Showing papers in "FEBS Journal in 1980"

Sugar-Lectin Interactions: How Does Wheat-Germ Agglutinin Bind Sialoglycoconjugates?

Abstract: The specific binding of N-acetylneuraminic acid to wheat-germ agglutinin is based on configurational similarities between N-acetylneuraminic acid and N-acetylglucosamine. The N-acetamido group and an adjacent hydroxyl group, both in an equatorial position are shown to be the main determinants. The N-acetylneuraminic acid - wheat-germ agglutinin interaction is increased by the removal of the last two carbons Cs and C9. The interaction between wheat-germ agglutinin and glycoconjugates containing N-acetylneuraminic acid is shown to be dependent on a charge effect and on an avidity effect. Succinylated wheat-germ agglutinin which is negatively charged at physiological pH, in contrast with wheat-germ agglutinin which is positively charged, does not bind cell surface glycoconjugates containing N-acetylneuraminic acid but does bind cell surface glycoconjugates containing N-acetylglucosamine. The use of wheat-germ agglutinin and of SUCcinylated wheat-germ agglutinin leads to the determination of the number of cell surface receptors containing N-acetylneuramipic acid

Heterogeneity of antigenic-side-chain length in lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2.

Abstract: Lipopolysaccharide from Escherichia coli 0111, its galE derivative when grown in galactose, E. coli 086, and Salmonella typhimurium LT2 all contain antigenic side chains and separate into more than 40 components by electrophoresis in gradients of polyacrylamide containing sodium dodecylsulfate. These components from E. coli 0111 are not interconvertible and show a heterogeneous size distribution when fractionated with Sephadex G-200. Isoelectric focusing of this mixture in pH 3.5–10 ampholines reveals a single component, ruling out extensive charge heterogeneity. The relative antigenic side chain lengths for the components, estimated using ratios of galactose in antigenic side chain to phosphate in the lipid-A–core oligosaccharide region, show that the size heterogeneity is due to differences in the number of antigenic side chain units per molecule and ranges from none to over 40. Preference for molecules of specific chain lengths, especially short ones, was observed. In contrast, the galE mutant grown without galactose does not synthesize antigenic side chains, and more than 90% of its lipopolysaccharide migrates as a single band at a position corresponding to the lowest-molecular-weight component from the above preparations. Lipopolysaccharide from E. coli PL2, a K12 strain lacking antigenic side chain, separates into two low-molecular-weight components on electrophoresis. These results confirm that the heterogeneity which we observe in lipopolysaccharide containing antigenic side chains, is due to the side chain rather than the lipid-A –core oligosaccharide region.

The optimal efficiency and the economic degrees of coupling of oxidative phosphorylation.

Abstract: A phenomenological theory considering the output characteristics of oxidative phosphorylation has been worked out by adopting the formalism of linear nonequilibrium thermodynamics. The linearity of oxidative phosphorylation in the range of the output forces of practical interest has been experimentally verified. The efficiency of oxidative phosphorylation is zero if either a load with a zero conductance (open-circuited situation) or a load with an infinite conductance (short-circuited situation) is attached to oxidative phosphorylation. In between these extreme conductances there exists a finite load conductance permitting oxidative phosphorylation to operate with optimal efficiency. The necessary and sufficient condition for optimal efficiency was found to be L33/L11=√1−q2 where L11 is the phenomenological conductance of phosphorylation, L33 the phenomenological conductance of the load and q the degree of coupling of oxidative phosphorylation driven by respiration. This condition was called conductance matching. Under the condition of conductance matching, four output functions of oxidative phosphorylation of practical interest were optimized. A maximal net rate of oxidative phosphorylation occurs at a degree of coupling qf= 0.78. A maximal output power of oxidative phosphorylation, i.e. net rate times established phosphate potential, results at qp= 0.91. The maximization of the function net rate times efficiency yielded an economic degree of coupling qecp= 0.95 for maximal ATP flow. Finally, maximization of the function output power times efficiency led to a degree of coupling qecp= 0.97. This last function simultaneously maximizes net rate of ATP production, developed phosphate potential and efficiency and reflects therefore the most economic solution to the output problem under the condition of conductance matching. In isolated rat livers perfused in a metabolic resting state, the condition of conductance matching is fulfilled. In addition, the degree of coupling of oxidative phosphorylation under these conditions corresponds to the economic degree of coupling qecp.

Lipopolysaccharide Heterogeneity in Salmonella typhimurium Analyzed by Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis

Abstract: Salmonella typhimurium strains deficient in uridine diphosphate glucose epimerase were specifically labelled in the lipopolysaccharide by growing them with [14C]galactose. Electrophoretic analysis of isolated cell envelopes in the presence of sodium dodecyl sulfate revealed at least 40 regularly spaced bands. This banding pattern was stable in boiling dodecyl sulfate, and did not correspond to the pattern of polypeptides. A similar pattern was obtained from the isolated lipopolysaccharide. Mutant bacteria whose lipopolysaccharide contains only the core portion of the molecule gave only the fastest moving band. Bacteria whose lipopolysaccharide has only one repeating unit gave only two bands, one apparently corresponding to the core, the other moving somewhat slower. Bacteria with wild-type lipopolysaccharide gave bands corresponding to both of these, plus a large number of progressively more slowly moving ones, most likely representing lipopolysaccharide molecules with increasing numbers of repeating units in the O side chain. The distribution of chain lengths was unequal so that 77% of molecules with side chains had from 19 to 34 repeating units. On the other hand, two thirds of all lipopolysaccharide molecules were devoid of O side chains. In addition to this major heterogeneity due to differences in chain lengths, a second heterogeneity was indicated by the double character of each band.

7-S collagen: characterization of an unusual basement membrane structure.

Abstract: A new type of collageneous structure, tentatively named 7-S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistance towards further degradation by bacterial collagenase at 20 degrees C. 7-S collagen showed an amino acid composition typical of basement membrane collagen and contained 22% carbohydrate mainly as glucosyl-galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360,000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with tm = 48 degrees C and 70 degrees C. The more stable domain could be isolated as an homogeneous fragment (Mr = 225,000) after a second digestion with collagenase at 37 degrees C. This fragment contained all the disulfide bonds (42 Cys/1,000 residues) of the original molecule. Electron microscopy showed a rod-like structure in agreement with the hydrodynamic properties of 7-S collagen. The dimensions of these peptides were 3 X 95 nm (long form) and 2.4 X 40-50 nm (short form). Complete reduction of 7-S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27,000-153,000 which differ from each other by Mr increments 25,000-27,000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross-linked or represent fragments produced during proteolytic treatments. Complete reduction of 7-S collagen under non-denaturing conditions lowered the thermal transiton of the triple helix to 48 degrees C but did not change its molecular weight except when exposed to dissociating solvents. 7-S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37 degrees C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross-reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7-S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.

Folding mechanism of the triple helix in type-III collagen and type-III pN-collagen. Role of disulfide bridges and peptide bond isomerization.

Abstract: The kinetics of triple-helix formation in type III pN-collagen, type III collagen and a quarter fragment of type III collagen was followed by optical rotation and circular dichroism. Kinetic intermediates were detected by trypsin digestion and polyacrylamide gel electrophoresis. The end products of refolding at 25 degrees C were identical to the native molecules according to their melting profiles, molecular weights and sedimentation behavior. Only at low temperatures (4-15 degrees C) were mismatched structures of lower stability formed. At 25 degrees C helix formation started exclusively at the set of three disulfide bridges which link the three chains at the carboxy-terminal end. The growth of the triple helix proceeds from this single nucleus at a rather uniform rate in a zipper-like fashion. This gives rise to zero-order kinetics over a large fraction of the conversion. Consequently the time of half conversion is proportional to the length of the molecule. From the appearance and disappearance of intermediates the growth of the triple helix could be observed directly. The rate of helix propagation is determined by the rate of cis leads to trans isomerization of peptide bonds. A model mechanism was devloped which quantitatively described the overall kinetics as well as the time course of the intermediates with a single set of parameters: the rate constant of cis leads to trans isomerization k = 0.015 s-1 and an average number of 30 tripeptide units in uninterrupted stretches of residues with all peptide bonds in trans configuration.

Lactose Carrier Protein of Escherichia coli Structure and Expression of Plasmids Carrying the Y Gene of the lac Operon

Abstract: The previously described hybrid plasmid pC7 which carries lacI+O+delta(Z)Y+A+ on a 12.3 X 10(6)-Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239-248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to d(A-A-T-T) and ligated to the vector pB322. lac Y-carrying inserts of various sized (Mr 1.5-4.7 X 10(6)) were obtained. Hybrid plasmid pTE18 (2300-base-pair insert) carries part of the I (repressor) gene, the promotor-operator region, part of the Z (beta-galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18-harbouring strains the Y-gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12-16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.

Localization of Individual Lysine-Binding Regions in Human Plasminogen and Investigations on Their Complex-Forming Properties

Abstract: After partial digestion of human plasminogen with elastase, followed by chymotryptic cleavage of one of the fragments produced, two polypeptides with molecular weights of approximately 10000 and with lysine-binding sites still intact were isolated by means of affinity chromatography and gel filtration. One fragment, which was completely sequenced (88 residues), was identified as the fourth kringle, whereas the other, according to partial sequence analysis represented the first kringle. Equilibrium, dialysis against 6-aminohexanoic acid yielded for the first kringle one high-affinity binding site (Ka= 60 mM−1) and for the fourth kringle one single low-affinity binding site (Ka= 28 mM−1). Moreover, interactions were detected between the first kringle and the N-terminal CNBr fragment of plasminogen and also fibrin. In these cases an additional lysine- binding site, though of low affinity, appears to be involved. Thus, the first kringle seems to play important roles, structurally by contributing to the maintenance of a compact structure of plasminogen through an intramolecular interaction with its N-terminal polypeptide region, and functionally by increasing the fibrin affinity of Lys-plasminogen (plasminogen lacking the first 76 residues) and plasmin.

Temperature Behaviour of Human Serum Albumin

Abstract: Structural alterations of albumin, their dependence on concentration and the role of free --SH groups at thermal denaturation, as well as the reversibility of thermally induced structural changes, were studied. Application of various physical methods provides information on a series of structural parameters in a major concentration range. Apart from changes of the helix content, heat treatment gives rise to beta structures which are amplified on cooling and which are correlated with the aggregation of albumin. With rising temperature and concentration the proportion of beta structures and aggregates increases. At degrees of denaturation of up to 20% complete renaturation is possibly in every case. The structure content is concentration-dependent even at room temperature. It may be that intermolecular interactions induce additional alpha-helix structures which are less stable, however, than the ones stabilized by intramolecular interactions. Unfolding of the pocket containing the free --SH group of cysteine-34 enables disulphide bridges to be formed leading to stable aggregates and irreversible structural alterations. Through binding of N-ethymaleimide to free --SH groups, which blocks the formation of disulphide bridges, it is possible to prevent aggregation and irreversible conformational changes. At temperatures below 65--70 degrees C, oligomers are formed mainly via intermolecular beta structures.

Two-Dimensional Gel Analysis of Histones in Acid Extracts of Nuclei, Cells, and Tissues

Abstract: Two-dimensional gel analysis of histones from extracts of nuclei, cells, and tissues is described. A discontinuous buffer system which concentrates the sample was used to increase resolution in both dimensions and also to allow the direct loading of HCl extracts of chromatin, nuclei, cells, and tissues. Stained one-dimensional gels are used as sample gels for the second dimension, cetyltrimethylammonium bromide being used to solubilize the proteins in the dye-protein complex. These methods enable one to purify proteins through acetic acid/urea/Triton, acetic acid/urea, and sodium dodecyl sulfate gels without eluting them from the gels. The method is also compatible with the use of protamine to displace histones from nuclei and nucleosomes separated in chromatin gels.

The Purine‐Nucleotide Cycle

Abstract: The contents of citric acid cycle compounds were measured in rat hind limb muscle in situ during rest, exercise, and recovery from exercise. The following changes in citric acid cycle intermediates were observed during exercise for 15 min. The contents of fumarate and malate increased fourfold over resting values. The contents of citrate, isocitrate, and succinate rose 68%, 48% and 87% respectively, but the increase in these intermediates was delayed relative to the increase in fumarate and malate. The content of 2-oxoglutarate did not change significantly. The content of oxaloacetate increased from less than 1 nmol/g dry weight in resting muscle to 8 nmol/g dry weight during exercise. The orthophosphate content doubled. The sum of the contents of citric acid cycle intermediates doubled. The ratios of the contents of malate/fumarate and citrate/isocitrate remained constant. The constancy of the citrate/isocitrate ratio indicates that the concentration of intra-cellular free Mg2+ is not affected by exercise. All metabolite levels returned to resting values during recovery. Comparison of these data with the rate of operation of the purine nucleotide cycle leads us to conclude that the increase in citric acid cycle intermediates can be accounted for by the operation of the purine nucleotide cycle.

Chloroplast Phosphoproteins. Evidence for a Thylakoid‐Bound Phosphoprotein Phosphatase

Abstract: 1 Isolated intact pea (Pisum sativum) chloroplasts incorporate [32P]orthophosphate into several thylakoid polypeptides in the light Transfer of the labelled chloroplasts to darkness results in rapid dephosphorylation of the polypeptides The most rapidly dephosphorylated phosphoproteins are the 26000-Mr doublet derived from the light-harvesting chlorophyll a/b binding complex 2 Incubation of isolated 32P-labelled thylakoids in buffer in the absence of stromal components also results in rapid protein dephosphorylation Again, the most rapidly dephosphorylated phosphoproteins are the 26000-Mr light-harvesting doublet Dephosphorylation of all thylakoid phosphoproteins is accelerated by addition of up to 10 mM MgCl2 3 The enzyme responsible for dephosphorylation is a phosphatase rather than a phosphotransferase or the thylakoid protein kinase acting in reverse The enzyme is specifically and totally inhibited by NaF and does not require phosphoryl group acceptors such as ADP Unlike the protein kinase, the phosphatase is indifferent to light and the electron transport inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea 4 The phosphorylated regions of the thylakoid phosphoproteins protrude from the outer surface of the membrane and are removed by trypsin treatment

Changes in Polygalacturonase Isoenzymes during the ‘Ripening’ of Normal and Mutant Tomato Fruit

Abstract: 1 Polygalacturonase activity is not detectable in mature green tomato fruits but appears as fruits begin to change colour and continues to increase during the ripening period There is a sequential appearance of two isoenzymes, polygalacturonase 1 and 2, during ripening These isoenzymes have been purified and their properties compared Polygalacturonase 1 has a Mr of 100,000, is 50% inactivated at 78 degrees C and has a density of 1343 g cm-3 in caesium chloride Polygalacturonase 2 has a Mr of 42,000, is 50% inactivated at 57 degrees C and has a density of 1300 g cm-3 in caesium chloride 2 Fruits from isogenic lines homozygous for the 'Neverripe' (Nr) mutation do not ripen normally and contain reduced amounts of polygalacturonase Only polygalacturonase 1 is produced in Nr fruit Tomatoes from isogenic lines homozygous for the 'ripening inhibitor' (rin) mutation do not ripen normally and produce very little detectable polygalacturonase 3 Although polygalacturonases 1 and 2 have different properties they both give rise to a single polypeptide on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl-sulphate (Mr = 46,000) A comparison of the major fragments produced by limited proteolysis of polygalacturonase 1 and 2 with chymotrypsin suggests that the polypeptides from the two isoenzymes are similar The same conclusion was reached from a comparison of polygalacturonase 1 and 2 by radioimmunoassay, using antibody prepared against polygalacturonase 2 and 125I-labelled polygalacturonase 2 4 The results from radioimmunoassay of extracts from green and ripening fruits suggest that the increase in polygalacturonase activity during ripening is due to net synthesis of protein

The Interaction of Bovine Pancreatic Deoxyribonuclease I and Skeletal Muscle Actin

Abstract: The rate of exchange of actin-bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 × 108 M−1 and 1.2 × 104 M−1 respectively. The depolymerisation of F-actin by DNAase I appears to be due to a shift in the G-Fequilibrium of actin by DNAase I. Inhibition of the DNA-degrading activity of DNAase I by G-actin is of the partially competitive type.

The Enhancement of Cytotoxicity of N‐Methyl‐N‐nitrosourea and of y‐Radiation by Inhibitors of Poly(ADP‐ribose) Polymerase

Abstract: Inhibitors of poly(ADP-ribose) polymerase show a synergistic potentiation of cytotoxicity with certain DNA-damaging agents. Non-toxic concentrations of 5-methylnicotinamide dramatically potentiate the cytotoxicity of N-methyl-N-nitrosourea as tested by the cloning ability of mouse leukaemia (LI210) cells. A dose-enhancement factor of about 10 is observed. This potentiation is dependent on the concentration of 5-methylnicotinamide. The methylxanthines theobromine, theophlline and caffeine also increase the cytotoxicity of methylnitrosourea. Thymidine, in the presence of sufficient deoxycytidine to overcome the perturbation of deoxynucleotide metabolism, also potentiates the cytotoxicity of methylnitrosourea. Nicotinate, which is not an inhibitor of poly-(ADP-ribose) polymerase, has no effect on methylnitrosourea toxicity. A very small, but consistent, enhancement of the toxicity of y-radiation by the same inhibitors has been observed. We suggest that this potentiation of cytotoxicity is mediated by inhibition of (ADP-ribose)n biosynthesis; and that the biosynthesis is stimulated by DNA damage. We therefore propose that (ADP-ribose)n takes part in cellular repair mechanisms, either by modifying chromatin structure or by a specific participation in DNA repair.

The presence of a new 3-oxoacyl-CoA thiolase in rat liver peroxisomes.

Abstract: A novel 3-oxacyl-CoA thiolase was found in rat liver. This thiolase, mitochondrial general 3-oxacyl-CoA thiolase and acetoacetyl-CoA thiolase were purified from the rat liver after the induction of these activities by the administration to rats of di(2-ethylhexyl)phthalate, which enhanced the peroxisomal beta-oxidation activity. The new 3-oxoacyl-CoA thiolase was distinguished from mitochondrial and cytoplasmic thiolases by the following: DEAE-cellulose chromatography, phosphocellulose chromatography, immunochemical titration, and substrate specificity. Subcellular fractionation of liver and sucrose-density-gradient centrifugation of the light mitochondrial fraction revealed that the new thiolase was mainly in peroxisomes.

Occlusion of divalent cations in the phosphorylated calcium pump of sarcoplasmic reticulum.

Abstract: The calcium pump of sarcoplasmic reticulum possesses high-affinity calcium-binding and ATP-binding sites. At 0 degrees C pH 6.8 and in the absence of calcium, about 3.5 nmol/mg of high-affinity ATP-binding sites are titrated with a dissociation constant, Kd, of 5 microM. In the presence of Ca2+, ATP phosphorylates the enzyme at a much lower concentration: K 1/2 = 100 nM. In the absence of ATP the calcium ions reversibly bind to the high-affinity calcium sites (6.5 nmol/mg); however the following is shown in this paper. 1. Phosphorylation of the enzyme in the presence of calcium leads to the immediate occlusion of the calcium ions bound to the high-affinity sites. 2. Two moles of calcium are occluded per mole of phosphoenzyme formed. 3. Occlusion can be reversed by ADP. 4. Transport is a slower process which occurs in the presence of Mg2+ at the same rate as the spontaneous decay of the phosphoenzyme. Experiments performed in the absence of magnesium reveal another divalent cation binding site which is probably directly involved in ATP and Pi binding. The nature of the cation bound to this site determines the stability and ADP-sensitivity of the phosphoenzyme.

Effect of ATPase Inhibitors on the Proton Pump of Respiratory-Deficient Yeast

Abstract: Diethylstilbestrol and dicyclohexylcarbodiimide inhibit the ATPase of the plasma membranes and the proton-pumping activity of the cells in a respiratory-deficient mutant of Saccharomyces cerevisiae. The effects of the inhibitors in vivo seem to be specific because neither the proton permeability nor the ATPase levels of the cells are affected. These results indicate that the yeast plasma-membrane ATPase corresponds to the proton pump of the cells. The fact that both inhibitors of the ATPase delay the fall of ATP levels which follows a block of fermentation indicates that ATPase function is one of the major ATP-consuming pathways in yeast. In addition, diethylstilbestrol prevents the fall of ATP levels produced by dinitrophenol, suggesting that this fall was caused by partial dissipation of the proton gradient and consequent stimulation of the proton-pumping ATPase.

The Subunit Composition of Mammalian Cytochrome c Oxidase

Abstract: Cytochrome c oxidase from rat liver mitochondria was separated into 12 different protein subunits by application of a highly resolving sodium dodecylsulfate/gel electrophoretic system of different compositions. The 12 protein subunits are shown to represent integral components of mammalian type cytochrome c oxidase for the following reasons. 1. All 12 subunits copurify through various purification procedures. 2. The subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. 3. All 12 subunits are present in the complex at one to one stoichiometric amounts. 4. A similar composition of 12 subunits was also found for cytochrome c oxidase from rat kidney, pig heart, rabbit liver and stone-marten liver. The difference between our results and all other published data on the subunit composition of mammalian-type cytochrome c oxidase, based on gel electrophoretic analysis, is due to the insufficient resolving power of previously used gel systems and the very similar molecular weight of subunits VIa, b, c, and VIIa, b, c.

Purification and Characterization of Multiplication‐Stimulating Activity

Abstract: Multiplication-stimulating activity (MSA) refers to a family of insulin-like growth factors that have been purified from serum-free medium conditioned by a Buffalo rat liver cell line (BRL-3A). Using Dowex ion-exchange chromatography, gel chromatography on Sephadex G-75, and preparative disc acrylamide gel electrophoresis, several polypeptides with the full biological multiplication-stimulating activity have been isolated. One of these polypeptides, designated MSA II-1, previously has been used to study the relationship of the activity to the insulin-like growth factors (somato-medins) purified from human plasma. Polypeptide II-1 is a single chain polypeptide of molecular weight 8700. Glycine is the COOH-terminal amino acid. Edman degradation of carboxymethylated MSA II-l did not reveal a free NH2-terminus. A polypeptide of lower molecular weight than MSA II-1 has also been purified. This polypeptide (MSA III-2) has been shown to be more potent than MSA II-1 in the rat-liver-membrane radioreceptor assay and in a competitive binding assay utilizing the rat-serum somatomedin-binding protein(s). The relationship of these various polypeptides has been investigated by gel filtration in guanidine hydrochloride and by acrylamide gel electrophoresis of the reduced and native polypeptides.

Transport of Proteins into Mitochondria

Abstract: The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner membrane. It is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is transferred into mitochondria in a posttranslational manner. The following results provide further evidence for such a mechanism and provide information on its details. 1. In homologous and heterologous translation systems th newly synthesized ADP/ATP carrier protein is present in the postribosomal supernatant. 2. Analysis by density gradient centrifugation and gel filtration shows, that the ADP/ATP carrier molecules in the postribosomal fraction are present as soluble complexes with apparent molecular weights of about 120 000 and 500 000 or larger. The carrier binds detergents such as Triton X-100 and deoxycholate forming mixed micelles with molecular weights of about 200 000-400 000. 3. Incubation of a postribosomal supernatant of a reticulocyte lysate containing newly synthesized ADP/ATP carrier with mitochondria isolated from Neurospora spheroplasts results in efficient transfer of the carrier into mitochondria. About 20-30% of the transferred carrier are resistant to proteinase in whole mitochondria. The authentic mature protein is also largley resistant to proteinase in whole mitochondria and sensitive after lysis of mitochondria with detergent. Integrity of mitochondria is a preprequisite for translocation into proteinase resistant position. 4. The transfer in vitro into a proteinase-resistant form is inhibited by the uncoupler carbonyl-cyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding. These observations suggest that the posttranslational transfer of ADP/ATP carrier occurs via the cytosolic space through a soluble oligomeric precursor form. This precursor is taken up by intact mitochondria into an integral position in the membrane. These findings are considered to be of general importance for the intracellular transfer of insoluble membrane proteins. They support the view that such proteins can exist in a water-soluble form as precursors and upon integration into the membrane undergo a conformational change. Uptake into the membrane may involve the cleavage of an additional sequence in some proteins, but this appears not to be a prerequisite as demonstrated by the ADP/ATP carrier protein.

Acetylation of prostaglandin endoperoxide synthetase with acetylsalicylic acid.

Abstract: Incubation of purified prostaglandin endoperoxide synthetase from sheep vesicular glands with aspirin results in a covalent binding of the acetyl group of acetylsalicylic acid to the protein. During this acetylation, the cyclooxygenase activity is lost, but not the peroxidase activity. The reaction is completed when almost one acetyl group is bound per polypeptide chain (Mr = 68 000). After proteolysis of [3H]acetyl-protein with pronase, radioactive N-acetylserine was obtained. Originally, however, the hydroxyl group of an internal serine residue in the chain is acetylated. The formation of N-acetylserine can be explained by a rapid O leads to N acetyl shift as soon as the NH2 group of serine is liberated. A radioactive dipeptide was isolated from a thermolysin digest of the [3H]acetyl-enzyme containing phenylalanine and serine, phenylalanine being its N-terminal amino acid. Automatic Edman degradation of native and acetylated enzyme showed that only one polypeptide sequence was present: Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-X-X-Tyr-. The N-terminal sequence has an apolar character.

The role of calcium ions, calmodulin and troponin in the regulation of phosphorylase kinase from rabbit skeletal muscle.

Abstract: Phosphorylase kinase has the structure (αβγδ)4 where the δ-subunit is identical to the calciumbinding protein termed calmodulin. In the presence of calcium ions the enzyme can interact with either a second molecule of calmodulin (termed the δ-subunit) or with skeletal muscle troponin-C leading to further enhancement of the activity [Shenolikar et al]. (1979) Eur. J. Biochem. 100, 329–337; Cohen et al. (1979) FEBS Lett. 104, 25–30; Picton et al. (1980) Eur. J. Biochem. (1980) III, 553–561]. In this paper the regulation of the different forms of phosphorylase kinase by calcium ions, the δ-subunit and troponin have been investigated. 1 The dephosphorylated form, termed phosphorylase kinase b, was found to be almost inactive below 1.0 μM calcium ions. The concentration required for half-maximal activation, co, 5, for calcium ions was 23 μM at pH 6.8 and 16 μM at pH 8.2. 2 At saturating calcium ions, phosphorylase kinase b was activated fivefold by saturating concentrations of δ-subunit or the troponin complex. The c0.5 for the δ-subunit and the troponin complex was 0.018 μM and 1.2 μM respectively at pH 6.8 and 0.012 μM and 1.5 μM respectively at pH 8.2. 3 The c0.5 for calcium ions required for the activation of phosphorylase kinase b by the δ-subunit was 20 μM at pH 6.8 and 11 μM at pH 8.2. These values were very similar to the c0.5 for calcium ions measured in the absence of the c0.5δ-subunit. Phosphorylase kinase b was almost inactive at 1.0 μM calcium ions even in the presence of the δ-subunit. 4 The c0.5 for calcium ions required for the activation of phosphorylase kinase b by the troponin complex was 4.0 μM at pH 6,8 and 2.0 μM at pH 8.2. At calcium ion concentrations below 3.0 μM the activity of phosphorylase kinase b was almost completely dependent on troponin. The activation was 20–30-fold at saturating concentrations of troponin. 5 Artificial thin filaments made by mixing actin, tropomyosin and the troponin complex at physiological concentrations were just as effective as troponin C or the troponin complex in the activation of phosphorylase kinase b. 6 No activation of phosphorylase kinase b by cardiac troponin-C was observed at concentrations of 2–20 μM. 7 No activation of phosphorylase kinase b by parvalbumin was detected, even at concentrations as high as 200 μM. Four different parvalbumins corresponding to two distinct evolutionary lineages of the protein were employed in these experiments. 8 Phosphorylase kinase a obtained by phosphorylation with cyclic-AMP-dependent protein kinase was 15-fold more active than phosphorylase kinase b at saturating calcium ions (0.08 mM). The c0.5 for calcium ions of the a-form was 1.6 μM at pH 6.8 and 0.6 μM at pH 8.2, 15–30-fold lower than phosphorylase kinase b. Consequently, phosphorylase kinase a was 150-fold more active than the b-form at 1.0 μM calcium ions. Phosphorylase kinase a displayed < 5% of its maximal potential activity at 0.1 μM calcium ions and had very little activity below 0.03 μM calcium ions. Phosphorylase kinase a could only be activated slightly by the δ-subunit (1.3-fold) or by the troponin complex (1.2-fold). 9 Phosphorylase kinase a obtained by limited proteolysis with trypsin was 20-fold more active than the b-form at saturating calcium ions (0.08 mM). The c0.5 for calcium ions of the a-form was 0.07 μM at μH 6.8 and 0.05 μM at pH 8.2, 300-fold lower than phosphorylase kinase h. Con- sequently, phosphorylase kinase a′ was 300–400-fold more active than phosphorylase kinase h at 1.O pM calcium ions. The properties of phosphorylase kinase a′ demonstrated that even a 2% degradation of phosphorylase kinase b would drastically effect plots of activity versus calcium ion concentration. Unlike the b-form and the a-form, phosphorylase kinase a′ did not have an absolute requirement for calcium ions and its activity in the absence of calcium ions was 25% of that measured at saturating calcium ions. The δ′-subunit and troponin had almost no effect on the activity of phosphorylase kinase a′. 10 The majority of the phosphorylase kinase, glycogen synthase and glycogen phosphorylase remained associated with the myofibrillar fraction when skeletal muscle from C3H/He-mg mice was homogenized in the presence of calcium ions. This did not occur when the muscle was homogenized in the presence of EDTA. Identical results were obtained for glycogen phosphorylase and glycogen synthase using skeletal muscle from ICR/IAn mice which lack the phosphorylase kinase protein. The interaction of phosphorylase kinase with troponin C is therefore not the basis for the calcium-dependent association of the enzymes of glycogen metabolism with the myofibrils. 11 The following conclusions have been drawn from this work. (a) The binding of three of four molecules of calcium ions to the δ′ -subunitis necessary before hosphorylase kinase b and a can be activated by this second molecule of calmodulin. (b) The binding of three or four molecules of calcium to each δ′-subunit appears to be required for the activation of phosphorylase kinase b. (c) The binding of only one or two molecules of calcium ions to each δ′-subunit appears to be required for the activation of phosphorylase kinase a or a′. (d) Troponin C is the dominant calcium-dependent regulator of phosphorylase kinase b at calcium ion concentrations in the micromolar range. It is proposed that troponin C rather than the δ′-subunit is the physiological activator of phosphorylase kinase b, and that this represents the most important mechanism for coupling glycogenolysis and muscle contraction. In contrast, the δ-subunit is the dominant calcium-dependent regulator of phosphorylase kinase a, and calmodulin therefore determines the regulation of phosphorylase kinase in its hormonally activatcd state.

Turnover of yeast fructose-bisphosphatase in different metabolic conditions.

Abstract: Earlier work demonstrated that addition of glucose to yeast growing on noncarbohydrate carbon sources sharply reduces the levels of fructose bisphosphatase. This report indicates that the decrease in the levels of fructose bisphosphatase is accompanied by a parallel decrease of cross-reacting material to specific antibody to fructose bisphosphatase. Use of the specific antibody shows that the loss of activity is irreversible and that its reapperance requires synthesis of protein de novo. The protein is highly stable during growth in ethanol (half life about 90 h). Addition of glucose increases the rate of degradation abut 200-fold. It is shown that the values of the rates of synthesis and degradation of fructose bisphosphatase vary with the metabolic situation of the yeast.

Characterization of Rat-Liver Microsomal Glutathione S-Transferase Activity

Abstract: Rat liver microsomes were shown to catalyze the conjugation of 1-chloro-2,4-dinitrobenzene with glutathione and this activity has been characterized. It cannot be removed from the microsomes by washing or other procedures which release loosely bound material from membranes. The microsomal glutathione S-transferase can be activated up to eight fold by treatment with N-ethylmaleimide. This activation also affects the apparent Km of the enzyme(s) for both glutathione and 1-chloro-2,4-dinitrobenzene. Upon subcellular fractionation of the liver the N-ethylmaleimide-activateable glutathione S-transferase distributes in the same manner as a marker for the endoplasmic reticulum and unlike markers for the other organelles and for the cytoplasm. Treatment of microsomes with proteases revealed that the enzyme is at least partially exposed on the cytoplasmic surface of the endoplasmic reticulum. Finally, three inducers of drug-metabolizing systems-i.e. phenobarbital, methylcholanthrene, and trans-stilbene oxide-all increase the activity of the cytoplasmic glutathione S-transferases, but they do not affect the microsomal activity. These and other considerations indicate that the microsomal glutathione S-transferase(s) is distinct from the cytoplasmic enzymes catalyzing similar reactions. The microsomal enzyme is likely to be involved in drug metabolism and the possibility of activating it through attack on a sulfhydryl group may represent an important physiological response to certain xenobiotics.

Structure and Activity of the Prosthetic Group of Methanol Dehydrogenase

Abstract: The reconstitutive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin resonance measurements suggest that the prosthetic group has not been modified during the isolation. This result, and the properties of the directly isolated prosthetic group and derivatives, confirm the suggestion that its structure is 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione. From the activity shown by derivatives of the prosthetic group and of structural analogues in the apoenzyme test it is concluded that the o-quinone structure is essential for activity. Hence the trivial name pyrrolo-quinoline quinone would be appropriate. The testing of the analogues also shows that the pyrrolo ring and the 9-carboxylic acid group are not essential for activity as they can be replaced by a pyridinol ring and a 9-hydroxy group respectively. The determination of the molar absorption coefficient of the prosthetic group (18400 M−1 cm−1 at 249 nm) enables its quantitative analysis. Thus it could be established that methanol dehydrogenase contains one prosthetic group per enzyme molecule. The consequences of this result in relation to already known properties of this ‘quinoprotein’ dehydrogenase are discussed.

Lipoprotein lipase. Mechanism of product inhibition.

Abstract: The rate at which lipoprotein lipase hydrolyzes triglycerides in lipoproteins and in synthetic emulsions decreases sharply with the amount of products formed unless albumin is present. Three factors which contribute to this inhibition as follows. (a) The fatty acids and the monoglycerides formed on hydrolysis locate at the lipid-water interface of the emulsion and, since they are substrates for the enzyme, act as competitive inhibitors of triglyceride hydrolysis. (b) The enzyme forms complexes with fatty acids. Therefore, as fatty acids accumulate in the system some of the enzyme will be sequestered into enzyme-fatty acid complexes. Albumin can prevent formation of such complexes since it has a higher affinity for fatty acids than the enzyme has. (c) Activator proteins do not enhance triglyceride hydrolysis unless a fatty acid acceptor is present. The strong inhibition of the enzyme itself and the loss of lipolysis-stimulating effects of activator proteins provide a feed-back regulation of the action of the lipase on lipoproteins at the capillary endothelium, ensuring that products are not generated more rapidly than they are utilized by the tissue.

Correlation of Enzymatic Activity and Anticoagulant Properties of Phospholipase A2

Abstract: Some highly purified phospholipases A from the venom of viperidae, crotalidae and elapidae were found to hve anticoagulant properties. All phospholipases which exhibited anticoagulant properties are characterized by a high isoelectric point, but not all strongly basic phospholipases are anticoagulant. Anticoagulant phospholipases hydrolyse highly packed monomolecular films of phospholipids without any lag time while non-anticoagulant phospholipases present considerable induction times indicative of a low penetrating power. When the ester linkages in the procoagulant lipids were replaced by the non-hydrolysable ether bonds, the mixture retained its clotting ability even in the presence of phospholipases, thus suggesting that anticoagulant phospholipases prevent clot formation by hydrolysis of phospholipids. This was confirmed by chemical modification of phospholipases, viz. alkylation of the active-centre histidine with 1-bromo-octan-2-one. This modification yielded proteins which had lost their anticoagulant properties but which retained a high affinity for phospholipids.

Detection and partial characterization of activity of chlorophyll synthetase in etioplast membranes.

Abstract: The esterification of chlorophyllide a was investigated in irradiated etioplast-membrane fractions (‘broken etioplasts’) from oat seedlings (Avena sativa L.). As a substrate, [1-3H]geranylgeraniol and its monophosphate and diphosphate derivatives were prepared by chemical synthesis. Geranylgeraniol and its monophosphate derivative are incorporated into chlorophyll only in the presence of ATP whereas the diphosphate derivative is incorporated also without ATP. The yield of esterified chlorophyll is 80–90% of chlorophyllide with saturating substrate concentrations. The term ‘chlorophyll synthetase’ is used to describe this enzyme activity which is different from chlorophyllase. Other substrates are phytol and farnesol either with ATP or as the diphosphate derivatives. The relative specificity of ‘chlorophyll synthetase’ for these substrates is geranylgeraniol: phytol: farnesol = 6:3:1. In these experiments in vitro, a new chlorophyll esterified with farnesol was detected which does not occur in intact plants. Geraniol and n-pentadecanol are no substrates for the enzyme. Proto-chlorophyllide which is present in non-irradiated etioplast membrane fractions is not esterified under the same conditions.