Showing papers in "FEBS Journal in 1981"

A Network Model for the Organization of Type IV Collagen Molecules in Basement Membranes

Abstract: Type IV collagen was solubilized from a tumor basement membrane either by acid extraction or by limited digestion with pepsin. The two forms were similar in composition and the size of the constituent chains but differed when examined by electron microscopy and in the fragment pattern produced by bacterial collagenase. The acid-soluble form showed after rotary shadowing strands mainly of a length of 320 nm which terminated in a globule, or two strands connected by a similar globule. The globule was identified as a non-collagenous domain (NC1) which under dissociating conditions could be separated into two peptides showing a monomer-dimer relationship. Higher aggregates of NC1 were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously 7-S collagen domain. The pepsin-solubilized form lacked domain NC1 and consisted mainly of four triple-helical strands (length 356 nm) joined together at the 7-S domain (length 30 nm). Common to both forms of type IV collagen was a small collagenase-resistant domain NC2 which was composed of collagenous and non-collagenous elements and located between the 7-S domain and the major triple helix. These data indicate that the collagenous matrix of basement membranes consists of a regular network of type IV collagen molecules which is generated by two different interacting sites located at opposite ends of each molecule. The 7-S collagen domain connects four molecules while the NC1 domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NC1) was estimated to be about 800 nm comprising the length of two molecules.

The Complete Amino-Acid Sequence of Hen Ovalbumin

Abstract: The complete amino acid sequence of hen ovalbumin, comprising 385 residues, has been determined. The sequence was deduced from the 17 cyanogen bromide fragments and from peptides derived by digestion with a number of proteolytic enzymes. The molecular weight of the polypeptide chain of ovalbumin is 42699. Ovalbumin has four sites of postsynthetic modification; in addition to the acetylated N terminus, the carbohydrate moiety is located at Asn-292, and the two phosphorylated serines are at residues 68 and 344. The 'signal sequence' of ovalbumin is between residues 234 and 252. The heptapeptide released during the conversion of ovalbumin to plakalbumin by subtilisin digestion corresponds to residues 346-352. The hen ovalbumin polymorphism characterised by an Asn leads to Asp replacement results from a mutation at residue 311. The amino acid sequence of ovalbumin deduced from these amino acid sequence studies is in complete agreement with the sequence of mRNA determined by McReynolds et al. [Nature (Lond.) 273, 723-728 (1978)].

The structure of the capsular polysaccharide (K5 antigen) of urinary-tract-infective Escherichia coli 010:K5:H4. A polymer similar to desulfo-heparin.

Abstract: The capsular polysaccharide was isolated from Escherichia coli 010:K5:H4; it could not be obtained from a uncapsulated (K5-) mutant. It contains N-acetylglucosamine and glucuronic acid in a molar ratio of 1:1. Acid hydrolysis of the acidic polysaccharide as well as Smith degradation and degradation by deamination of the carboxyl-reduced polysaccharide suggested that the polysaccharide is composed of a disaccharide repeating unit. The data obtained by methylation analysis and nuclear magnetic resonance spectroscopy indicated that the repeating sequence of the capsular polysaccharide is the 4-beta-glucuronyl-1,4-alpha-N-acetylglucosaminyl unit. This structure is similar to that of desulfo-heparin.

Biosynthesis of the Light-Harvesting Chlorophyll a/b Protein

Abstract: 1 Antibodies raised against the 26000-Mr polypeptides of the light-harvesting chlorophyll a/b proteins of pea leaves specifically immunoprecipitated two 32000 -Mr polypeptides synthesized when pea leaf poly(A)-containing RNA was translated in vitro. On the basis of immunochemical relatedness and by comparison of their partial tryptic digestion products, the 32000 -Mr products formed in vitro are identified as precursors to the authentic polypeptides of the light-harvesting chlorophyll a/b complex. 2 The specificity of the immunoprecipitation permitted the development of an assay for the cellular levels of translationally active light-harvesting protein mRNA in plants exposed to different light regimes. Low levels of the mRNAs were detectable in dark-grown plants. Exposure to continuous illumination caused these levels to increase by at least ten-fold and led to the appearance of large quantities of the light-harvesting chlorophyll a/b complex. In plants exposed to intermittent illumination (2 min of white light every 2 h for 2 days), the light-harvesting complex did not accumulate, although levels of mRNA specifying the polypeptides of the complex were high (50% of those in continuously illuminated plants). 3 Messenger RNAs encoding the light-harvesting proteins were detected in polysomes of intermittently illuminated leaves. These polysomes were active in a wheat-germ 100000 ×g supernatant ‘run-off’ system, to form light-harvesting protein precursors, under conditions when only nascent polypeptide chains initiated in vivo were elongated and terminated. These results demonstrate that the inability of intermittently illuminated leaves to accumulate the light-harvesting proteins is not due to a selective inhibition of the translation of the corresponding mRNAs. 4 Intermittently illuminated leaves were labelled with [35S]methionine in darkness, and incorporation of radioisotope into the light-harvesting proteins and their precursors was assayed immunologically. No pool of untransported or unprocessed 32000 -Mr precursor polypeptides could be detected in the soluble fraction (cytoplasm and stroma). However, low levels of the mature 26000 -Mr polypeptides were detected in the membrane fraction. It is concluded that the newly synthesized light-harvesting chlorophyll a/b proteins fail to accumulate in intermittently illuminated leaves because they undergo rapid turnover. The site of light-harvesting protein breakdown is probably the thylakoid membrane, and the cause of breakdown is probably the absence of chlorophyll a and chlorophyll b molecules that are required for eventual stabilization of the proteins within the photosynthetic membrane.

Systematic application of two-dimensional 1H nuclear-magnetic-resonance techniques for studies of proteins. 2. Combined use of correlated spectroscopy and nuclear Overhauser spectroscopy for sequential assignments of backbone resonances and elucidation of polypeptide secondary structures.

Abstract: This paper describes a new nuclear magnetic resonance approach for the determination of secondary structure in globular proteins. To illustrate the practical application of the new procedure, two-dimensional correlated spectroscopy and two-dimensional nuclear Overhauser enhancement spectroscopy were used to obtain individual assignments for all the backbone protons of the beta-sheet secondary structures in the basic pancreatic trypsin inhibitor. First, combined connectivity diagrams of these two methods recorded in both 2H2O solution and H2O solution of the inhibitor were employed to obtain sequential, individual resonance assignments for the separate strands in the beta sheet. Second, a 2D nuclear Overhauser enhancement spectrum recorded with a long mixing time was used to determine how the separate, extended polypeptide strands are linked by hydrogen bonds in the sheet structures. By combination of these results with the identifications of the amino acid side-chain resonances described in the preceding paper, the beta-sheet structures can, without reference to data on the spatial structure obtained with other techniques, be localized in the amino acid sequence. This investigation confirms results on limited regions of the beta sheet in the inhibitor obtained previously with one-dimensional nuclear magnetic resonance experiments and demonstrates that the entire beta-sheet structure seen in single crystals of the inhibitor is preserved in aqueous solution.

The Amino-Acid Sequence of the α Subunit in Bovine Brain S-100a Protein

Abstract: The brain specific S-100 protein is a mixture of two components S-100a and S-100b with a subunit composition αβ or β2 respectively. The amino acid sequence of the β subunit has been previously determined. This paper presents the sequence of the α subunit in the S-100a protein. The α subunit consists of 93 amino-acid residues and has a relative molecular mass of 10400. The sequence shows extensive homology (58%) with that of the β-subunit and shares an apparent calcium binding site in the C-terminal half of the molecule, suggesting a close evolutionary relationship between these subunits.

Purification of glycogen synthase kinase 3 from rabbit skeletal muscle. Copurification with the activating factor (FA) of the (Mg-ATP) dependent protein phosphatase.

Abstract: Glycogen synthase kinase 3 was purified 80-100000-fold from extracts of rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate, chromatography on phosphocellulose and Affigel Blue, and affinity chromatography on (glycogen synthase)-Agarose. 0.08 mg of protein was isolated from 5000 g muscle corresponding to a yield of 8%. The preparations were estimated to be 50%pure, and the results suggest that glycogen synthase kinase 3 is a monomeric enzyme, Mr=54000 ± 3000. Glycogen synthase kinase 3 phosphorylated three serine residues in glycogen synthase, all contained within nine amino acids in the same tryptic peptide. The rate of formation of the mono-, di- and triphosphorylated derivatives of the peptide showed that the three sites were not phosphorylated either randomly or in a simple sequential manner. The results could be fitted to a model in which either of two serine rersidues were phosphorylated initially, thephosphorylation of one of these residues being liked to an extremely rapid phosphorylation of the third serine. Glycogen synthase kinase 3 phosphorylated glycogen synthase much more effectively than all other proteins tested. Casein was phosphorylated at a very low rate and with a much higher Km. There was no significant phosphorylation of glycogen phosphorylase, phosphorylase kinase, protein phosphatase inhibitors 1 and 2, l-pyruvate kinase, acetyl-CoA vstnocylsdr, ATP-citrate lyase, and histones H1 and H2B. The enzyme was also unable to catalyse the inactivation of hydroxymethylglutaryl-CoA reductase. Glycogen synthase kinase 3 underwent an ‘autophosphorylaaaation’ reaction following incubation with Mg-ATP and up to 4 mol of phosphate could be incorporated per mol of enzyme. The activating factor (FA)of the (Mg-ATP)-dependent protein phosphatase was found to copurify with glycogen synthase kinase 3 throughout the isolation procedure.It also proved impossible to separate the two activities by chromatography on CCCM-Sephadex or hydroxyapatite, or by gel filtration on Sephadex g-100. The two activities had similar Km values for ATP and GTP. The results suggest that glycogen synthase kinase 3 and factor FA activities reside in the same protein. Neither the catalytic subunit of cyclic-AMP-dependent protein kinase nor pphosphorylase kinase were able to activate the (Mg-ATP)-dependent protein phosphatase. The implications of these findings for the regulation of glycogen metabolism, and the mechanism of activation of the (Mg-ATP)-dependent protein phosphatase are discussed.

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers.

Abstract: In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.

Actin typing on total cellular extracts: a highly sensitive protein-chemical procedure able to distinguish different actins.

Abstract: Based on the finding that the amino-terminal tryptic peptide of actin is a reliable marker for actin divergence, we describe in detail a highly sensitive protein-chemical procedure for actin typing. The method is performed on non-radioactively labelled cells and tissues and six actins can be identified unambiguously in warm-blooded vertebrates. The method is quantitative and gives directly the ratio of the different actins present in the specimens. It does not require previous purification of actin and can be used on total cellular extracts without any prior fractionation. The procedure can be extended to actins not previously characterized by amino acid sequence analysis and makes certain predictions possible about the partial amino acid sequences of the amino-terminal tryptic peptides, mostly sufficient for a correlation with DNA sequences derived from cloned actin genes. This is done as an example for the cytoplasmic actin present in Schneider L-2 Drosophila melanogaster cells. Although the method is currently used routinely on 105 cells, modifications are discussed, which should allow the analysis to be performed with even higher sensitivity.

Studies on the Biosynthesis of Laminin by Murine Parietal Endoderm Cells

Abstract: The biosyntnesis and processing of the polypeptides A (Mr=450×103), B1 (Mr=450×103), B2 (Mr=230×103) and C (Mr=150×103) of the extracellular matrix protein, laminin, were studied in murine parietal endoderm cells labeled with [35S]methionine. Various lines of evidence suggest that the A chains are not precursors to the smaller B chains. Firstly, in pulse-chase experiments, radioactivity in cytoplasmic A and (B1+ B2) chains declines with the same half-life of about 70 min. Secondly, peptide maps generated by digestion of A and (B1+ B2) chains with Staphylococcus aureus V8 protease are different. Finally, rabbit antibodies to isolated, denatured (B1+ B2) chains do not cross-react with reduced and alkylated A chains. A, B1, B2 and C polypeptides are all glycosylated by an intracellular process involving the addition of tunicamycin and endo β-N-acetylglucosaminidase H. sensitive N-linked oligosaccharide side chains. Further glycosylation probably occurs around the time of secretion. Disulphide bonding of some A and B chains can be observed in the cytoplasm within 10 min of adding [35S]methionine. However, it appears that some free A and B2 chains are present in the cytoplasm and that free A chains exist in the medium. The relationship between the 150×103-Mr C glycoprotein and the A and B components is discussed. Although B and C chains generate different peptide maps after digestion with S, aureus V8 protease, antibodies raised against isolated, denatured C chains cross-react with reduced and alkylated B (but not A) chains. This suggests that B and C chains may share some antigenic determinant(s).

On the Hydrophobic Nature of Signal Sequences

Abstract: A number of signal sequences, prokaryotic as well as eukaryotic, have been analyzed in terms of gross amino acid composition and hydrophobicity. It is shown that the amino acid composition of the hydrophic core can be well reproduced in a computer simulation of signal sequence ‘evolution’ with selection operating on the mean hydrophobicity of the sequence and the non-occurrence of charged residues. The calculated hydrophobicities are interpreted in terms of a model in which the hydrophobic part of the signal sequence partitions directly into the membrane interior, thereby making further translocation of the growing nascent chain possible.

The protochlorophyllide holochrome of barley (Hordeum vulgare L.). The effect of light on the NADPH:protochlorophyllide oxidoreductase.

Abstract: During the illumination of etiolated barley plants a rapid decline of the NADPH: protochlorophyllide oxidoreductase is observed. Within the first 5 min of continuous light approximately 90% of the enzyme activity present in dark-grown barley plants disappears and, at the same time, the amount of enzyme protein is diminished by more than 60%. No stable polypeptide fragments have been found which might be formed during the light-induced degradation of the enzyme protein. The rate of enzyme protein synthesis is not drastically affected at the beginning of the illumination period. During the subsequent light-dependent chloroplast development a phytochrome-induced decline in the rate of protein synthesis, concomittant with a continuous light-dependent degradation of the enzyme protein, leads to a progressive decrease of the concentration of the enzyme. After 6 h of continuous light, when the rate of chlorophyll accumulation is at its greatest, only traces of the enzyme protein are visible and the enzyme activity is no longer detectable within the plants. In contrast to previous concepts of chlorophyll biosynthesis in higher plants, our results present evidence that the NADPH: protochlorophyllide oxidoreductase functions only for a short time period after the onset of light.

The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Phytochrome-induced decrease of translatable mRNA coding for the NADPH: protochlorophyllide oxidoreductase.

Abstract: During the illumination of dark-grown barley plants light induces a rapid decrease of a translatable mRNA which codes for a polypeptide of Mr 44000. This component was identified as a precursor of the NADPH:protochlorophyllide oxidoreductase. The precursor has an Mr larger than the authentic protein by approximately 8000. The light-induced change in the level of translatable mRNA can be induced by a 15-s red-light pulse followed by 5 h of darkness. The red-light effect is reversed by a subsequent far-red-light treatment. It is concluded that the light-induced decline of translatable mRNA for the NADPH:protochlorophyllide oxidoreductase is controlled by phytochrome. The significance of this finding for present concepts of light-dependent control of chloroplast development and chlorophyll synthesis is discussed.

Effect of Funiculosin and Antimycin A on the Redox-Driven H+-Pumps in Mitochondria: on the Nature of ‘Leaks’

Abstract: The effect of antimycin A and funiculosin, two inhibitors which block electron transfer in the b-c1 complex, on electron flow and electrochemical potential difference of H+ ions in mitochondria at static head (state 4) is investigated. In addition, the respiratory control ratio is determined as the ratio between uncoupler stimulated and static-head electron flow. Malonate, a competitive inhibitor of succinic dehydrogenase, is used for comparison. All three inhibitors cause an extensive depression of static-head electron flow but only a limited decrease in the electrochemical potential difference of H+ ions. With the antimycin-type of inhibitors, the respiratory control ratio slightly increases up to about 50% inhibition of electron flow and then steeply declines. With malonate, a strong decrease of the respiratory control ratio is observed in a concentration range where the electron flow is inhibited less than 10%. It is shown that the data do not compiy with the generally accepted hypothesis of a leak conductance being regulated by the electrochemical potential difference of H+ ions. They can be interpreted in terms of not tightly coupled redox-driven H+-pumps. A non-vanishing electron flow at static head then arises predominantly from molecular slipping in the pumps, and the (constant) leak conductance yields only a minor contribution.

Cytochrome P-450 Monooxygenase Activities in Human and Rat Liver Microsomes

Abstract: Microsomes were prepared from human livers obtained from renal donors of various ages and both sexes. Their drug-metabolizing capacity was measured and compared to that of rat liver microsomes. The following parameters were investigated: cytochrome P-450, cytochrome B5, NADPH-cytochrome c reductase, epoxide hydrolase, aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, p-nitroanisole-O-demethylase, ethoxycoumarin-O-deethylase, steroid-16 alpha-hydroxylase. In addition, the metabolism of benzo(a)pyrene, progesterone, pregnenolone, testosterone, dehydroepiandrosterone and estradiol was studied in detail in vitro. The inhibitory effect of metyrapone and alpha-naphthoflavone on 7-ethoxycoumarin-O-deethylase was measured. The microsomal proteins of both species were separated by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The following conclusions were drawn from the results obtained. Human liver microsomes can be stored under optimal conditions for the measurement of a large variety of enzymic activities. Human liver microsomes are able to metabolize the various xenobiotics used as substrates with a rate similar to that of female rat liver microsomes. No sex-linked difference in enzymic activity was observed in human microsomes. Significant differences in benzo(a)pyrene and steroid metabolism were registered when human and rat liver microsomes were compared. The monooxygenase activities, the sensitivity to in vitro alpha-naphthoflavone and metyrapone, the results of steroid metabolism, and slab gel electrophoresis are strong indications for multiplicity of human liver cytochrome P-450.

Purification and characterisation of a membrane-bound substance-P-degrading enzyme from human brain.

Abstract: A membrane-bound enzyme which degrades substance P (an undecapeptide) has been purified from human brain. The properties of this enzyme suggest that it may be involved in the physiological inactivation of the peptide by neural tissues. Enzyme activity was extracted from a membrane fraction of human diencephalon with a non-ionic detergent, Brij 35, and activity was monitored by measuring the disappearance of added substance P using radioimmunoassay, bioassay or radiochemical assay. The enzyme was purified about 1000-fold by chromatography on DEAE-cellulose, hydroxyapatite and Sephadex gel filtration columns. To identify the cleavage sites in substance P, the peptide was incubated with the purified enzyme and the breakdown products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis. The results suggested that the enzyme preparation was functionally homogeneous and it cleaved substance P between Gln6–Phe7, Phe7–Phe8 and Phe8-Gly9, with no exopeptidase action. The enzyme had a pH optimum in the range 7–9 and was strongly inhibited by metal-chelating agents, but not affected by most other peptidase inhibitors; it can thus be classified as a neutral metallo-endopeptidase. The enzyme was thermolabile and had a molecular weight of 40000–50000 as estimated by gel filtration, density-gradient ultracentrifugation and sodium dodecylsulphate gel electrophoresis. The highly purified substance-P-degrading enzyme could be distinguished from previously described peptidases for which substance P is a substrate. An important feature was that substance P was the preferred substrate among various other neuropeptides tested.

The primary structure of Escherichia coli RNA polymerase. Nucleotide sequence of the rpoB gene and amino-acid sequence of the beta-subunit.

Abstract: The combined structural study of proteins and of their corresponding genes utilizing the methods of both protein and nucleotide chemistry greatly accelerates and considerably simplifies both the nucleotide and protein structure determination and, in particular, enhances the reliability of the analysis. This approach has been successfully applied in the primary structure determination of the β and β′ subunits of Escherichia coli DNAdependent RNA polymerise and of their structural genes, yielding a continuous nucleotide, sequence (4714 base pairs) that embraces the entire rpoB gene, the initial part of the rpoC gene and the intercistronic region, together with the total amino acid sequence of the β subunit, comprising 1342 residues, and the N-terminal sequence of the β′ subunit (176 residues).

Energy is Required for Maturation of Exported Proteins in Escherichia coli

Abstract: It has been established in numerous cases that proteins which are exported from Escherichia coli are synthesized on membrane-bound polysomes in precursor forms which are proteolytically cleaved to generate the mature species. Here we present evidence that at least one step in the export of proteins requires energy. Energy requirements for processing of the precursors of both the M13 coat protein [Date, T., Zwizinski, C., Ludmerer, S., and Wickner, W. (1980) Proc. Natl Acad. Sci. USA, 77, 827-831; Date, T., Goodman, J. M., and Wickner, W. T. (1980) Proc. Natl Acad. Sci. USA, 77, 4669-4673] and the B subunit of heat-labile enterotoxin [Palva, T., Hirst, T. R., Hardy, S. J. S., Holmgren, J., and Randall, L. L. (1981) J. Bacteriol. in the press] have been demonstrated previously. An energy requirement for the proteolytic processing of an additional five exported proteins is reported here. Studies utilizing an uncA mutant suggest that the form of energy required is proton-motive force. Thus an energized membrane is probably essential for export of most periplasmic and outer membrane proteins.

The Major Components of the Mouse and Human Genomes

Abstract: Main-band DNA from mammals and birds can be resolved by density gradient centrifugation techniques into three or four families of fragments of different dG + dC contents. These major DNA components are similar in their buoyant densities and relative amounts in all species tested and are observed in DNA preparations ranging in Mr from 2 × 106 to over 200 × 106. In the present work, the four major components of mouse and human DNAs were prepared and characterized in several basic properties: relative amounts, dG + dC contents, buoyant densities and compositional heterogeneity. The results obtained lead to the following conclusions: (a) the major DNA components of mouse and man form at least 85% and possibly the totality of the main bands of these DNAs; (b) they have very low compositional heterogeneities over a wide molecular weight range; (c) they derive from very large chromosomal DNA segments of fairly homogeneous base composition, for which the name ‘isochores’ is proposed. A comparison of the compositional heterogeneity of main-band DNAs from warm-blooded and cold-blooded vertebrates confirms our previous conclusion that these DNAs are characterized by a different sequence organization.

Assembly of Cytochrome c. Apocytochrome c Is Bound to Specific Sites on Mitochondria before Its Conversion to Holocytochrome c

Abstract: Transport of apocytochrome c across the outer mitochondrial membrane and conversion to holocytochrome c were studied in vitro. Apocytochrome c was synthesized in a cell-free homogenate from Neurospora crassa. Transfer in vitro was accomplished in a reconstituted system consisting of the postribosomal supernatant of the cell-free homogenate and of isolated and purified mitochondria from Neurospora. The reconstituted system has the following characteristics: * 1. Apocytochrome c is rapidly cleared from the supernatant and holocytochrome c appears in the mitochondria with the same kinetics. More than 80% of the apocytochrome c employed is converted to holocytochrome c. No transient accumulation of apocytochrome c is found in mitochondria. * 2. The heme group becomes covalently linked to apocytochrome c in the reconstituted system as demonstrated by analysis of tryptic peptide maps of the apoprotein and holoprotein. * 3. Deuterohemin added to the reconstituted system but not deuteroporphyrin inhibits the formation of holocytochrome c. This inhibition is reversed by protohemin. * 4. In the presence of deuterohemin about half of the apocytochrome c remains in the supernatant; the other half becomes associated with the mitochondria. The latter portion is tightly bound and is specifically released upon incubation of the mitochondria with excess apocytochrome c. It is converted to holocytochrome c after addition of protohemin. We conclude from these observations that apocytochrome c is transported across the outer mitochondrial membrane via receptor sites. In the presence of the heme analogue deuterohemin, binding to the receptor sites on the cytoplasmic surface of the outer mitochondrial membrane still takes place but translocation does not. The latter step is apparently coupled to the covalent linkage of the heme group. We suggest that the formation of the thioether bonds between apoprotein and heme is catalysed by an enzyme in the intermembrane space and that deuterohemin can compete with protohemin for binding to the enzyme. Finally, the data indicate that it is the heme group and not the porphyrin group which is coupled to the apoprotein.

Purifieation of a Soluble, Sodium-Nitroprusside-Stimulated Guanylate Cyclase from Bovine Lung

Abstract: A soluble, sodium-nitroprusside-stimulated guanylate cyclase as been purified from bovine lung by DEAE-cellulose chromatography, ammonium sulfate precipitation, chromatography on Blue Sepharose CL-6B and preparative gel electrophoresis. Apparent homogeneity was obtained after at least 7000-fold purification with a yield of 3%. A single stained band (Mr 72000) was observed after gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme migrated as one band also under non-denaturing conditions in acrylamide gels (5-12%). The mobility of this band corresponded to an Mr of 145000. The enzyme sedimented on sucrose gradients with an S20, w of 7.0 S. Gel filtration yielded a Stokes' radius of 4.6 nm. These data suggest that the enzyme has an Mr of approximately 150000 and consists of two, presumably identical, subunits of Mr 72000. Sodium nitroprusside stimulated the purified enzyme 15-fold and 140-fold to specific activities of 8.5 and 15.7 mumol of cGMP formed min-1 mg-1 in the presence of Mn2+ and Mg2+, respectively. Formation of cGMP was proportional to the incubation time and to the amount of enzyme added. The stimulatory effect of sodium nitroprusside was half-maximal at about 2 microM, was observed immediately after addition and could be reversed either by dilution or by removal of sodium nitroprusside on a Sephadex G-25 column. The purified enzyme in the absence of catalase was stimulated by sodium nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine and 3-morpholino-sydnonimine and in the presence of catalase by sodium nitrite and sodium azide. In the presence of Mn2+ and sodium nitroprusside, the purified enzyme catalyzed the formation of cAMP from ATP at a rate of 0.6 mumol min-1 mg-1.

Nucleotide sequence coding for the respiratory NADH dehydrogenase of Escherichia coli. UUG initiation codon.

Abstract: The nucleotide sequence of the structural gene coding for the respiratory NADH dehydrogenase of Escherichia coli has been determined by the chain-termination method. The reading frame for the protein starts with the unusual initiation codon UUG and predicts an amino acid sequence of 434 residues (Mr= 47304). The reading frame was confirmed by protein chemical studies including determination of the N-terminal sequence of the protein. The product made in vivo was found to have threonine as its N-terminal residue, indicating that the initiating N-formylmethionine had been removed by post-translational processing.

Effect of trypsin treatment of rat adipocytes on biological effects and binding of insulin and insulin-like growth factors: further evidence for the action of insulin-like growth factors through the insulin receptor.

Abstract: Trypsin-treatment of isolated rat adipocytes abolishes the metabolic effects not only of insulin, but also of the insulin-like growth factors: in trypsin-treated cells, concentrations of these hormones that are otherwise maximally effective no longer stimulate 3-O-methylglucose transport and lipogenesis or inhibit epinephrine induced lipolysis. Concomitantly, the trypsin-treated adipocytes no longer display specific insulin binding. In contrast, the characteristics of the binding of the insulin-like growth factors are not grossly affected by prior trypsinization of the adipocytes. These findings add further support to the concept that the insulin-like growth factors act on glucose metabolism and antilipolysis via the insulin receptor of the adipocyte.

A General Secondary‐Structure Model for Procaryotic and Eucaryotic RNAs of the Small Ribosomal Subunits

Abstract: A consensus on the folding of the Escherichia coli 16-S ribosomal RNA is emerging and several complete nucleotide sequences of small ribosomal subunit RNAs, covering diverse types of organisms and organelles, are now available. We therefore investigated the extent of both nucleotide sequence and secondary structure conservation that may exist between the E. coli 16-S RNA and other ribosomal RNAs. All the RNA molecules examined could be folded into secondary structure schemes that illustrated remarkable preservation of many structural motifs as well as striking nucleotide sequence conservation compared with the E. coli molecule. This study presents a unitary scheme for the structural organization of the small ribosomal subunit RNAs. The evolutionary constraints on both primary and secondary structures most likely reveal the basic role of some restricted RNA regions in the function of the ribosome.

A Role in vivo for Penicillin-Binding Protein-4 of Staphylococcus aureus

Abstract: The degree of cross-linking of the peotidoglycan of Staphylococcus aureus H and mutants lacking penicillin-binding proteins 1 and 4 was studies. No major changes were ovserved in organisms lacking protein 1 whereas oloss of protein 4 was accompanied by a marked reduction in the degree of cross-linked and the absence of a membrane-bound ‘model’ transpeptidase activity. A smimlar effect was achieved when cultures of the staphylococci were treated with the β-lactam antibiotic cefoxitin. At low concentrations (0.05μg ml-1) cefoxition shows highest affinity for protein 4 to which it appears to bind irreversibly. Treatment of the mutant lacking protein 4 with this concentration of the antibiotic did not affect the degree of cross-linkage. The possibility that the decrease in cross-linkage was a consequence of DD-carboxypeptidase activity on peptidoglycan precursors was investigated. Although both S. aureus H and the mutants possessed such activity it was insensitive to benzylpenicillin and cefoxition and the role of this enzyme(s) in peptidoglycan biosynthesis remains unknown. We conclude that in vivo protein 4 acts as a transpeptidase involved in the secondary cross-linking of peptidoglycan and this activity is necessary to achieve the high degree of cross-linkage observed in the peptidoglycan of staphylococci.

Changes in Cytoplasmic pH and in Membrane Potential in Thrombin-Stimulated Human Platelets

Abstract: The response of human platelets to stimulation by a specific aggregant such as thrombin has been postulated to proceed sequentially via induction of response at the membrance, followed by execution of shape change, secretion, and aggregation of the platelets. We have shown earlier that the platelet response includes a depolarization of the membrane which starts within less than 5s and is thrombin-dose-dependent up to 4.5 nM α-thrombin, This depolarization may be measured by the distribution of either a fluorescent or a tritium-labeled lipophilic cation. We present here an adaptation of techniques for intracellular pH measurements to the humanj platelet. These show that stimulation with thrombin also induces a rapid change in the platelet trans-membrane pH gradient as measured using either a weak base or a fluorescein derivative as a probe. The pH gradient undergoes a time-dependent and thrombin-dose-dependent change which parallels that eshibited by the membrane potential and by serotonin secretion.

Thionins: Plant Peptides that Modify Membrane Permeability in Cultured Mammalian Cells

Abstract: Thionins, which are high-sulphur polypeptides present in the endosperm of wheat and related species, have been found to prevent growth and to inhibit macromolecular synthesis in cultured mammalian cells. Baby hamster kidney (BHK) cells were markedly more sensitive to thionins than the other cell lines tested (monkey CV1, mouse L, human HeLa). A thionin concentration of 5 μ/ml (1 μM) completely blocked translation in BHK cells. It was later found that omission of both calcium and magnesium ions from the medium strongly enhanced the inhibitory effects of thionins (BHK cells, 80% inhibition, 0.5 μ/ml). Several lines of evidence indicate that thionins might act at the membrane level. Indeed, both the 86Rb+ content and the nucleotide pool of BHK cells were drastically decreased at thionin concentrations that inhibited translation. In addition, thionin concentrations that did not affect macromolecular synthesis in these cells, allowed inhibition of translation by antibiotics, such as hygromycin Bthat are not able to cross the cell plasma membrane by themselves. Our results suggest that the inhibition of protein, RNA and DNA synthesis in BHK cells might be a consequence of membrane leakiness induced by thionin treatment. In this respect, particularly striking was the parallelism found between 86Rb+ leakage and inhibition of protein synthesis by treatment with different genetic variants of thionins (α1 purothionin, α2 purothionin, β purothionin from wheat; hordothionin from barley), as well as with the viscotoxins, which are homologous polypeptides from the European mistletoe.

The Fatty Acid Composition of Subcellular Membranes of Rat Liver, Heart, and Brain : Diet-Induced Modifications

Abstract: The influence of diets having different fatty acids composition on the fatty acid content of (the phospholipids) of rat liver mitochondria and microsomes, heart mitochondria, brain mitochondria and microsonies has been analyzed. It has been found that each organelle has its own peculiar composition in fatty acids. This composition may be profoundly influenced by the diet, but to different degrees in different organelles. Those of brain are most resistant. The changes observed are rather rapid, being generally already maximal after three weeks of treatment. The parallel between fatty acid composition of diets, and the changes observed in the organelles, is not strict, and is probably influenced by the metabolic competition among oleic acid, linoleic acid, linolenic acid. Unusual fatty acids like crucic acid. trans-oleic acid. and trans-linoleic acid can also become incorporated into the membranes of cell organelles.

Amino Acid Utilisation in Isolated Hepatocytes from Rainbow Trout

Abstract: The utilisation (conversion to CO2 and/or glucose) of a series of amino acids by isolated trout hepatocytes was investigated and compared to the utilisation of lactate and palmitate In fed fish, several amino acids (alanine, serine, asparagine and glycine) and lactate produced CO2 at considerably higher rates than palmitate During starvation plus exercise, the rate of CO2 production from palmitate increased while that from lactate and most of the amino acids decreased Gluconeogenesis from amino acids in fed fish was lower than from lactate Serine and asparagine were the most effective substrates; alanine gave lower rates of incorporation During prolonged starvation plus exercise, the rates of gluconeogenesis from amino acids increased twofold and, simultaneously, there was a corresponding increase in phosphoenolpyruvate carboxykinase activity in liver It is concluded that several amino acids (dietary or released from muscle protein) are potentially major oxidative substrates in trout In addition, amino acids appear to have the capability to maintain supplies of glucose during a period of prolonged starvation and exercise No evidence could be found to support the contention that alanine is the most important glucogenic amino acid