein with an absorption spectrum typical of flavoproteins Its molecular weight is 148000, and its estimated FAD content is approximately 1 mole per mole enzyme The purified enzyme has essentially the same broad substrate specificity as crude preparations In addition to phenol it also hydroxylates the three isomeric diphenols and a number of phenol derivatives to their corresponding o-diols Km values for the three substrates of the holoenzyme are all of the order 10 μM : Km (phenol) = 18 μM, Km(NADPH) = 71 μM The absorption spectrum of the holoenzyme is modified in the presence of phenol FAD could be resolved from the purified enzyme by preparative disc electrophoresis Reassembly of the holoenzyme required SH-groups The enzyme activity is unaffected by chelators of iron and copper but it is inhibited by heavy metals The inhibition by p-chloromercuribenzoate is readily reversed by dithiothreitol Among oxidizing agents, hydrogen peroxide and peroxidase depressed the enzyme activity, whereas catalase was without effect Among reducing agents ascorbate depressed enzyme activity Sodium dithionite and sodium borohydride bleached the enzyme with concomitant loss of activity After reduction with dithionite, the enzyme was rapidly re-oxidized, re-gaining its original activity After reduction with borohydride re-oxidation was very slow However, the enzyme could be re-activated by incubation with FAD The enzyme is very sensitive to inorganic salts, nitrogen bases and detergents Chloride is quite deleterious whereas phosphate seems to stabilize the enzyme

Phosphorylation of very-lysine-rich histone in Physarum polycephalum. Correlation with chromosome condensation.

Abstract: Physarum polycephalum histones have been analysed by acrylamide gel electrophoresis. Two of the five major bands had electrophoretic mobilities identical with the mobilities of two bands from calf thymus histone. The P. polycephalum pattern is qualitatively the same at all stages of the synchronous mitotic cycle. Quantitative changes in the relative proportion and relative mobility of the very-lysine-rich histone are reported. In particular, a dramatic increase in phosphate content of this histone occurred in late G2 phase with a peak where chromosome condensation is seen to be occurring in the phase contrast microscope. Phosphate content is low during S phase and the peaks of RNA synthesis.

Further evidence for the pool function of ubiquinone as derived from the inhibition of the electron transport by antimycin.

Abstract: The role of ubiquinone (Q) in the respiratory chain is studied by analyzing the inhibition by antimycin of the electron transport in uncoupled submitochondrial particles. As a result, the antimycin inhibition curve of substrate oxidation is quantitatively explained. 1 The participation of Q in the electron transport is required for the occurrence of nonlinear antimycin inhibition curves as demonstrated with external hydroquinones of different redox potentials as hydrogen donors. The respiration is non-linearly inhibited with a hydroquinone of sufficiently low redox potential to reduce the mitochondrial Q (menadiol) whereas a linear inhibition curve is found with hydroquinones of higher redox potentials (durohydroquinone, dimethoxy-dimethylbenzohydroquinone). If Q is extracted, the inhibition curve also with menadiol becomes linear. This demonstrates the relation of the nonlinearity of the inhibition curve to the function of Q as a distributor of hydrogen between parallel cytochrome chains. 2 The electron transport through the cytochrome segment is inhibited linearly with increasing amounts of antimycin as demonstrated by the linear titration of three different functions: (a) the oxidation rate of Qred on addition of oxygen to anaerobic submitochondrial particles, (b) the repiratory activity with added hydroquinones, (c) the acceptor activity as defined by the ratio of the respiration rate to the redox state of Q (Kroger, Klingenberg and Schweidler, 1973). 3 The ratio of donor to acceptor activity to Q (Vred/Vox) is the main parameter in controlling the shape of the antimycin titration curve. This curve becomes more nonlinear with decreasing this ratio. Also the redox state of Q is nonlinearly dependent on the amount of antimycin and controlled by the donor/acceptor activity ratio. 4 A theoretical hyperbolic relation of the rate of substrate oxidation to the amount of antimycin is derived, involving the ratio donor to acceptor activity and the linear inhibition by antimycin of the electron transport through the cytochrome segment. With independently measured donor/acceptor activity ratios, antimycin titration curves are calculated which give a good fit to the experimental titrations over the range from highly nonlinear to almost linear curves. The observed nonlinearity is thus recognized to result from a hyperbolic relationship. Also the calculated titration curves for the redox state of Q are in agreement with the experiments. 5 The deviation of the experiment from the calculated curves near equimolarity between the amount of antimycin and inhibition sites can be accounted for by the dissociation of antimycin from the site of inhibition.

Respiratory Control and the Proton Electrochemical Gradient in Mitochondria

Abstract: The relationship between the rate of electron transport and the proton electrochemical gradient ΔH across the inner membranes of rat liver mitochondria was investigated. ΔH was calculated from ΔPH as measured by 14C-labeled 5,5-dimethyl-2, 4-oxazolidine dione distribution and from Δφ as measured by 42K distribution (in the presence of valinomycin). When mitochondria are in state 3, i.e. during phosphorylation, ΔH was only 5 m V less than in state 4, while respiration increased 3.5-fold. In the presence of uncoupler, respiration was the same as in state 3, but ΔH was reduced by 22 m V with dinitrophenol, 28 m V with KCI + valinomycin and 60 m V with gramicidin. In the presence of valinomycin, when uncoupling increases by increasing KCl concentration, there was a linear relationship between ΔH and the rate of respiration. These results are in agreement with the suggestion that in uncoupled systems the respiratory rate is controlled by ΔH. However, the phosphorylation reaction controls the respiratory rate directly and not through its effect on ΔH. This conclusion is not compatible with the chemiosmotic model of oxidative phosphorylation.

Purification and characterization of adenosine triphosphate: ribonucleic acid adenyltransferase from Escherichia coli.

Abstract: A primer-dependent poly (A)-polymerizing activity using ATP as substrate (ATP: RNA adenyltransferase) was isolated in high yield from Escherichia coli and purified to apparent homogeneity. For this purpose an assay system had to be used which restricted a variety of infering enzyme activities. Since the enzyme aggregates to macromolecular cell components or precipitates when kept in low salt conditions (<0.4 M NaCl), it was necessary to perform the entire purification procedure in high salt conditions. This was accomplished by using a high salt ribosomal supernatant, a polyethylenglycol-dextran-NaCl phase partition step and a very efficient final high salt phosphocellulose chromatography. At 0.5 M NaCl the enzyme has a molecular weight of approximately 58000. Dodecylsulfate gel electrophoresis shows a single polypeptide chain of molecular weight × 50000. The enzyme has a high preference for ATP as substrate. Manganese ions show higher activity as cofactors than magnesium ions. All classes of natural RNAs are used as primers. The free 3′terminal hydroxyl group of the RNA is required. The enzyme is unable to catalyze a pyrophosphorolysis or a phosphorolysis reaction. It is shown that ATP: RNA adenyl-transferase preferentially synthesizes rather long chains of poly(A) attached to the RNA primers. No relation between the enzyme protein and subunits of the DNA-dependent RNA polymerase could be detected.

Isolation and Characterisation of Two Calf‐Thymus Chromatin Non‐Histone Proteins with High Contents of Acidic and Basic Amino Acids

Abstract: The group of calf thymus chromatin non-histone proteins which have a high electrophoretic mobility have been fractionated by CM-cellulose ion-exchange chromatography. The fractions thus obtained have been characterised by polyacrylamide gel electrophoresis, amino acid analysis, N-terminal amino acid analysis, and by their molecular weights. Using the CM-cellulose chromatographic method, two proteins which have a high content of basic and acid amino acids have been isolated in a pure form.

The Primary Structure of an Acidic Protein from 50-S Ribosomes of Escherichia coli which is Involved in GTP Hydrolysis Dependent on Elongation Factors G and T

Abstract: The primary structures of the ribosomal proteins A1 (= L7) and A2 (= L12) have been elucidated. Comparison of the two amino acid sequences confirms earlier studies by us (1972) which indicated that the two proteins are identical except that A1 possesses an N-terminal acetyl group. Sequencing of tryptic and chymotryptic peptides was accomplished primarily by automatic solid-phase Edman degradation of 50- to 70-nanomole peptide samples. A large tryptic peptide T1Phe, which could not be sequenced by this method, was fragmented with elastase and its sequence mainly derived by conventional methods. The sequence of the first 51 residues of the nonacetylated A2-protein was obtained by Edman degradation in the Beckman sequencer. A-protein comprises three distinct regions: I (residues 1—55), which is negatively-charged and hydrophobic, II (56—81), which is positively charged, and III (82—120), which is negatively charged and hydrophilic. α-Helix promoting residues are located primarily in regions I and III. ɛ-N-Monomethyllysine, which occurs in 50% of the A1 and A2 chains is located in the more flexible region II at position 81. The distinctive clustering of hydrophobic and charged residues is quite remarkable and suggests that in situ certain of these regions may contain a binding site for components involved in peptide chain elongation.

Cytochrome c: a thermodynamic study of the relationships among oxidation state, ion-binding and structural parameters. 1. The effects of temperature, pH and electrostatic media on the standard redox potential of cytochrome c.

Abstract: The standard redox potential at I= 0.01, 25°C and pH 7.0 has been determined for the following species of cytochrome c: horse heart, baker's yeast isoenzyme-1, Candida species yeast, tuna heart and turkey heart. The thermodynamic parameters of the redox reaction were evaluated and found to be identical, within experimental error, in the five species investigated. The effect of pH on the standard redox potential of horse heart cytochrome c was studied in the pH range 7.0–11.2. A single heme-linked ionization of the oxidized protein was observed in this pH range, with a dissociation constant of 3 × 10−9 at I= 0 and 25°C. The effects of the electrostatic media on the standard redox potential of horse heart cytochrome c depend on the nature of the anions employed. For non-binding medium at 25°C, the observed potential dependence on the ionic strength is given by the equation: E0obs= 0.274–0.336 √I/(1 + 6√I). In binding medium, specific binding of ions to the protein takes place.

Biohydrogenation of sterols by Eubacterium ATCC 21,408--Nova species.

Abstract: Biohydrogenation of Δ5-steroids was studied in vitro by incubating various steroids with a pure culture of Eubacterium 21,408 in a brain-thioglycollate medium under anaerobic conditions. 1 Eubacterium 21,408, a strictly anaerobic bacterium, was isolated from rat cecal contents. It differed from any previously described Eubacterium species by its requirements for a Δ5-3β-hydroxy steroid. 2 Eubacterium 21,408 reduced the 5,6-double bond of cholesterol, campesterol, β-sitosterol and stigmasterol, exclusively yielding the corresponding 5β-saturated derivatives. Similarly, the 4,5-double bond of allocholesterol was reduced to yield coprostanol. Neither the 7,8-double bond in 7-dehydrocholesterol or in lathosterol, nor the 22,23-double bond in the side chain of stigmasterol were reduced. 3 In the absence of a 3-hydroxyl function (e.g. in cholest-5-ene), when the 3-hydroxyl function was in the 3α-position (e.g. in epicholesterol) or was substituted (e.g. in 3β-chloro-cholest-5-ene and in cholesteryl esters), no saturation of the 5,6-double bond was observed. 4 When incubated with Eubacterium 21,408, the carbonyl group of 4-cholesten-3-one, was reduced to the corresponding 3β-hydroxyl group along with saturation of the 4,5-double bond. The bacterium also reduced the 3-oxo group of cholestanone and coprostanone to a 3β-hydroxyl group.

Outer membrane of salmonella. Sites of export of newly synthesised lipopolysaccharide on the bacterial surface.

Abstract: The UDP-galactose-4-epimeraseless strain 1195 of Salmonella typhimurium bears incomplete lipopolysaccharide, and only after addition of galactose to the growth medium it produces wild-type lipopolysaccharide. It was demonstrated in a double-label experiment that already existent, incomplete lipopolysaccharide is not completed after galactose addition but that only the newly synthesised lipopolysaccharide carries the wild-type specificity. It was thus possible to locate newly synthesised lipopolysaccharide with specific anti-wild-type, ferritin-conjugated antibodies in freeze-etch preparations and ultra-thin sections. With the freeze-etch technique new lipopolysaccharide was found to appear in about 220 patches per cell, 30 s after 50 mM galactose had been added to the medium. After growth for 2–3 min under these conditions new lipopolysaccharide was found to be evenly spread over the entire cell surface. In ultra-thin sections of plasmolysed cells 86% of the ferritin patches, indicating newly synthesised lipopolysaccharide, was found located over sites where the cytoplasmic and the outer membrane adhere to one another. It is discussed that new lipopolysaccharide emerges from these sites and that lateral movement of lipopolysaccharide in the outer membrane results in an even distribution on the bacterial cell surface.

Activation of human plasminogen by an insoluble derivative of urokinase. Structural changes of plasminogen in the course of activation to plasmin and demonstration of a possible intermediate compound.

Abstract: Activation of plasminogen was performed by perfusing a plasminogen solution through a small column loaded with insolubilized urokinase. The effluent was immediately collected in an excess of plasmin inhibitor (p-nitrophenyl-p-guanidino-benzoate). This provides a practically instantaneous inhibition of the plasmin formed and autocatalytic degradation is thus reduced to a minimum. Different degrees of activation are obtained by changing the perfusion rate. Dodecylsulfate-polyacrylamide electrophoresis, amino acid and NH2-terminal amino acid analyses show that the activation of human plasminogen involves cleavage of at least two peptide bonds, the first event being the release of a peptide of 64 amino acid residues from the NH2-terminal end. An intermediate inactive product with a molecular weight of about 86000 and methionine as NH2-terminal amino acid is formed. Cleavage at the second site results in formation of a molecule with two chains connected by disulfide bridges. One chain which has a molecular weight of about 63000 is very sensitive to proteolytic degradation in its NH2-terminal part. However, there is evidence that methionine is the true NH2-terminal amino acid of this fragment. The other chain has a molecular weight of about 25000 and has valine as NH2-terminal amino acid.

Initiation of Aerobic Oxidation of Sulfite by Illuminated Spinach Chloroplasts

Abstract: Spinach chloroplasts induced aerobic oxidation of sulfite on illumination. The oxidation of sulfite was not found in KCl- and Tris-treated chloroplasts and was inhibited by 3(3,4-dichloro-phenyl)-1,1′-dimethylurea, suggesting that the reaction is induced through the electron transport system in chloroplasts. The oxidation rate of sulfite was much higher than the rate of the Hill reaction. In addition, inhibition by several sugars indicates that the oxidation of sulfite proceeds as a chain reaction. Although catalase did not affect the reaction, ferredoxin plus NADP, cytochrome c and superoxide dismutase prevented the oxidation of sulfite. We thus propose that a superoxide anion formed by the univalent reduction of oxygen by illuminated chloroplasts is the initiator of sulfite oxidation.

Lipid Requirement of Membrane‐Bound ATPase

Abstract: 1 The (Na++ K+)-stimulated ATPase of human erythrocyte ghosts is completely inactivated by treatment with pure phospholipase A2 or phospholipase C. 2 These phospholipases also cause considerable or even complete loss of Mg2+-dependent ATPase activity. 3 Enzymatic hydrolysis of sphingomyelin does not significantly influence either of the ATPase activities. 4 Conversion of the glycero-phospholipids into phosphatidic acid by phospholipase D causes a drastic decrease in the Mg2+-dependent ATPase activity, whereas the (Na++ K+)-stimulated activity is increased. 5 Enzymatic decarboxylation of the phosphatidylserine fraction of the ghosts does not influence the (Na++ K+)-stimulated ATPase, unless the last 13% of this fraction is converted into phosphatidyethanolamine, when there is a complete loss of this particular ATPase activity. On the other hand, the Mg2+-dependent activity tends to be increased by this treatment. 6 The (Na++ K+)-stimulated ATPase activity of erythrocyte ghosts treated with phospholipase C and subsequently extracted with dry ether, can be completely reconstituted by the addition of phosphatidylserine. Phosphatidylcholine, phosphatidylethanolamine and phospholipid mixtures are found to be ineffective. Phosphatidic acid produced a partial restoration of activity. 7 It is concluded that the (Na++ K+)-stimulated ATPase activity strongly depends upon the presence of phosphatidylserine, of which only a minor fraction in the erythrocyte membrane is directly involved. It cannot be concluded from the experiments described in this paper that the Mg2+-dependent ATPase also requires a specific phospholipid. However, a direct involvement of either cholesterol or sphingomyelin in this activity can be precluded.

Products of mitochondrial protein synthesis in Neurospora crassa. Determination of equimolar amounts of three products in cytochrome oxidase on the basis of amino-acid analysis.

Abstract: Cytochrome oxidase isolated from N eurospora crassa was resolved into seven protein eompo­ nents by eleetrophoresis in polyaerylamide gels eontaining sodium dodeeylsulfate. The apparent molecular weights were determined tobe 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the eomponents 1, 2, 3, 4, 5, 6, and 7, respectively. The components 1, 2 and 3 are synthesized on mitochondrial ribosomes as shown by the incorporation of radioactive amino aeids in the pres­ enee of cyeloheximide. Amino-acidanalysis of the isolated components 1, 2 and 3 revealed a high content of apolar amino acids and a low eontent of basic amino aeids compared to an average amino-aeid eomposi­ tion of components 4-7. Components 1, 2 and 3 eontribute 27.9°/0 , 18°/0 and 14.2°/0 to the whole eytoehrome oxidase protein. This was calculated from the contributions of the single eomponents to the totalleueine eontent of the enzyme and the leueine eontents (nmol leueine per mg protein) of the single eomponents as determined by amino-aeid analysis. Equimolar relations of the components 1, 2 and 3 are found by dividing the amounts of protein by their apparent molecular weights. A stoi­ chiometry of 1:1:1 results assuming a minimal molecular weight of 150000 for the whole cyto­ chrome oxidase protein. On the basis of the heme a content a molecular weight of about 70000 per heme group was determined, using an absorption coeffieient L1e605 (redueed minus oxidized) of 12 mM- 1 cm- 1 • It is concluded that the smallest structural unit of eytochrome oxidase contains two heme groups.

Subcellular Location of Superoxide Dismutase in Spinach Leaves and Preparation and Properties of Crystalline Spinach Superoxide Dismutase

Abstract: Localization of superoxide dismutase in spinach chloroplasts was demonstrated by differential and sucrose-density centrifugations. About half to two thirds of the enzyme in chloroplasts was released by osmotic shock. In peroxisomes, no superoxide dismutase was detected. Disc electrophoresis of chloroplast and supernatant fractions revealed a single band of superoxide dismutase activity corresponding to the purified enzyme. Superoxide dismutase was purified from spinach leaves and prepared in a crystalline state. s020, w was 2.75 S and the molecular weight was approximately 32000 daltons. The enzyme was composed of two subunits of equal size and contained 2 atoms of copper and 2 atoms of zinc per molecule. In addition, visible and ultraviolet spectra, electron paramagnetic resonance spectrum and amino acid composition were similar to these properties of the enzyme isolated from mammalian tissues, Neurospora, green pea and yeast. A possible function of superoxide dismutase in chloroplasts is discussed.