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In 2000 ml of pooled human male plasma unconjugated 3β, 17β-dihydroxyandrost-5-ene as diacetate was identified. The identification was based on paper chromatography, acetylation followed by thin-layer chromatography and final mass spectrometry, Furthermore aliquot parts of the extract after paper-chromatography were subjected to a protein-binding assay to assure the 17β-hydroxyl configuration. [ABSTRACT FROM AUTHOR]

Peptide B, the N-terminal fragment of thioredoxin obtained by cyanogens bromide treatment was aminoethylated and digested with pepsin. Eight peptides were separated by high voltage paper electrophoresis and chromatography and characterized with respect to amino acid sequence. The results account for all 37 residues peptide B. The N-terminal sequence of peptide B was determined as: Ser-Asp-Lys-Ile-Ile-His-Leu-Thr-Asp-Asp-Ser-Phe. [ABSTRACT FROM AUTHOR]

The fate of the hydrogen atoms at C-3 and C-4 of uridine diphospho-D-glucuronic acid (UDPGlcUA) in the course of its conversion to uridine diphospho-apiose (UDP-api) was investigated. The specifically labeled UDP-GlcUA substrates were prepared enzymatically from D-[3-³H]glucose and D-[4-³H]glucose by the combined action of hexokinase, phosphoglucomutase, UDPGpyrophosphorylase, and UDPG dehydrogenase. UDP-[U-14C, 3-³H]GlcUA, UDP-[U14C, 4-³H]GlcUA, and UDP-[U14C, 3,4-³H2]GlcUA were separately converted to UDP-Api by an enzyme preparation from Lemna minor. In order to obtain an apiose (3-C-hydroxymethyl-D-erythrofuranose) derivative which was reasonably stable and which could be produced without passing through the aldehydo form, the apiosyl radical of UDP-Api was enzymatically transferred to 7-glucosylapigenin by an enzyme preparation from parsley. The product of this reaction, apiin (7-O-[β-D-apiofuranosyl-(1→2)-β-D-glucosyl-5,7,4'-trihydroxyflavone), was subjected to mild periodate oxidation to convert the branch hydroxymethyl group of the apiosyl residue to formaldehyde. Approximately 14-16% of each of the substrates was converted to UDP-Api. The transfer of the apiosyl moiety to 7-glucosylapigenin was essentially quantitative in all cases. The isolated apiin was shown to have retained 53.5% of the tritium from the substrate labeled at C-3, 99% from the substrate labeled at C-4, and 73% from the substrate labeled at C-3,4. All of the tritium and one-fifth of the 14C of apiin produced from the variously labeled substrate was accounted for in the formaldehyde-dimedone derivative. These results establish the occurrence of a hydride shift from C-4 of UDP-GlcUA to C-3' of apiose in the biosynthesis of UDP-Api from UDPGlcUA. [ABSTRACT FROM AUTHOR]

Tryptic and chymotryptic digestion of peptide B, the N-terminal fragment obtained by cyanogen bromide cleavage of thioredoxin, provided the information required to determine the contiguity of the peptic fragments studied previously. The complete sequence of the 37 residues of peptide B could thus be established. Peptide B contains the active center of thioredoxin. Its amino acid sequence was found to be: -Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys-Met-. This sequence supports earlier conclusions dragon from fluorescence studies of thioredoxin, which indica ted a close relationship between one or both tryptophan residues and the disulfide bridge of the active site. Peptide maps of the tryptic digests of carboxymethylated thioredoxin and of the two cyanogen bromide fragments are described. Amide nitrogen analyses of thioredoxin and the two cyanogen bromide fragments demonstrate that all seven amide groups of thioredoxin are localized in the C-terminal fragment (peptide A). [ABSTRACT FROM AUTHOR]

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [ABSTRACT FROM AUTHOR]

Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

Porcine secretin is a heptacosapeptide with the amino acid sequence His-Ser-Asp-Gly-Thr- Phe -Thr- Ser -Glu -Leu -Ser -Arg -Leu -Arg -Asp-Ser -Ala -Arg -Leu -Gin -Arg -Leu -Leu-Gln-Gly-Leu- Va1NH2. Fourteen of its twenty-seven amino acids occur in the same position as they do in porcine glucagon. [ABSTRACT FROM AUTHOR]

Extracts of the hypobranchial body of two prosobranchiate molluses of the Muricidae family were examined for their content of choline esters, as well as of precursors and metabolites of these esters. The hypobranchial gland of Concholepas concholepas contained, in addition to large amounts of urocanylcholine (murexine) and senecioylcholine, appreciable amounts of free choline, free urocanic acid and ethyl urocanate. All these compounds, and especially the choline esters, were predominantly located in the dye-secreting area of the hypobranchial gland. As much as 3% of the wet weight of the purple area was accounted for by murexine and seneciocylcholine. The only choline ester found in the hypobranchial body of Thais cholate was senecioylcholiine; derivatives of urocanic acid were lacking. The occurrence of ethyl urocanate in the hypobranchial gland of Concholepas concholepas demonstrates that not only choline but also other alcohols may be esterified by urocanic acid. [ABSTRACT FROM AUTHOR]

For the monosialo-lactoisohexaosyl-ceramide, a ganglioside from human spleen, the following structure was established: NeuNAcα2 → 3Galβ1 → 4GlcNAcβ1 → 3Galβ1 → 4GlcNAcβ1 → 3Galβ1 → 4Glc → Cer. [ABSTRACT FROM AUTHOR]

Semliki-Forest-virus 42-S virion RNA is translated in a mouse ascites cell-free system. One of the major polypeptides made in vitro has been identified as Semliki-Forest-virus capsid protein by co-electrophoresis on dodecylsulfate -- polyacrylamide gels and by tryptic peptide analysis on paper. Another major product of the cell-free system has been characterised as envelope protein by peptide analysis but it does not co-electrophorese on gels with envelope proteins from purified virions. It is possible that the nascent, non-glycosylated polypeptide is partially degraded by proleolytic enzymes present in the cell-free extract, or alternatively that fragments of envelope protein are produced by premature termination on the viral RNA. Several other polypeptides are synthesised in the cell-free system in variable amounts but these have not been identified. The genetic content and the mechanism of translation of Semliki-Forest-virus 42-S RNA are discussed. [ABSTRACT FROM AUTHOR]

Previous reports [1-5], we have described some of the features of the primary structure of bovine αs1-casein. In the present work, the complete amino acid sequence has been established and the salient features of this phosphoprotein have been discussed. In the polyeptide chain, the region containing the phosphopeptides Tm1 (T1-T2), Tm1T2 and Tm1T1 [1,4,5], had only been slightly studied as yet. Because of the difficulties encountered in the breakdown of these phosphopeptides, hydrolysis with endopeptidases and exopeptidases were performed on both native and dephosphorylated peptides. It was confirmed that peptide Tm1T2 contains three hydroxy-amino acids (2 Ser, 1 Thr), but, instead of three [1], two phosphorus atoms were found with purified preparations. Partial acid hydrolysis and dephosphorylation using an orthophosphoric-monoester phosphohydrolase indicate that the two seryl residues m this peptide are O-phosphorylated. The remaining gap in the sequence of the central part of peptide Tm1T2 was bridged by studying two fragments obtained by papain digestion of the dephosphorylated preparation. Instead of four serines, postulated earlier from the results of the amino-acid composition of peptide Tm1T1 [1], five were shown to be present after detailed analysis of the fragments. Since the five phosphate groups could be ready removed by an orthophosphoric-monoester phosphohydrolase, the five seryl residues could be O-phosphorylated. The fragments obtained after hydrolysis with thermolysin of both native and dephosphorylated peptide Tra1T1 were further degraded using classical methods for the determination of the amino acid sequence. In the light of all the results obtained on this phosphoprotein (αs1-casein B), the total number of amino acid residues has been corrected to 199, instead of 198, as reported previously [1-5], and the molecular weight has been calculated to be 23616. The following amino-acid composition; Asp7, Asn8, Thr5, Ser8, SerP8, Glu25, Gln14, Pro17, Gly9, Ala9, Val11, Met5, Ile11, Leu17, Tyr10, Phe8, Lys14, His5, Trp2, Arg6, indicates that there is a higher number of acidic than basic residues in this protein. On the basis of the intrinsic dissociation constants of titratable groups in proteins [6], the negative net charge of the molecule was estimated to be 22 at pH 6.5 and 28 at pH 8.6. Since Bigelow's parameter for thc average hydrophobicity [7] of this protein is 1170, it could be considered to be a hydrophobic protein The high amount (8.5%) and uniform distribution of prolyl residues indicate that this protein has limited structuralcoiling possibilities. The polypeptide chain contains three hydrophobic regions, viz. 1-44, 90-113 and 132-199. The first two are characterized by the fact that basic residues predominate over acidic residues. The third region, where most of the aromatic residues are located, contains very few basic residues and is therefore of more acidic character. Two regions, 45-89 and 114-131, are hydrophilic. The former contains more than half of the total acidic residues, in particular seven of the total eight phosphoseryl residues. Another remarkable feature is the concentration of amino acids with carboxylic side chains, particularly glutamic acid, in the vicinity of two clusters of phosphoseryl residues. It may be noticed that the phosphoseryl residues m αs1-casein are arranged m a manner similar to those in β-casein [8]. In particular, in αs1-casein, the 62-70 region containing four phosphoseryl residues, is similar to the 13-21 region in β-casein [8]. It is interesting to point out that ail but one of the phosphoseryl residues always occur m position n with relation to a glutamyl or a phosphoseryl residue, which is in position n + 2. From this, it would appear that there is an enzymatic phosphorylation of the polypeptide chum (carried out by a phosphoryl kinase which may require a negative charge in the phosphorylation site) rather than a direct incorporation of phosphoserine into the polypeptide chain during protein synthesis. The location of the amino-acid substitutions that characterize the αs1-Casein D and the β-casein C variants has provided evidence for an enzymatic phosphorylation of casein [9]. The problem of the phosphorylated sites in αs1- and β-caseins will be discussed in a forthcoming paper. The repeating amino-acid sequences located in the two regions of αs1-casein, viz. 70-84 and 110-125, are also of interest. The primary structure of αs1-casein given here is that of the genetic variant B. The three other variants A, C and D have also been studied. The C and D variants differ from the B variant by the substitutions of Gly/Glu in position 192 [10] and ThrP/Ala in position 53 [9], respectively. The A variant is characterized by a deletion of thirteen amino-acid residues from position 14 to position 26 in the hydrophobic NH2-terminal part of the polypeptide chain [11]. Because of this deletion, there m an important change in the physico-chemical properties of αs1-casein [12, 13]. Some important features of the amino-acid-sequence determination should be noted. Firstly, the great value of maleic anhydride as a reversible-blocking reagent for amino groups of proteins [14], and the usefulness of thermolysin as a specific enzyme for degrading polypeptides [15]. Secondly, the difficulties encountered during the study of the phosphopeptides: these peptides were difficult to purify since they have similar anionic characters and only react slightly with ninhydrin during paper revelation; other difficulties have been the high destruction of phosphoserine during hydrolysis with HCl 5.7 N, the poor yield in the substractive Edman degradation when the NH2-terminal residue is a phosphoserine, and the failure of endopeptidases and exopeptidases to split off peptide bonds that are close to phosphate groups. Fortunately, alkaline phosphatase has proved to be a useful tool for removing phosphate groups, thus avoiding most of these difficulties. Since the amino-acid sequence of αs1-casein is now known, the degradation of this protein by coagulating and other proteolytic enzymes during cheese ripening may be studied. In particular, bitter peptides originating from αs1-casein may be located. As we have already briefly noted [4], it is obvious that one of the three bitter peptides isolated by Matoba et al. [16] from a tryptic hydrolysate of whole casein, was the segment 23-34 of the polypeptide chain of αs1-casein. In addition, the knowledge of the primary structure will be of help in understanding many aspects of the relations between structure and physical properties in this phosphoprotein. [ABSTRACT FROM AUTHOR]

4-Deoxy-D-arabino-hexose has been identified as a constituent of the four Citrobacter sero-types 4, 23, 27 and 36a. Its structure was evaluated by the aid of mass spectrometry and gas liquid chromatography in comparison with known deoxyhexoses. [ABSTRACT FROM AUTHOR]

A linear lattice of equivalent binding sites with nearest neighbor cooperativity is used as a basic model for a linear biopolymer displaying cooperative binding of small ligands. Complicating effects due to the dimerization of free ligands are taken into account. The theory implies two types of intrinsic binding processes: (1) the binding of an isolated ligand (nucleation), and (2) the binding of a ligand in the immediate neighborhood of an already bound one (aggregation). The ratio of the respective association constants determines the degree of cooperativity. It is generally shown how the equilibrium properties of the system can be derived by means of the matrix method. Sonic properties of special practical significance, such as the equilibrium concentrations of free and bound monomer ligands, are discussed in more detail. On the basis of a pertinent kinetic model chemical relaxation of strong cooperative bindinng is also examined. The paper gives particular emphasis to the formulation of quantitative relations which are most useful for an experimental application of the the oretical results. Appropriate procedures to plot measurable data and to evaluate basic parameters are proposed. They are applied to specific experimental systems in a subsequent set of papers. [ABSTRACT FROM AUTHOR]

Valyl-transfer ribonucleic acid was degraded by polynucleotide phosphorylase from Escherichia coli in the presence of phosphate or arsenate. A compound that was identified as 2′&(3′)-O-valyl-adenosine 5′-pyrophosphate was isolated from the phosphorolytic digest of valyl-tRNA while 2′(3′)-O-valydenosine 5′-monophosphate was isolated from the arsenolytic digest. It was concluded that valyl-tRNA can be phosphorolyzed by polynucleotide phosphorylase. The rate of phosphorolysis of aminoacylated tRNA was lower than that of uncharged tRNA. N-blocked derivatives of valyl-tRNA and phenylalanyl-tRNA were also phosphorolyzed by polynucleotide phosphorylase. Some possible uses of nucleoside diphosphates substituted in their suger moiety are suggested. [ABSTRACT FROM AUTHOR]

1. The arginine-requiring mutant of Chlamydomonas reinhardi, isolated by Ebersold in 1956 and called arg-1, shows no activity of the enzyme acetyl-glutamate-reductase, for this reason we propose to call this mutant arg C1. By analogy with this we call the arginine-requiring mutant arg-2, isolated by Eversole in 1956, arg G2. 2. Under circumstances in which the synthesis of the last enzyme of the arginine pathway, the argininosuccinate lyase, shows strong repression and derepression, we could not find any regulation of the synthesis of the 2nd up to and including the 6th enzyme of this pathway. 3. A more detailed study on the 6th enzyme of the pathway, the ornithine transcarbamylase, also failed to show any regulation of the synthesis. The findings of 2 and 3 support our hypothesis on the regulation of the arginine biosynthesis in Chl. Reinhardi, which we presented in the preceding paper. [ABSTRACT FROM AUTHOR]

The results obtained with tea plants treated with 14C-maleic hydrazide indicate that tea plants can metabolize maleic hydrazide to some extent. According to auto-radiographic studies, two radioactive spots were found on X-ray films after exposure and development. Based on two-dimensional paper chromatography, possible ring cleavage, and infrared spectroscopic techniques, the possible metabolic products are lactic acid, succinic acid, maleimide and hydrazine. The biological activity of the metabolic products was tested by utilizing the Avena first internode test. Based on this investigation, it was noted that each radioactive area contained compound(s) that possess(es) some growth promoting properties. However, the results obtained with unknown number one (U1) indicate that it possesses greater growth-promoting properties than unknown number two (U2). Finally, the approximate concentrations of U1 and U2 were determined by comparison with known concentrations of IAA and in relation to the amount of growth produced during the 19 ½-hour period of incubation. [ABSTRACT FROM AUTHOR]

The maximal potential activity of UDP-glucuronyltransferase is constrained by the structure of the phospholipid environment in intact microsomal membranes. This constraint can be relieved by treatment of microsomes with phospholipase A. As shown by the data in this paper, however, relief of constraint is associated with a loss of specificity in the binding of UDP-sugars at the UDP-glucuronic acid site of UDP-glucuronyltransferase. As a result, several UDP-sugars which have no effect on the activity of the untreated enzyme act as inhibitors of the unconstrained, phospholipase A-treated form of UDP-glucuronyltransferase. In addition to the loss of specificity of substrate binding, the phospholipase-A-treated form of UDP-glucuronyltransfease cannot be activated by UDP-N-acetylglucosamine, which is a positive K-type of allosteric effector for the untreated form of the enzyme. UDP-N-acetylglucosamine, in fact, is an inhibitor of the phospholipase-A-treated form of the enzyme. In contrast to the effects of other UDP-sugars, inhibition by UDP-N-acetylglucosamine seems to result from the binding of UDP-N-acetylglucosamine at an allosteric site rather than the active site. Inhibition of the phospholipase-A-treated form of UDP-glucuronyltransferase by UDP-N-acetylglucosamine and other UDP-sugars is additive. [ABSTRACT FROM AUTHOR]

With the help of an automatic protein sequenator, we have verified a prediction made by us in a recent paper, namely that residue 16 in Candida krusei cytochrome c is a glutamine and not a glutamic acid. Neurospora crassa cytochrome c now remains the only mitochondrial cytochrome c which apparently does not have a glutamine at this position. In the course of this investigation, from two strains of Candida krusei we isolated two cytochromes which differ in amino acid composition. One of them seems to correspond to that described in the literature. Two of the differences have been located. [ABSTRACT FROM AUTHOR]

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

The isolated α-chain of sea anemone collagen was cleaved at methionyl residues with cyanogen bromide. Eleven unique peptides were isolated by ion-exchange and molecular-sieve chromatography, eight of them in approximately equimolar amounts. Characterization of the eleven peptides with regard to amino-acid composition and molecular weight demonstrated that the isolated peptides account for all of the amino acids and for the molecular weight of the α-chain within experimental error. Since the molecule of sea anemone collagen contains ten methionyl residues, the data of the present paper confirm earlier analytical and preparative results that this invertebrate collagen is made up of three identical α-chains. In comparison with known collagens, on the basis of quantitative and qualitative features of amino-acid compositions, the cyanogen bromide peptides of sea anemone collagen indicate genetical relationship of sea anemone collagen to basement membrane collagen. [ABSTRACT FROM AUTHOR]

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

Commercial lima bean inhibitor was fractionated into four apparently homogeneous components as previously described by Jones, Moore and Stein in 1963. Reduced and alkylated component IV was hydrolyzed with trypsin and the resulting peptides were separated by ion exchange chromatography on Dowex 50 X2 or gel filtration on Bio-Gel P-6. Where necessary, further purification of the peptides was carried out by paper chromatography, paper electrophoresis or a combination of both. Seven peptides were obtained in pure form and their compositions are reported here. The amino acid sequence of six of these peptides was determined, using classical methods. When allowance is made for the occurrence of two homologous peptides, presumably resulting from heterogeneity m the original protein preparation, the tryptic peptides isolated, account for the complete composition of the protein. In an attempt to obtain overlapping sequences the tryptic digest was also performed on the reduced, alkylated and guanidinated protein. Five tryptic peptides, including one containing homoarginine as the carboxy-terminal residue, were isolated in pure form from the digest; their amino acid compositions are reported. [ABSTRACT FROM AUTHOR]

Two monomeric haemoglobins of the insect Chironomus thummi thummi are described which, though their O2-binding curves are exactly hyperbolic (Hill coefficient n = 1.00), do exhibit a Bohr effect (haemoglobin III, 0.28 at 25 °C; haemoglobin IV, 0.42 at 25 °C). Thus, these haemoglobins can exist in two conformational states differing in O2-affinity. The states are characterized also by different ΔS values: the acid conformation with low affinity possessing a low, the alkaline conformation with high affinity a higher ΔS value. In this paper the Bohr effect is regarded from generalizing aspects, i.e. as an interaction between the proton-binding site and the 6th coordination point of the iron which can therefore be studied as well with oxidised haemoglobin. In the case of the oxidised form of haemoglobin the allosteric interaction between the proton-binding site and the coordinated H2O was the basis of our experiments. Absorbance and ellipticity of the oxidised form of haemoglobin measured at different wavelengths as a function of pH result in titration curves differing in the position of their inflection points. On the basis of the circuit process described previously [21], the percentage distribution of the constituent conformation isomers according to pH and the allosteric pK values (pKa2 = 7.30; pKa1 = 7.00) were calculated. In electron spin resonance spectroscopy the heterotropic allosterism is revealed by a change in the symmetry of the intramolecular electric field of the H2O-derivative, The acid conformation with rhombic symmetry (g⊥1 ≠ g⊥2) is transformed into the alkaline conformation with axial symmetry. The acid conformation is further characterized by five hyperfine structure lines in the g⊥1 signal, showing the d-electrons to interact with two N-nuclei in the z-direction. The hyperfine structure lacking in the alkaline conformation indicates a larger distance of the proximal imidazole from the iron. The pH-dependent variation of this distance controls the affinity of the sixth ligand. A possible molecular mechanism of the Bohr effect, of haemoglobin III is described, considering the interactions, revealed by the atomic model [8]. It is shown that the Bohr effect mechanism in Chironomus haemoglobin is based on thc same conditions of tertiary structure as in the case of vertebrate haemoglobins [7]. These conditions of tertiary structure, however, are realized with completely differing primary structures. [ABSTRACT FROM AUTHOR]

The isolation of oligosaccharides produced by the action of phage-induced enzymes on the bacterial exopolysaccharide colanic acid is reported. The products from colanic acid produced by different strains of Escherichia coli and Salmonella typhimurium were hexasaccharides which differed in their acyl substituents. All contained fucose, glucose, galactose and glucuronic acid in the molar ratio 2:1:2:1. They appeared to have the same carbohydrate structure as that postulated for the repeating unit of colanic acid. All the hexasaccharides contained acetate. Pyruvate was also found in most preparations. The phage-induced enzymes only converted 30-35 % of the polymer to oligosaccharides. They also hydrolysed de-acetylated polysaccharide but, were inactive against carboxyl-reduced material and against a number of other bacterial exopolysaccharides. [ABSTRACT FROM AUTHOR]

Several laboratories have shown that the two carbon atoms C-28 and C-29 of the ethyl or ethylidene side chains of phytosterols are introduced on to a C-24 unsaturated side chain by two methylation steps. A scheme based on previous work suggests the various intermediates which can be formed by stabilisation of the carbonium ion 3. We have shown previously that in the biosynthesis of 5α-stigmastan-3β-ol (11α)and 5α-stigmast-22-en-3β-ol (12α), sterols of the myxomycete Dictyostelium discoideum, routes h and i could be excluded. In the present paper we describe the in vivo conversion, by the myxomycete, of differently labelled lanosterol (13), stigmastanol (11c), and 5α-stigmast-22-en-3β-ol (12c), into the sterols 12a and 11a, and discuss the possible biosynthetic routes for the two sterols. After incorporation of a mixture of [26,27-14C2]lanosterol and [24-³H]lanosterol (³H/14C = 5.60 ± 0.25), the isolated sterols 11a and 12a were converted into 11b (³H/14C = 5.54 ± 0,28) and 14b (³H14C = 5.61 ± 0.28). By ozonisation followed by oxidation in neutral or basic medium. 12a was converted into the esters 15 (³H/14C @ 5.54 ±0.28) and 16 (³H/14C 5.55 ± 0.3), respectively. These results show that during the C-24 methylation. H-24 is not lost: the hydrogen which is lost is located @9' neither at C-24 nor at C-23 and must therefore be at C-25. This excludes routes a and b for sterol 12. After incorporation of a mixture of 126,27-14C2]lanosterol and [23-³H]lanosterol (³H/14C = 4.36 ± 0.1) the isolated sterols 11a and 12a were converted into 11b (³H/14C = 4. 12 ± 0.2) and 14b (³H/14C = 3.32 ± 0.14); a fraction of 12b was degraded into 15 (³H/14C = 2.28 ± 0.31) and 16 (³H/14C= 3.44 ± 0.15). These results show that during the conversion of 13 into 12a, 50% of the H-23 atoms migrate from C-23 to C-24 and 25% remain on C-23 ; the other 25% are eliminated. During the conversion of 13 into 11a both H-23 atoms are retained. These results excluded route g for the two sterols. Since we have demonstrated that the conversion of 11c into 12a is possible in high yields (4 6%) with an efficiency eight times greater than the conversion of 12c into 11a, we propose that the sterol 11a is synthesized in vivo according to route e with migration of H-23 from C-23 to C-24. The sterol 12a is biosynthetized either by route d, f, the 22,23 double bond being introduced during the alkylation step, or by desaturation of the sterol 11a, the C-22 C-23 double bond being introduced after the C-24 alkylation. [ABSTRACT FROM AUTHOR]

The analytical active-enzyme-centrifugation method permits the determination of the hydrodynamic properties of enzymes while they are fully active on their substrates: the centrifugation can be performed at the very dilute concentrations used in kinetic studies (&aysmp; 1 µg/ml): furthermore a purified enzyme preparation is not necessary and in many cases a crude extract is sufficient. This method is beginning to be used in several laboratories even though few of its experimental aspects have been published. This paper reports the details for carrying out, an activeenzyme-centrifugation run, discusses the experimental conditions anti details with artifacts and ways, which are easy, to overcome them. [ABSTRACT FROM AUTHOR]

The active thiol group of lombricine kinase from Lumbricus terrestris muscle was labelled with N-ethyl-[l-14C]maleimide. The resulting inactivated N-ethyl-[1-14C]succinimido enzyme was then subjected to tryptic hydrolysis. The peptide containing the labelled essential thiol group was isolated and found to contain: Leu-Gly-Tyr-Ile-Thr-[14C]Cys-Pro-Gly-Ser-Asn-Leu-Gly-Thr-Leu-Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP: guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle. In addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight ... 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits. .AUV- [ABSTRACT FROM AUTHOR]

This paper gives a presentation of ADP and AMP inhibition of phosphoglycerate kinase with MgATP2- and 3-phospho-D-glycerate as substrates at high and low Mg2+ concentrations and pH 7.8. The enzyme seems to contain at least two nucleotide binding sites, one presumably binding to MgATP2- and the other to ADP3-. The ADP3- binding site might bind MgADP1- also. AMP2- competes for the same form of the enzyme, probably the same site, as MgATP2-. ADP3- and MgADP1- are competive inhibitors and AMP2- is a non-competitive inhibitor of 3-P-glycerate. Values of the inhibition constant, Ki, for ADP3- at low Mg2+ level and MgADP1- at high Mg2+ level are 0.2 and 0.02 mM, respectively. The latter value is about ten times less than the expected Michaelis constant for corresponding substrate in the reverse reaction. Ki for AMP is about 2.0 mM at both low and high Mg2+ concentrations but the inhibition is stronger at a high than at a low Mg2+ level, probably caused by conformational and/or other -differences of the enzyme at these two metal ion concentration. The main catalytic reaction suits a pattern that is consistent with a rapid equilibrium random mechanism. [ABSTRACT FROM AUTHOR]

In this paper an efficient procedure is described for the isolation of polysomes from tissue culture cells. optimal yields, especially of the heavier classes of polysomes can only be obtained at a rather high KCl concentration in the presence of nonionic detergent. Evidence is provided that the lower polysomal yield after extraction at low salt concentration is not due to breakdown of the polysomes by RNAase. Furthermore, the yield of polysomes is pH dependent only at low salt concentration, while at high salt concentration no influence of the pH is observed. Refreshing of growth medium several hours before harvesting the cells, increases the ratio of polysomes to 80 S monomers. [ABSTRACT FROM AUTHOR]

In order to investigate the core structure of the lipopolysaccharide from an Escherichia coli 0100 strain, two derivatives were prepared from this strain by introducing genetic material from Salmonella abony. One, an SR hybrid, contained a lipopolysaccharide with short side chains consisting of simple repeating units of Salmonella group B specificity attached to the E. coli core. The other, an R form, contained only the E. coli core and no O side chains, due to a mutation in the Salmonella rfb chromosomal region, which determines the synthesis of the O repeating units. By partial acid hydrolysis of the SR and R lipopolysaccharides, oligosaccharide fragments were obtained which made it possible to reconstruct the hexose region of the core (see formula below). Methylation studies performed on the dephosphorylated polysaccharides confirmed and extended these results. It was found that only about 60% of the distal glucose units of the core chains were substituted by N-acetylglucosamine residues. Further, the heptose region was shown to form a branched trisaccharide. On mild acid hydrolysis of both lipopolysaccharide preparations a Gal-α1,7(8)-KDO disaccharide was formed, indicating that a galactose unit is linked to the 2-keto-3-deoxyoctonate (KDO) portion of the hpopolysaccharide. After Smith degradation of the SR lipopolysaccharide, the oligosaccharide Gal-β1,4-Glcglycerol was identified which derived from the linkage region between the Salmonella type repeating unit and the E. coli core. Only the core stubs substituted by N-acetylglucosamine residues carried an O-specific unit. On the basis of the these findings the partial structure of the E. coli 0100 core polysaccharide with a Salmonella group B-specific unit attached to it is proposed as given below. The relationships to core structures identified in lipopolysaccharides of other representatives of Enterobacteriaceae are discussed. [ABSTRACT FROM AUTHOR]

Constitutionally equivalent groups that occur in a single molecule are classified on the basis of their topic relationships by comparing their environments. The proposed classification extends earlier suggestions of Mislow and Raban. The existence of homotopic groups (those indistinguishable under any conditions) is related to the symmetry properties (gyrosymmetry) of rigid as well as flexible finite moieties. Stereoheterotopic groups (those that are distinguishable but occur in constitutionally equivalent environments) may be associated with various steric elements which will be fully discussed in a second paper. Each of these elements, the center, the axis, the plane and the torsional element, is divided into four mutually exclusive classes: the chiral and achiral elements of stereoisomerism; the prochiral and proachiral elements of prostereoisomerism. Of these classes, only the various centers of stereoisomerism and of prostereoisomerism are discussed in this report. Some modifications in our earlier proposals for the naming of individual stereoheterotopic groups are introduced. The utility of these new and revised terminologies for biochemical discussions is indicated. [ABSTRACT FROM AUTHOR]

Semihemoglobins α and β are derivatives of human hemoglobin which possess the usual chain composition (α2β2 or αβ) but which carry the heine prosthetic group on only one kind of chain (α or β), the complementary chain (β° or α°) being heme-free. In this paper, new results on two compounds of this type, namely, semiHbαD (αβ°) and semilHbβ (α°β) have been presented; existing data on other earlier preparations have also been reviewed. Methods for preparation of semiHbαD and semiHbβ have been described-. Some of their properties including optical absorption, circular dichroic spectra, oxygen equilibrium and kinetics of carbon monoxide binding have been measured; these properties are then compared with those possessed by intact hemoglobin (β2β2), isolated chains (α,β) and the berne-free apoprotein (α°β°). 1. The contribution of the heine moiety to the optical absorption and circular dichroic spectra of the semihemoglobins are found to be intermediate between those of hemoglobin and those of the corresponding isolated chain. This result indicates that the effect of interaction between the unlike chains of these molecules are transmitted as far as the heine prosthetic group. The structure of the heme-free chain is also modified as the result of this interaction as revealed by titration of sulfhydryl groups, although circular dichroic spectra in the far ultraviolet are not conclusive. 2. The oxygen affinity and the Bohr effect of the semihemoglobins are again intermediate between those of hemoglobin and isolated chain, the semiHbβ being closer to hemoglobin than semiHbαD. The same is true concerning the velocity of carbon monoxide binding. 3. The kinetics of fixation of carbon monoxide to semihemoglobins is strongly biphasic. It is suggested that this property could reflect the existence of semihemoglobins as two types of filmers (αβ) but differing among themselves on account of different contact regions brought into play. (α1β1 and α1β1 in the nomenclature of Perutz.) 4. The significance of the fact that semiHbα is obtained in multiple forms is considered. 5. The ensemble of the results are discussed in the context of chain-chain interactions. [ABSTRACT FROM AUTHOR]

The Cbl-1 mutant of Bacillus subtilis 168 WT is the result of a spontaneous mutation presenting a pleiotropic character. In this paper, the results of the morphological and chemical analyses of the cell walls isolated from both strains are compared. When either the whole cells or the isolated cell walls of both strains were fixed with osmium tetroxide, the examination by electron microscopy showed extensive morphological differences in structure due to the partial destruction of the mutant cell walls. The chemical investigations performed on the isolated cell walls revealed surprising differences in chemical structure. In the cell walls of the mutant, teichoic acid was completely missing and was replaced by protein. A major protein-containing fraction accounting for about 50% of the material solubilized by autolysis of the mutant cell walls was isolated. Polyacrylamide gel electrophoresis showed that this fraction was composed mainly of a single protein of high molecular weight When denatured by sodium dodecyl sulfate treatment in the presence of β-mercaptoethanol, the protein yielded by gel electrophoresis two major bands with molecular weights of approximately 105000 and 155000, respectively. The molecular weight of the Cbl-1 cell wall protein determined by high speed sedimentation equilibrium was 255 600. [ABSTRACT FROM AUTHOR]

The biosynthesis of melittin, the main toxin of bee venom, was studied in vivo by feeding radioactive amino acids to honey bees. Extracts from venom glands were analyzed for the presence of labeled melittin and other components. Radioactivity was first incorporated into another peptide which is considered to be a precursor of melittin. This conclusion is based on the observed labeling kinetics demonstrating the transient formation of this peptide. Furthermore, the structural similarity between melittin and this component could be established. Evidence is presented showing that it differs from melittin at the amino end. The precursor peptide could not be detected in the ejected venom. [ABSTRACT FROM AUTHOR]

A direct reading, six channel conductivity recording apparatus has been constructed and used to assay enzymes, including some of those found in muscle. This paper gives the essential theory of the method, and indicates that it is a highly versatile assay system. It may be applicable whenever ionic substrates are involved, but this is more easily tested by experiment than by calculation. The manipulation involved in an assay is probably less than in any other method. Conductimetry could be used as a kinetic method for some enzymes (for example amidases), but suffers from restrictions in the amounts of non-reacting ionic species which can be tolerated. Improvements in the temperature control of the apparatus could allow much higher sensitivity to be used. [ABSTRACT FROM AUTHOR]

Ornithine carbamoyltransferase from Halobacterium salinarium is a typical extremely halophilic enzyme. It is rapidly and irreversibly inactivated when exposed to a salt-free environment and shows high activity at NaCl concentrations above 4 M. The present paper describes the purification of the enzyme from cell free extracts, by acetone fractionation, gel filtration, sucrose gradient centrifugation and chromatography on calcium, phosphate gels. All purification procedures were carried out on 4.3 M NaCl solutions to prevent irreversible inactivation of the enzyme. Ornithine (0.1 M) stabilizes the enzyme and was included throughout the purification. The purified enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis and by analytical ultracentrifugation. Enzyme activity was measured as citrulline formation from ornithine and carbamoylphosphate in the presence of 4.3 M NaCl, at 37°. Specific activity of the purified enzyme was about 1 µmole citrulline formed per minute per µg protein or an about 50-fold increase from that of cell-free extracts. Halophilic enzymes which can not be reactivated from a salt-free environment have not previously been extensively purified. [ABSTRACT FROM AUTHOR]

1. 1. Acapsular (K-) forms were isolated from Escherichia colt strain E56b (serotype O8 :K27(A): H-). A number of different R strains were obtained from the K- forms by selection of spontaneous mutants and by phage selection. 2. The lipopolysaccharides of these R strains (consisting of core and lipid A) were subjected to mild acid degradation and, after removal of insoluble lipid A, the mixtures were fractionated by gel chromatography. Results of chemical analyses showed that the core oligosaccharides of different R mutants differed with respect to their sugar constituents as well as to the molar ratios of these constituents. A group of R mutants did not contain phosphate in their core oligosaccharides (P- mutants). Enzyme determinations indicated that one R mutant had a block involving UDP glucose pyrophosphorylase, whereas the enzymes of activation and interconversion of component sugars were intact in the other R mutants. 4. By allelic exchange (mating with S-form Hfr strains)it could be demonstrated that with one exception the chromosomal site of the S→ R mutation of all mutants maps close to mtl (mannitol utilization). In Salmonella the rfa locus maps fun the same region. All R mutants had an unimpaired (his-linked) rfb locus. 5. The groups of mutants which differed in the sugar composition of their core oligosaccharides could also be differentiated serologically and by their sensitivity to phages. There was serological cross reactivity between the lipopolysaccharides of some R mutants with R mutants of Salmonella minnesota which also have a very incomplete core. It is concluded that the lipopolysaccharides of the R mutants described in this paper have incomplete core structures which result from blocks at successive stages in the synthesis of the coli R1 core. in analogy to Salmonella these E. coli R mutants shoed, with one exception, be termed rfa type mutants. [ABSTRACT FROM AUTHOR]

Chemical investigation of the glycosidic constituents of Oospora virescens (Link) Wallr. resulted in the isolation of several glycosides. We have shown recently that two of them, named virescenoside A (I) and B (II) are β-D-altropyranosides of virescenol A and B; these diterpenic aglycones have been found to have the structure of isopimaradien-α,3β-19-triol (JV) and isopimaradien-3β,19-diol (V), respectively. In this paper we describe the isolation and structure of virescenoside C, a new metabolite of Oospora virescens. The results obtained show that virescenoside C (IIIa), C26K40O7, m. p. 160-162°, [α]D -71,4Y°, is a β-D-altropyranoside of virescenol C, which is found to have the structure of 3-keto 19-hydroxy isopimaradiene (VIa). These results are based on physical evidence (infra-red, nuclear magnetic resonance and mass spectra) and particularly on the chemical correlation with virescenol B and virescenoside B. Virescenoside C seems to be the third example of an altroside found in nature, [ABSTRACT FROM AUTHOR]

1. The distribution of ribosomal particles between the soluble and membrane fractions of exponential-phase cells of Bacillus licheniformis was studied. The cells were very gently lysed by a combined treatment with lysozyme and Brij 58 in 10 mM. Tris-buffer, pH 7.6, with 60 mM K+, 10 mM Mg2+, 0.5 mM spermine and 0.75 mM spermidine. The proportion of the ribosomal material present in the membrane fraction was estimated after solubilization of the membranes by the combined action of Brij 58, deoxyribonuclease and lipase. 2. Under these conditions of lysis 96% of the total ribosomal material, consisting of subunits (12%) monosomes (20%) and polyribosomes (68%) and most of the cellular DNA, is found to be membrane-bound. 3. In the soluble fraction, containing only 4% of the total cellular amount of the ribosomal material, only subunits and 70S particles and no polyribosomes are found. 4. DNA does not seem to be involved in the binding of the ribosomal material to the membranes. 5. The results will be discussed considering the question of whether the distribution described in this paper represents the true distribution in vivo of ribosomes. [ABSTRACT FROM AUTHOR]

The specific extracellular material with endotoxie properties (free endotoxin) was isolated from liquid culture of Shigella sonnei phase I. Bio-Gel P-300 filtration and subsequent ultracentrifugation at 105 000 × q was used to purify and separate free endotoxin into two fractions: the sediment and the supernatant. The sediment fraction was identified as lipopolysaccharide-protein complex of the molecular weight of several millions, containing the same constituents as cell lipopolysaccharide of Shigella sonnei phase I, prepared by phenol—water extraction. This substance was a better endotoxin than cell lipopolysaccharide: it showed strong toxicity (L.D.50 = 15 µg), pyrogenicity and elicited local and general Shwartzman reaction.The supernatant fraction represents phase I specific substance practically free of heptose, 2-keto-3-deoxyoctonic acid and phosphorus. It contained glucose, galactose and glucosamine as does phase I lipopolysaccharide, and also galactosamine and small amounts of mannose. Phase I specific substance was very active serologically and revealed very weak endotoxie properties (L.D.50 ≥ 1800 µg). The homogeneity of the supernatant fraction was proved in sedimentation experiments, free electrophoresis and rechromatography on Bio-Gel P-300 and Sephadex G-200 columns. The examination of hydrodynamic properties (sedimentation coefficient, limiting viscosity number, diffusion constant and partial specific volume) of the supernatant fraction showed that this substance has a linear structure with molecular weight 90800, and diameter and length of the molecule of about 15 Å and 800 Å, respectively. [ABSTRACT FROM AUTHOR]

Phage-induced fucosidases prepared from cell lysates of a Klebsiella aerogenes strain have been shown to hydrolyse the polysaccharide of Escherichia coli K27. The major hydrolysis product of one enzyme was identified as an acetylated tetrasaccharide containing equimolar amounts of galactose, glucose, glucuronic acid and fucose. The reducing terminus was characterised as fucose. A minor product of enzymic hydrolysis was the corresponding unacetylated tetrasaccharide. This oligosaccharide was also obtained from alkali-treated polysaccharide. Neither tetrasaccharide could be hydrolysed further with glycosidases. The product of hydrolysis with the second enzyme was probably an octasaccharide comprising two repeating units of the E. coli polysaccharide. It also contained equimolar amounts of the constituents and approximately 50% of the fucose was reduced with borohydride, From the results it is concluded that both enzymes require the sequence ....... α-glucuronosyl — 1 → 3 fucosyl-glucose for substrate activity. The presence of the O-acetyl groups was not essential for enzymic hydrolysis. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

The sialic acid-free M and N substances and glycopeptides from their pronase digest were treated with alkaline borohydride and products of degradation were fractionated. Galactose and N-acetylgalactosamine were split off during the degradation in the form of disaccharide: galacto- syl-N-acetylgalactosaminitol, and it was accompanied by the release of amino acid components. The conclusion has been drawn that a part of carbohydrate moiety of M and N substances represents trisaccharides: N-acetylneuraminyl—galactosyl—N-acetylgalactosamine, linked to serine and threonine residues in the peptide chain by an alkali-labile O-glycosidic bond. The other type of oligosaccharides present in M and N substances is linked to peptide chain by an alkali-stabile bond and includes mannose, fucose, N-acetylglucosamine, the rest of sialic acid and galactose. The degraded M' and N' glycoproteins (M and N glycoproteins free of N-acetylneuraminic acid) did not inhibit the hemagglutination by Vicia graminea phytoagglutinins. The degraded native M and N substances were inactive towards both anti-M and anti-N rabbit immune sera, and anti-N phytoagglutinins from V. graminea, but they were still as good inhibitors of viral hemagglutination as untreated substances. [ABSTRACT FROM AUTHOR]

Multiple species of Escherichia coli transfer RNA for valine and leucine were separated from each other by countercurrent distribution. Each transfer RNA species was charged with the corresponding radioactive amino acid and then allowed to transfer its label into hemoglobin peptides in the ribosomal system from rabbit reticulocytes. Earlier experiments had shown that the minor tRNAIIbLeu would only label one position, no. 48 of the α-hemoglobin chain under these conditions. In this paper we report the coding properties of the other separated leucine tRNAs and of multiple valine tRNAs with the hemoglobin messenger. The leucine codons in the α-chain messenger can be divided into three classes with respect to their tRNA response : (a) position 48 is recognized only by tRNAIIbLeu. (b) The majority of the leucine codons can be recognized by both the two major leucine tRNAs. (c) Some leucine codons can only be recognized by one of the major leucine tRNAs. Similarly, two classes of valine codons can be distinguished in the α-chain messenger. [ABSTRACT FROM AUTHOR]

Earlier investigations had shown that the C-methyl group at C-3 of L-mycarose (2,6-dideoxy- 3-C-methyl-L-ribo-hexose) originates from the S-methyl group of L-methionine. In the present paper the question was investigated whether this methylation involves transfer of the intact methyl group. A tylosin producing strain of Streptomyces [radiae was grown in the presence of L-[methyl-14C]- methionine and D,L-[methyl-2H3]methionine. The dilution value of 14C into mycarose isolated from tylosin after 6 days of fermentation was 1.47. Mycarose was converted to its 1-ethylthio- glycoside. In the mass spectrum of the latter compound the molecular ion and all fragment ions which contain the branched C-methyl group had additional peaks of 3 mass units higher than the corresponding peaks of the unlabeled thioacetal. From the peak intensities the amount of trideuterated species present was calculated to be 31 ± 3%. Molecular species with one or two deuterium atoms were present to less than 3%. The structure of the fragment ions was confirmed by high resolution mass spectrometry. The results prove that methylation had taken place with transfer of the intact methyl group of methionine to C-3 of the hexose chain. [ABSTRACT FROM AUTHOR]

1. This paper examines in detail the activation, of bovine and porcine trypsinogens and of bovine chymotrypsinogens A and B by trypsin and aspergillopeptidase A. Kinetic data have also been obtained (Km and kcat) for the hydrolysis catalyzed by these proteases of several model peptides with sequences related to the N-terminal sequence of bovine trypsinogen. 2. The N-terminal sequence of (aspartyl)4 residues is not necessary for the recognition of the strategic Lys-Ile bond of trypsinogen. 3. We have shown previously that there are two binding sites for Ca2+ on trypsinogen. One of these sites is constituted by the 2 aspartyl residues which are the nearest neighbours of the important Lys-Ile bond. The saturation of the site by Ca2+ improves the formation of the trypsinogen-trypsin complex; Ca2+ has no effect on the rate of decomposition of this complex. 4. The comparison of the kinetic data for the activation of trypsinogens and chymotrypsinogens A and B on one hand and the comparison of kcat and Km of different model peptides, among them the synthetic nonapeptide Val-(Asp)4-Lys-Ile-Val-Gly, on the other hand, implies that the 4 aspartyl residues confer no special reactivity to the Lys-Ile bond but conversely have a very negative effect. For example kcat = 1.6 sec-1 for chymotrypsinogen B but only 2.5 and 5.5 x 10-3 sec-1 for bovine and porcine trypainogens. The values of Km are similar. For the peptide Val-(Ala)2-Lys-Ile-Val-Gly kcat = 7.0 sec-1 but kcat between 10-4 and. 10-2 sec-1 has been found for the peptide Val-(Asp)4-Lys-Ile-Val-Gly. This very negative effect is observed only with trypsin. In the activations by aspergillopeptidase A, trypsinogen is a much better substrate than chymotrypsinogen.The implications of this exceptionally slow hydrolysis of the Lys-Ile bond are discussed. The problem of the formation of inert proteins in particular appears to be closely related to the very poor quality of this bond as a substrate for trypsin. A mechanism is given for the formation of inert proteins; a similar mechanism also explains the degradative autolysis of trypsin. [ABSTRACT FROM AUTHOR]

An investigation has been made of the acid soluble protein fractions of wool that has been reduced with benzylmercaptane and treated with methyliodide. When this modified wool was shaken in dilute formic acid (50 v/v) at room temperature for 1 hour, 7% of a protein material was dissolved. After dialysis and subsequent lyophylisation the protein material was isolated, and shown to be heterogeneous by paper electrophoresis. Fractionation of this protein on Sephadex G-75 in dilute acetic acid gave more than six components of relatively high content of tyrosine and phenylalanine. One of these fractions, obtained in good yield, was purified by rechromatography. For further separation the protein fraction was subjected to ion exchange chromatography on Dowex 1 (×2) columns. By stepwise elution with buffers of decreasing pH-values, containing acetic acid and 8 M urea, four subfractions were obtained. The amino acid compositions of reduced wool, of the protein obtained by formic acid extraction, of the fraction after gel filtration on Sephadex G-75 and of one subfraction on Dowex 1 (×2) are given. In all three fractions an increase in glycine, tyrosine and phenylalanine content was observed with relatively low amounts of basic and acidic amino acids. The subfraction obtained after the final step of separation on Dowex 1 (×2) was further characterised by endgroup determinations. Dnp­glycine was the only N-terminal amino acid with traces of Dnp­alanine as contaminant. Hydrazinolysis resulted in tyrosine on the C-terminus. After digestion of the subfraction by trypsin and chymotrypsin a number of peptides with different chain lengths was obtained, indicating that, tyrosyl residues had accumulated and that larger arginyl­peptides were present. Sedimentation measurements by ultracentrifugation indicated that the subfraction had a weight average of apparent molecular weight in the range of 9,000 to 13,000. This is in agreement with the molecular weight of 16,400 derived from the amount of Dnp­glycine and the calculated one from the amino acid composition. The analytical results show that the subfraction represents a single polypeptide chain containing only one N-terminal residue and about hundred aminoacids of which about fifty are glycine, tyrosine and phenylalanine. [ABSTRACT FROM AUTHOR]

From Salmonella R lipopolysaccharides of the chemotypes Rc, Rd1, and Rd2 oligosaccharides were prepared and their structure studied by the following methods: quantitative sugar analyses; periodate oxidation followed by borohydride reduction in order to transform the L-glycero-D-mannose residues into D-mannose residues; methylation of original and transformed oligosaccharides, methanolysis and gas chromatographic identification of the methylated heptoses or mannoses; the action of glycosidases; serological tests. The results of the study are in agreement with the formulae given below for the different oligosaccharides. The anomeric form of the glucose unit linked to heptose, as well as that part of the oligosaccharides which contains the 2-keto-3-deoxy-octonate residues [KDO] remain uncertain. Chemotypes Rd2: IIeptose pα 1 → [KDO]x; Rd1: Heptose pα 1 → 3 Heptose pα 1 → [KDO]x; Rc: Glucose 1 → 3 Heptose pα 1 → 3 Heptose pα 1 → [KDO]x. [ABSTRACT FROM AUTHOR]

In this paper the thermal stability of poly (A + U) and poly (A + 2U) in acid solution is studied. It is known that at low pH poly A associates with itself to form a double protonated hellcat structure, poly (A + A). The dissociation Tm's of poly (A + U) and poly (A + 2U) do not vary with pH as long as they are higher than TmA [the dissociation temperature of poly (A + A)]. TmA itself varies with the pH (it increases as the pit is lowered) and thus, there are pH values at which TmA equals the dissociation Tm'S of the complexes poly (A + U) and poly (A + 2U). Below such pH's, Tm2-3, Tm3-1, Tm2-1 decrease as the pit becomes more acid. In contrast, Tm3-2 does not exhibit such a variation. The transition corresponding to this last Tm [the partial dissociation of poly (A + 2U) into poly (A + U)] does not change the concentration of ‘free’ poly A. We conclude that the change in thermal stability of the complexes in this region of pH is due only to their competition with poly (A + A) and that poly (A + U) and poly (A + 2U) are not modified. At low ionic strength, the transitions observed change with the pH and, by identifying them by means of two independent methods (continuous variation method, and comparison of spectral variations) we have shown that the stability diagram (temperature versus pH) formally resembles the stability diagram (temperature versus ionic strength) at neutral pH (Fig.3), when pH is decreased and/or ionic strength is increased, poly (A + 2U) is relatively more stable than poly (A + U): The formation of poly (A + A) lowers the stability of both poly (A + U) and poly (A + 2U), but, since the proportion of adenylic residues is 1/3 in this last complex, against 1/2 in the former, poly (A + 2U) is less destabilized, and is more sensitive to the diminution of electrostatic repulsion brought about by the sodium ion concentration. These effects are summarized in a three-dimensional diagram temperature-pH-ionic strength (Fig. 9). Some of the transitions occuring in acidic solution appear to be irreversible, or very slowly reversible. Such is the case of the formation of poly (A + U). We have shown that this effect is due to the structure poly (A + A). When the dissociation temperature (TmA) of this structure is higher than that of poly (A + U), it is necessary to Porto two A — U pairs replacing one A — A pair in order to obtain the free energy decrease. Therefore, such a reaction cannot occur in two steps, and this suggests a third order mechanism. Another example is given by the rearrangement of poly (A + U) into poly (A + 2U) and poly (A + A). This observation leads us to a general rule concerning the possibility of reorganization in a system of polynucleotides where several associations are possible [for instance it answers the question as to why poly G cannot expel poly I from poly (I + C)]. [ABSTRACT FROM AUTHOR]

The enzymatic synthesis of the polyribonucleotide ApUp(ApUp)n by the use of RNA polymerase is described. Snake venom and spleen phosphodiesterase degrade this polymer at a very slow rate compared to poly r(A—U). The dinucleotide resulting from digestion with pancreatic ribonuclease is resistant to spleen phosphodiesterase. [ABSTRACT FROM AUTHOR]