Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60-85%) and the gamma-subunit of nerve growth factor (by 55-93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the gamma-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), (IV), and (VI)], papain [(I), (II), (IV), and (VI)], subtilisin [(V) and (VI)], alpha-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 x 10(-2) M, while, at a concentration of 1 x 10(-3) M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

New Proctolin Analogues: Synthesis and Biological Investigation in Insects

We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X¹-Tyr-Leu-Pro-Thr-OH, where X¹ = D-Arg (I), N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn(iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) and D-Lys(Nic) (IX). In analogues I–IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) or homo-Arg (compound VII). Analogues I–IX were evaluated for myotropic activity on the in vitro heart preparation of Tenebrio molitor, whereas peptides II, V, and VII–IX were tested for contractile activity on the isolated foregut of locust Schistocerca gregaria. Peptide II and III showed full cardiotropic activity in T. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.     

The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI

At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).     

Specific and nonspecific glucanases from Trichoderma viride

Six endoglucanases (Endo I, II, III, IV, V, and VI), three exoglucanases (Exo I, II, and III), and a beta-glucosidase (beta-gluc I) isolated from a commercial cellulase preparation of Trichoderma viride origin were examined as to their activities on xylan ex oat spelts. Endo I, II, and III as well as Exo II and III showed no activity toward xylan and were classified as specific glucanases. Less specificity was found for the endoglucanases Endo IV, V, and VI, Exo I, and beta-gluc I, whose enzymes were able to hydrolyze xylan. With respect to product formation these xylanolytic cellulases fit the classification of xylanases generally accepted in the literature. Kinetic experiment with xylan, CM-cellulose, and p-nitrophenyl-beta-D-glucoside revealed that Endo IV, V, an VI and Exo I prefer to hydrolyze beta-1, 4-D-glucosidic linkages. beta-Gluc I showed no clear substrate preference.     

New Proctolin Analogues: Synthesis and Biological Investigation in Insects

Summary We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X¹-Tyr-Leu-Pro-Thr-OH, where X¹=d-Arg(I),N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn (iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) andd-Lys(Nic) (IX). In analogues I–IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) orhomo-Arg (compound VII). Analogues I–IX were evaluated for myotropic activity on thein vitro heart preparation ofTenebrio molitor, whereas peptides II, V, and VII–IX were tested for contractile activity on the isolated foregut of locustSchistocerca gregaria. Peptide II and III showed full cardiotropic activity inT. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.     

Phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase 1: Evidence of phosphorylation sites specific for casein kinase 1

Casein kinase 1 phosphorylated rabbit skeletal muscle glycogen synthase at both seryl and threonyl residues. With glycogen synthase phosphorylated up to 7.5 mol phosphate/mol subunit, about 26% of the phosphate was present in the N-terminal cyanogen bromide fragment (CB1) and 74% in the C-terminal fragment (CB2). Both fragments contained phosphothreonine (11 to 14%) in addition to phosphoserine. When ³²P-labeled glycogen synthase was totally digested with trypsin and chromatographed on reversephase high-performance liquid chromatography, seven phosphopeptides were observed. Peptide I eluted in the vicinity of the peptide containing site 1a, peptide II coincided with sites 4 + 5, peptides III and IV eluted in the region corresponding to sites 3a + 3b + 3c, peptide V appeared slightly after the peptide containing site 1b and peptide VII behaved as the peptide containing site 2, whereas peptide VI did not coincide with any of the known phosphopeptides. Limited trypsinization prior to analysis by HPLC led to the disappearance of peaks V and VI without altering peaks I to IV and VII. Only peaks I and VII remained when limited chymotrypsinization was performed prior to HPLC analysis. Chromatography on HPLC of the fragments derived from complete trypsinization of CB2 showed the presence of peaks II to VI. Phosphoamino acid analysis of the different peptides demonstrated the presence of quantitative amounts of phosphothreonine in peptides V, VI, and VII. These results indicate that multiple phosphorylation sites for casein kinase 1 must exist in both the N-terminal and C-terminal regions of glycogen synthase, some of which would only be labeled by casein kinase 1.     

Transformation of arylamines into direct-acting mutagens by reaction with nitrite

Arylamines including aniline (I), 1-naphthylamine (II), 2-naphthylamine (III), 2-aminofluorene (IV), 1-aminoanthracene (V) and 1-aminopyrene (VI) were treated with 4 equivalent amounts of nitrite at pH3 and 37 degrees C for 4 h. The reaction mixtures of I, IV, V and VI showed mutagenicity to Salmonella typhimurium TA98 and TA100 strains without metabolic activation. The numbers of His+ revertant colonies to TA98 strain were 110/0.05 mumole I, 970/0.055 mumole IV, 620/0.10 mumole V and 870/0.02 mumole VI. These arylamines were converted into mutagens with diazoquinone, diazonium and nitro functions depending on their structures. The mutagen from I was p-diazoquinone (I2). The mutagen from IV was highly unstable fluorene-2-diazonium salt (IV1). The mutagens from V were N3O3-introduced anthracene (V1-1) and 1-nitroanthracene (V2), and those from VI were unidentified nitro-introduced compound (VI1) and 1-nitropyrene (VI2).     

Identification of four additional myoinhibitory peptides (MIPs) from the ventral nerve cord of Manduca sexta.

Four new myoinhibitory peptides were isolated and identified from the ventral nerve cord of adult Manduca sexta. The new peptides are related to two previously identified myoinhibitory peptides also isolated from adult M. sexta, Mas-MIP I and Mas-MIP II. The sequences of the new peptides are APEKWAAFHGSWamide (Mas-MIP III), GWNDMSSAWamide (Mas-MIP IV), GWQDMSSAWamide (Mas-MIP V), and AWSALHGAWamide (Mas-MIP VI). Mas-MIPs III-VI were found to inhibit spontaneous peristalsis of the adult M. sexta anterior hindgut (ileum) in vitro.     

Metabolism of n-Alkylcyclohexanes with an even number of carbon atoms in the side chain by Micrococcus sp. RCO-4M

The metabolism of n-alkyleyclohexanes with an even number of carbon atoms (6, 8, 10, 12, 14) in the side chain by Micrococcus sp. RCO-4M was investigated. Evidence for the formation of cyclohexanecarboxylic acid (I), cyclohexaneacetic acid (II), 1-cyclohexenecarboxylic acid (III), 6-hydroxyhexanoic acid (IV), adipic acid (V), and trans-4-hydroxycyclohexaneacetic acid (VI) is presented. The presence of products (II), (IV) and (V) especially represent the complete degradation of n-alkylcyclohexanes with an even number of carbon atoms in the side chain by a single organism. The occurrence of a newly-identified product (VI) suggests that a new metabolic pathway for n-alkylcyclohexanes with even-carbon-number side chain operates in this organism.     

Mutagenicity of the anticancer drug, caracemide, and related compounds for salmonella

Caracemide, MeCON(CONHMe)(OCONHMe) (I), is a novel anticancer drug. Since it was derived from acetohydroxamic acid (II), a known mutagen, its potential metabolites and related compounds were synthesized and tested for mutagenicities in S. typhimurium TA98 and TA100. These compounds were: MeNHCONH(OCONHMe) (III), MeCONH(OCONHMe) (IV), MeCONOH(CONHMe) (V), MeNHCOONH2 X HCl (VI), MeNHCONHOH (VII), MeNHCOON(CONHMe)2 (VIII), and NOH(CONHMe)2 (IX). The mutagenicities in the absence of rat liver homogenate were: (VI) much greater than (IV) greater than (II), (III), (V). The other compounds were not mutagenic. (I) was mutagenic only in the presence of rat liver homogenate. The doses required to demonstrate mutagenicities of these compounds were from 0.05 to 5 mumoles/plate. The major hydrolytic products at 25 degrees C, pH 7, were (III), (IV), and (V) from (I); (II) and (III) from (IV); and (II), (III), (VII) and MeNHCONH(OCOMe) (X) from (V). (III) was stable at pH 7. Treatment of (IV) with HCl yielded (VI). Hydrolysis of (III) or (V) with ammonia yielded (VII). These results suggest that caracemide may be activated enzymatically or nonenzymatically by deacetylation or decarbamoylation, and its anticancer activity may be related to the reactivity of its metabolites with DNA. The synthetic procedures and characterizations of new compounds (IV), (V) and (X) are described.     

Adsorption and kinetic behavior of purified endoglucanases and exoglucanases from Trichoderma viride

Adsorption on crystalline cellulose of six endoglucanases (Endo I, II, III, IV, V and VI; 1, 4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) and two exoglucanases (Exo II and III; 1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.92), purified from a commercial cellulase preparation of Trichoderma viride origin, was studied. Endo I, III, and V adsorbed strongly on Avicel cellulose, while adsorption of Endo II, IV, and VI was much lower. Also, the two exoglucanases could be divided into one enzyme (Exo III) that had a high adsorption affinity and another enzyme (Exo II) that adsorbed only moderately. Adsorption data fitted the Langmuir-type adsorption isotherm. However, adsorption was only partially reversible with respect to dilution. No relation could be found between adsorption affinity and degree of randomness in cellulose hydrolysis, measured as the diversity of released hydrolytic products. Kinetic measurements indicated that only part of the adsorbed enzyme molecules are hydrolytically active.     

Quantitative isolation of radiolabeled metabolites without chromatography: measurements of the biosynthesis of purines, pyrimidines, and urea in isolated hepatocytes

Poly(U) Sepharose column chromatography was used to characterize the poly(A) RNA in RNA fractions differentially extracted from mammalian cells and subcellular components.. RNA fraction A was phenol extracted at pH 5.2 and 4°C and fraction B was phenol extracted from the residual material by elevating the extraction temperature and pH. With labeled RNA from HeLa cells, six peaks were isolated using a decreasing discontinuous KCl gradient (peaks I through IV), 1% sodium dodecylsulfate (peak V), and 90% formamide (peak VI). Peaks I through IV in fraction A were 0.8 to 2.3% polyadenylic acid; peak V was 2.9%; and peak VI, 16.7%. In fraction B RNA, peaks I through IV were 6 to 7.6% polyadenylic acid; peak V, 7.7%; and peak VI, 19.5%. After 24 h labeling of human myeloma cells to achieve a steady state, and subsequent subcellular fractionation, peak III was localized in RNA fraction B from the chromatin; this peak was not found in the polysomes. These and other observations suggest that poly(U) Sepharose chromatography combined with a discontinuous elution scheme is a very sensitive procedure for monitoring metabolic changes in poly(A) RNA subpopulations with time, subcellular location, and RNA extraction procedure.     

Subunit arrangement in beef heart complex III

Beef heart mitochondrial complex III was separated into 12 polypeptide bands representing 11 different subunits by using the electrophoresis conditions described by Sch?gger et al. [(1986) Methods Enzymol. 126, 224-237]. Eight of the 12 polypeptide bands were identified from their NH2-terminal sequences as obtained by electroblotting directly from the NaDodSO4-polyacrylamide gel onto a solid support. The topology of the subunits in complex III was explored by three different approaches. (1) Protease digestion experiments of submitochrondrial particles in the presence and absence of detergent showed that subunits II and VI are on the M side of the inner membrane and subunits V and XI on the C side. (2) Labeling experiments with the membrane-intercalated probes [125I]TID and arylazidoPE indicated that cytochrome b is the predominant bilayer embedded subunit of complex III, while the non-heme iron protein appears to be peripherally located. (3) Cross-linking studies with carbodiimides and homobifunctional cleavable reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and VI+VII. The cytochrome c binding site was found to include subunits IV, VIII, and X. The combined data are used to provide an updated model for the topology of beef heart complex III.     

Oxidation of phenolic arylglycerol beta-aryl ether lignin model compounds by manganese peroxidase from Phanerochaete chrysosporium: oxidative cleavage of an alpha-carbonyl model compound.

Manganese peroxidase (MnP) oxidized 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(4-(hydroxymethyl)-2-methoxyphenoxy) -1,3-dihydroxypropane (I) in the presence of MnII and H2O2 to yield 1-(3,5-dimethoxy-4-hydroxyphenyl)- 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-1-oxo-3-hydroxypropane (II), 2,6-dimethoxy-1,4-benzoquinone (III), 2,6-dimethoxy-1,4-dihydroxybenzene (IV), 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-3-hydroxypropanal (V), syringaldehyde (VI), vanillyl alcohol (VII), and vanillin (VIII). MnP oxidized II to yield 2,6-dimethoxy-1,4-benzoquinone (III), 2,6-dimethoxy-1,4-dihydroxybenzene (IV), vanillyl alcohol (VII), vanillin (VIII), syringic acid (IX), and 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-3-hydroxypropanoic acid (X). A chemically prepared MnIII-malonate complex catalyzed the same reactions. Oxidation of I and II in H2(18)O under argon resulted in incorporation of one atom of 18O into the quinone III and into the hydroquinone IV. Incorporation of one atom of oxygen from H2(18)O into syringic acid (IX) and the phenoxypropanoic acid X was also observed in the oxidation of II. These results are explained by mechanisms involving the initial one-electron oxidation of I or II by enzyme-generated MnIII to produce a phenoxy radical. This intermediate is further oxidized by MnIII to a cyclohexadienyl cation. Loss of a proton, followed by rearrangement of the quinone methide intermediate, yields the C alpha-oxo dimer II as the major product from substrate I. Alternatively, cyclohexadienyl cations are attacked by water. Subsequent alkyl-phenyl cleavage yields the hydroquinone IV and the phenoxypropanal V from I, and IV and the phenoxypropanoic acid X from II, respectively. The initial phenoxy radical also can undergo C alpha-C beta bond cleavage, yielding syringaldehyde (VI) and a C6-C2-ether radical from I and syringic acid (IX) and the same C6-C2-ether radical from II. The C6-C2-ether radical is scavenged by O2 or further oxidized by MnIII, subsequently leading to release of vanillyl alcohol (VII). VII and IV are oxidized to vanillin (VIII) and the quinone III, respectively.     

Polycondensation of thermal precursors of amino acids and characterization of constituent amino acids.

As a model reaction of polyamino acid formation from non-amino acid precursors, diammonium citraconate (I), ammonium citraconamate(II) and ammonium itaconamate(III) were converted to polyimide type polymers by thermal polycondensation by heating at 130–210°C. The imide type polymer was partially hydrolyzed to the corresponding peptide type polyamino acid. The polymer was composed of α-methylaspartic acid (IV), threo- and erythro-ß-methylaspartic acid (V) and α-(aminomethyl) succinic acid (VI). On the other hand, IV was thermally polycondensed to the corresponding polymer. It was found that the amino acid composition of the polymer was similar to that of the polymer prepared from I, II and III. The formation and isomerization of amino acids during the thermal polycondensation are described.     

Synthesis of plasmin substrates and relationship between their structure and plasmin activity

The plasmin substrates, D-Ile-Phe-Lys-pNA (I), 3-MV-Phe-Lys-pNA (II), Ile-Phe-Lys-pNA (III), D-Pro-Phe-Lys-pNA (IV), CP-Phe-Lys-pNA (V) and Pro-Phe-Lys-pNA (VI), were synthesized by the conventional solution method and the kinetic parameters of their amidolysis by plasmin were determined. It was found that the free amino group of the D-amino acid in substrates (I) and (IV) made a contribution to an increment in affinity between the substrate and plasmin.     

Galactosylation of N-linked oligosaccharides by human beta-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells

Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.     

Photobiological behaviour of bacteria and phages supplemented with aza-analogoues of nucleic acid bases.

The photochemical stability of anomalous nucleic acid bases of the azatype, 5-azacytosine (I), 5-azacytidine (II), 6-azacytosine (III), 6-azacytidine (IV), 6-azathymine (V), 6-azauracil (VI), and 8-aza-adenine (VII) to U. V. light of the wavelength 254 nm differs from the U. V. stability of the normal constituents. Changes of the U.V. inactivation of Escherichia coli K12 C600, E. coli B, Bacillus cereus, as well as E. coli phages gamma cb2 and gamma b2b5 supplemented with azaderivatives prior to irradiation were investigated. It was found that I, II, III, IV, and VII are more, V and VI less sensitive to U. V. light compared with corresponding natural nucleic acid bases. Their changed U. V. sensitivities are reflected in the survival curves after U. V. -irradiation in as far as azabases are incorporated into the nucleic acids in vivo. This explains the increase in U.V. sensitivity of E. coli K12 C600, E. coli B, and B. cereus supplemented with I, II, III, IV, and VII and the decrease in U.V. sensitivity of Streptococcus faecalis supplemented with V (the latter information was taken from Gunther and Prusoff 1967). The lack of any significant influence on inactivation curves of E. coli K12 C600 by V and VI, and on E. coli phages gamma cb2 and gamma c2b5 by II, is discussed in terms of too small incorporation rates. No discrimination was put forward with respect to DNA and RNA incorporation.     

Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.     

Evidence that chicken hypothalamic luteinizing hormone-releasing hormone is [Gln8]-LH-RH

As previously reported, chicken LH-RH has been purified in a yield of ca. 2.5 micrograms from 2,000 chicken hypothalmi, and we have estimated its chemical structure to be [Gln8]-LH-RH (1). Based on our previous observation (1), [Gln8]-LH-RH and [Glu8]-LH-RH have been synthesized by the solution method for the purpose of the structural confirmation. Synthetic [Gln8]-LH-RH was confirmed to be identical to natural chicken LH-RH on a HPLC system. For further structural confirmation, chymotryptic and thermolytic peptides from synthetic [Gln8]-LH-RH and natural chicken LH-RH were also identified. Thus, these studies support the view that chicken LH-RH has the following structure; pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2, [Gln8]-LH-RH. The gonadotropin releasing potency of synthetic [Gln8]-LH-RH was also in a good agreement with that of natural preparation.