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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]

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]

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]

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]

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]

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 complexes of poly A and poly U are interesting models for studying the structure of nucleic acids. In the first part of this paper, the thermal stability of these complexes has been studied as a function of both pH and ionic strength in alkaline solution. The thermal stability is measured by the half transition temperatures, or Tm's. There are four different kinds of transitions: Tm2-1 refers to the dissociation of the two-stranded complex, poly (A + U); Tm2-1 to the dissociation of the three-stranded complex, poly (A + 2U) [4]. Tm8-2 is the temperature of half dissociation of poly (A + 2U) into poly (A + U) [1], and Tm2-3 is the temperature of rearrangement of 2 poly (A + U) into poly (A + 2U) [3] (Fig. 1). It is found that Tm2-1, and Tm8-1 decrease as pH increases above neutrality (Fig. 2 and 3), but that Tm2-8 does not vary (Fig. 3). This effect is readily explained if it is assumed that the ionisation of uracil brings a change in the state of the free poly U, but that the complexes themselves are not modified. In the second part of the paper a simple thermodynamic formulation is given which represents satisfactorily the relationship between the four equilibria, as observed at neutral pH (Fig. 1). A more complex treatment is then presented. It includes quantitation of electrostatic interactions, according to the theory of Kotin [18] and of the free enthalpy term corresponding to the organisation (stacking) of poly A. This leads to an excellent representation of the experimental data (Fig. 5). In alkaline solution, one can calculate the free enthalpy change associated with the ionisation of poly U at the Tm. According to our observations, it is likely that this is the only factor responsible for the decrease of Tm. Therefore, we can write that the sum of the free enthalpy of complex formation in the same conditions, but at pH 7, and of the free enthalpy of ionisation is zero. This leads to a determination of ΔG both as a function of temperature and ionic strength: the variation of ΔG with these factors is shown to be in good agreement with our detailed model. However, the absolute values are different, probably because of uncertainties concerning the data for poly A stacking enthalpy and entropy. Apart from providing evaluation of the thermodynamic parameters for double and triple helix formation, our results lead us to emphasize the importance of the thermodynamic state of the unassociated polynucleotides. This may be particularly relevant when one considers the partially complementary secondary structures of RNA's. [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]

The antibiotic, chlorothricin, inhibits the reaction catalyzed by pyruvate carboxylase from Bacillus stearothermophilus. With steady-state kinetic measurements the following effects can be observed. (a) Inhibition of the overall reaction is competitive with respect to CoASAc, the allosteric activator of this enzyme, and non-competitive with respect to both MgATP and pyruvate (substrates). (b) Chlorothricin raises the Hill coefficient, n, of CoASAc from 2 to nearly 3. On the other hand, interaction of the antibiotic with enzyme is characterized by n = 3. (c) The Michaelis-Menten-type kinetic of MgATP is changed to a kinetic showing substrate inhibition, while the substrate activation of pyruvate carboxylase by pyruvate is suppressed upon the addition of chlorothricin. The data presented in this paper strongly suggest that the conformation of site 1 of pyruvate carboxylase responsible for the regulation of the overall enzyme activity is influenced by chlorothricin and CoASAc in an antagonistic manner. A model is proposed which assumes the existence of hybrid enzyme forms. [ABSTRACT FROM AUTHOR]

This paper reports some physico-chemical and functional properties of two of the isolated hemoglobin components from trout (Salmo irideus) blood (Hb trout I and Hb trout IV). Both hemoglobins in the oxygenated state are tetrameric at pH between 7 and 7.8 and protein concentration above &assymp; 0.1 mg/ml. The tetramer is extremely stable towards dissociation into subunits and the stability constants, estimated by differential gel filtration, are between 10 and 50 times larger than that of human hemoglobin in phosphate buffer. A very small degree of dissociation is also suggested by the very small extent of hybrid formation even in concentrated KI or triethanolamine. The dichroic properties of both Hb trout I and Hb trout IV show distinct features in the wavelength region between 300 and 200 nm. Hb trout IV resembles human hemoglobin in the shape of the CO spectrum and in the values of the reduced ellipticity (θ); furthermore upon deoxygenation the value of θ at 222 nm becomes more negative by about 50%. On the ether hand Hb trout I shows a different circular dichroism spectrum and in particular the ellipticity at 222 nm is lower than that of Hb trout IV. Moreover the spectrum is insensitive to the presence of heme ligands. Hb trout IV binds human haptoglobin (Hp) as shown by quenching of the fluorescence of the tryptophanyl residues of Hp upon addition of hemoglobin. The apparent stoichiometry of complex formation corresponds to two Hb tetramers per Hp molecule (while for human hemoglobin the stoichiometry corresponds to &assymp; one tetramer per Hp). A clear cut effect of Hp binding is observed on the kinetics of O2 and especially CO combination as studied by flash photolysis. The effect of several third components on the O2 equilibrium of both Hb trout I and IV has been investigated. In general the effect of adding ions or other molecules to a solution of lib trout I is very minor. On the other hand Hb trout IV is more susceptible to changes in solvent composition. Of particular importance is the action of ATP, the physiological organic phosphate, which is without effect on Hb trout I, while it has a clear effect on Hb trout IV. The addition of ATP shifts the Root effect, the characteristic property of Hb trout IV, to higher pH values, thereby acting as one of the allosteric effectors. [ABSTRACT FROM AUTHOR]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

Two hybrid species of haemoglobin M Iwate exist: α2Mmetβmetβdeoxy and α2Mmetβ2deoxy. These species differ in their ligand and effector binding properties. The α2Mmetβmetβdeoxy hybrid is characterized by a Bohr effect, while the Hill coefficient is n = 1.00. The energy of the interaction between the Bohr protons and the ligand-bindlng site corresponds to the interaction energy found in monomeric haemoglobins and amounts to 0.55 kcal/mol. One molecule of 2,3-bisphosphoglycerate per mole is bound at neutral pH, while at alkaline pH two co-operative molecules are bound. Binding of 2,3-bisphesphoglycerate reduces the ligand affinity and shifts the pK value of the Bohr proton binding site to the alkaline region. The α2Mmetβdeoxy hybrid shows a pH-dependent co-operativity of the ligand-binding sites (n = 1.8 at pH 9). The co-operativity is weakened by binding of protons or 2,3-bisphosphoglycerate. The latter lowers the ligand affinity, but in the alkaline region no influence is observed. Furthermore the Bohr effect curve is shifted to the alkaline region by this compound but the amplitude remains unchanged. The results of the present paper are interpreted in terms of intra-chain and β-β inter-chain intereactions, the latter being responsible for a consecutive change of the ligand affinity in a “frozen” T-state molecule. [ABSTRACT FROM AUTHOR]

We have further characterized the polynucleotide ligase purified from cultures of the human heteroploid line EUE [Spadari, Ciarrocchi and Falaschi, Eur. J. Biochem. 22, 75 (1971)]. The 350-fold purified enzyme gives positive response with four different assays; the rates with the different substrates are different, but the variations are identical to those observed with the purified ligase induced by T4-phage. The enzyme can reconstitute the transforming activity of Bacillus subtilis DNA inactivated by pancreatic DNAase. The human cell enzyme is unable to use a hybrid substrate where an interrupted polydeoxynucleotide is annealed to a polyribonucleotide, whereas in the same conditions the T4 enzyme gives appreciable activity. The partial dependence of the purified enzyme on proteins present in a boiled crude extract, reported in the previous paper, seems due to an aspecific protective effect of the soluble proteins of cell extracts. The enzyme does not show any appreciable sequence specificity in the phosphodiester bond it can form. The purified enzyme can be fractionated into two molecular forms, one having a molecular weight of 190000, the other 95000; fresh extracts of EUE cells contain almost exclusively the high molecular-weight form; ageing of the extract or purification lead to the appearance of the second peak, without variations in the total activity. This could correspond to the conversion of a dimer structure into a monomer. The “dimer” has a pH optimum close to 8.1, whereas the “monomer” has its optimum at 7.5; this explains the bimodal pH curve previously described in the purified enzyme. [ABSTRACT FROM AUTHOR]

Binding of tryptophan, tryptamine and ATP to tryptophanyl-tRNA synthetase was studied by equilibrium dialysis experiments. There arc two binding sites per mole of enzyme both for tryptophan and for tryptamine. Ks for tryptophan is 0.95 μM and for tryptamine is 1.8 μM. In the case of ATP no binding could he measured over the range of concentrations examined. The Scatchard plots of tryptophan and tryptamine binding do not shown any cooperativity between the subunits. Dissociation of the dimeric enzyme was studied at very low protein concentration. Upon dilution of the enzyme both the [32P]PPi-ATP isotope exchange activity and the tRNA charging activity are lost simultaneously. Kinetic evidence demonstrates that the inactivation is primarily due to the dissociation of the active direct into inactive monomers. The dissociation is strongly promoted by alkaline pH and is inhibited by the presence of tryptophan or tryptophanyladenylate but not by that of tRNA. The dissociation constant of the dimer-monomer equilibrium at pH 8.5 is 15 nM at 25 °C. The dissociation form is more susceptible to denaturation than the dimeric species. The results reported in the paper suggest that even if there are not binding interactions between the subunits, the active conformation of the enzyme depends on their associated state. [ABSTRACT FROM AUTHOR]

The primary structure of porcine luteinizing hormone α-subunit was established by determination of the amino acid sequence of its tryptic peptides, which were aligned by homology with the known sequence of ovine luteinizing hormone α-subunit. Its polypeptide chain is shorter by six amino acid residues at its amino-terminus, when compared to the ovine α-subunit. Furthermore four amino acid replacements are observed in the porcine as compared with the ovine chain. Heterogeneity of both carbohydrate prosthetic groups of porcine luteinizing hormone α-subunit is indicated by the different sugar compositions of the glycopeptides isolated in the course of this work. [ABSTRACT FROM AUTHOR]

The sequences of the 119 amino acids of bovine and porcine luteinizing hormone β-subunits were determined, 17 amino acid replacements being observed between these species. Variability in the primary structure was observed for porcine β-subunit, in that position 10 is occupied by both arginyl and glutamyl residues. No free amino terminal residue was detected for both porcine and bovine β-polypeptide chains and both exhibited carboxy-terminal heterogeneity. The homology of the β-subunits of luteinizing hormone from various species and the β-subunit of the bovine thyroid-stimulating hormone is discussed as the subunit of both hormones can combine with a similar α-subunit. [ABSTRACT FROM AUTHOR]

The αA2 chain of bovine α-crystallin was fragmented by means of cyanogen bromide treatment and by tryptic, chymotryptic and thermolytic digestions. Twenty tryptic peptides were obtained from the S-aminoethylated αA2 chain, accounting together for the complete sequence. The direct Edman degradation and the dansyl-Edman technique were used to determine the sequences of the tryptic peptides. The order of the tryptic peptides was deduced from overlapping peptides obtained by cyanogen bromide treatment, tryptic digestion of the maleylated chain and chymotryptic and thermolytic digestion of the S-aminoethylated chain. The sequence of the αA2 chain comprises 173 residues and corresponds to a molecular weight of 19832. [ABSTRACT FROM AUTHOR]

The object of this paper is to analyse the effects of the coenzymes NAD(P)+ and NAD(P)H, and the effectors ADP and GTP, on the polyhexameric structure of pig-liver dehydrogenase. The linear polymerisation model proposed by Eisenberg for the native quaternary structure of this protein is valid with any effector; the observed variations of the degree of polymerization are explained by the modification of the apparent association constant of the hexamers. The appendices I and II define the free and associated areas and give, the theoretical foundations of the variation of the association constant of hexamers in terms of the binding of the ligands to the protemers. The increase in the degree of polymerization of the glutamate dehydrogenase with the binding of NAD(P)H is explained by a higher affinity of the coenzymes for the protomers which have an associated area compared to the protomers which have a free area. No variation is observed with NAD(P)+, ADP, or GTP alone. The formation of the protein · GTP · NAD(P)H ternary complex leads to a complete depolymerization when the two ligands are in saturating concentrations. The systematic study of the variations of polymerization in terms of increasing concentration of NAD(P)H at constant concentration of GTP, or in terms of increasing concentration of GTP at constant concentration of NAD(P)H shows that the interaction between the two opposite protomers of two consecutive hexamers is responsible for the sigmoidal shape of the depolymerization curves. The reversibility of this effect by ADP is assigned to a competition between the binding of ADP and the binding of GTP. [ABSTRACT FROM AUTHOR]

The intracellular distribution of ATP, 2,3-bisphosphoglycerate(P2-glycerate) and Mg2+ was calculated for the oxygenated and deoxygenated human erythrocyte for the normal range and that of pathophysiological variations based on the association constants of the relevant complexes. The data indicate that about 20% of ATP is bound both in the oxygenated and deoxygenated cells, while 39 and 73% of P2-glycerate is bound under these conditions. An increase of the free Mg2+ concentration from 0.7 to 1.1 mM is produced by complete deoxygenation of haemoglobin. The calculations would indicate that during deoxygenation of haemoglobin the hexokinase reacts to the increased concentration of the activator Mg2+ and the decline of the inhibitor P2-glycerate with an elevation of its activity which corresponds to experimental data on intact erythrocytes. The P2-glycerate formation rate in deoxygenated cells is stimulated about 2.5 times in comparison to oxygenated cells as estimated from the free concentration of P2.glycerate and the kinetic constants of bisphosphoglycerate mutase. An assessment of the possible influence of other anions including bicarbonate shows that the distribution of the species of ATP, P2-glycerate and Mg2+ are changed by less than 20%. Together with the data presented in the accompanying paper, the results given here indicate that the constants and estimates are approximately valid for intracellular conditions. [ABSTRACT FROM AUTHOR]

A NADPH-cytochrome c reductase (called Fraction II) has been solubilized with Triton X-100 from porcine liver microsomes and purified. 1. Fraction II can reconstitute with a cytochrome P-450 fraction (called Fraction I) solubilized with Triton X-100 from porcine kidney cortex microsomes to exhibit laurate ω-hydroxylation activity. 2. When the purified Fraction II is subjected to disc electrophoresis on polyacrylamide gel in the presence of 0.1% Triton X-100, it shows a single hand. In contrast, on electrophoresis in the absence of Triton X-100, a polydisperse state is obtained. Fraction II is estimated to have a molecular weight of about 400000 by gel-filtration with detergent, and it does not contain iron. 3. Either gel filtration on a Sephadex G-100 column or treatment with trypsin at 4 °C results in a conversion of Fraction II to a form, which migrates at a much faster rate on disc gel electrophoresis than dose Fraction II. This fast-moving form (called Fraction IIf) is indistinguishable from either steapsin or trypsin-solubilized NADPH-cytochrome c reductase, judging from several criteria including electrophoretic and sedimentation behaviors, and molecular size. 4. Fraction IIf is reconstitutively inactive in the hydroxylation, unless supplemented with spinach ferredoxin-NADP reductase. Incubation of Fraction IIf with trypsin at 30 °C results in a remarkable enhancement in the reconstitution activity in the presence of ferredoxin-NADP reductase. The results in this paper along with an earlier report [11] suggest that Fraction II may contain two different electron carrier components, possibly participating in the electron transfer from NADPH to cytochrome P-450. [ABSTRACT FROM AUTHOR]

Degradation studies performed on the original or dephosphorylated core oligosaccharides derived from lipopolysaccharides of Salmonella Ra, Rb, and RcP+ mutants revealed the presence of a branched heptose trisaccharide, -3(HepIII-1,7-)-HepII-1,3-HepI-. The branching heptose III is linked to position 7 of heptose II. Heptose II is also substituted (at either position 2, 4, or 6) by a phosphate group. Other previously identified substituents of the heptose units are a pyrophosphoryl-ethanolamine group linked to position 4 of heptose I and the hexose oligosaccharide of the core linked to position 3 of heptose II. The degree of substitution of the heptose main chain with heptose III varies in different lipopolysaccharides from 20 to 90%. A lipopolysaccharide of chemotype RcP- was analyzed and found not to contain the third heptose unit. [ABSTRACT FROM AUTHOR]

N-terminal acetylation of the lens protein α-crystallin has been studied in a lens cell-free system. This system is capable of synthesizing de novo α-crystallin, a protein with acetylmethionine as N-terminus. When [35S]Met-tRNAfMet is used as radioactive precursor only the α-crystallin chains are labeled in the N-terminal position. This phenomenon enables the study of the process of acetylation. Newly synthesized polypeptides could be separated according to chain length. Electrophoretic analysis of thermolytic peptides revealed that acetylation of the N-terminus occurs while the chain is still on the ribosome. It is concluded that the N-terminal acetylation takes place after chain initiation and before completion of the polypeptide chain. [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]

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]

The de novo synthesis of α-amylase in barley endosperm and isolated aleurone layers is induced by 3',5'-cyclic purine mononucleotides and gibberellic acid. The induction of α-amylase by cyclic purine mononucleotides is prevented by 2,4-DNP, inhibitors of RNA and protein syntheses, CCC, AMO-1618 and phosfon. The induction of a-amylase formation by 3',5'-cyclic purine mononucleotides, but not by gibberellic acid, is also blocked by inhibitors of DNA synthesis. Extracts from cyclic AMP-treated endosperm halves exhibit a characteristic gibberellin-like activity which is detectable within 12 hours from the addition of the cyclic AMP. On paper chromatograms this gibberellin-like activity is located at the Rf typical for GA3. Its formation is prevented by inhibitors of DNA synthesis, CCC and AMO-1618. Glucose inhibits the formation of α-amylase induced by gibberellic acid. Glucose has no effect on the cAMP-induced gibberellin biosynthesis. The evidence shows that the cyclic purine mononucleotides induce DNA synthesis, which results in gibberellin biosynthesis, which in turn activates the synthesis of α-amylase. [ABSTRACT FROM AUTHOR]

Certain 6-acylaminopurines have been shown to exhibit activity in several cytokinin bioassays. The active compounds included 6-N,2'-O-dibutyryladenosine 3' : 5'-cyclic monophosphate, but adenosinc 3' : 5'-cyclic monophosphate was inactive. The metabolites formed from [2,8-3H]6-benzoylaminopurine by radish seedlings and excised radish cotyledons were investigated. When compared with zeatin, this amide showed considerable stability in vivo. Conversion to 6-benzylaminopurine and its riboside was not detected but slight degradation to adenine was indicated. The principal metabolite was an unidentified compound. [ABSTRACT FROM AUTHOR]

Glycine-2-C14, hypoxanthine-8-C14, and urea-C14 were fed to both leaf disks and sterile root cultures of Symphytum uplandicum (Nyman). In both tissues, hypoxanthine-8-C14 was largely converted to allantoin, in which the distribution of radioactivity suggested its formation by way of a symmetrical intermediate. In leaf disks, the conversion of glycine-2-C14 to allantoin was at most very slight. In roots, it was partially converted to allantoin in either tissue. It is concluded that in roots allantoin is synthesised from glycine through purine intermediates. [ABSTRACT FROM AUTHOR]

The present paper describes the effect of (+)-biotin on the metabolism of a freshly isolated strain of Desulphovibrio desulphuricans. In proliferating pure cultures of this Zürich strain on a synthetic substrate with ammonium chloride, lactate and sulphate an oxido-reduction process takes place according to the stoichiometric relation established by Baars (1930). In the medium mentioned (+)-biotin was shown to have favourable action when added in comparatively small amounts, whereas in presence of greater amounts of the vitamin a marked inhibition was observed. The maximum stimulation is induced at a concentration of 25 jig. of biotin per litre, while a pronounced inhibition sets in when the vitamin concentration is increased from 100 to 250 μg. per litre. It is proposed that the beneficial effect of biotin might be related to the action of the vitamin in the synthesis of aspartic acid and other amino acids from pyruvic acid, carbon dioxide and ammonia through Wood-Werkman condensation, amination and transamination. [ABSTRACT FROM AUTHOR]

The study of the structure of the peptides arising from native, oxidized or reduced and maleinized bovine growth hormone on incubation with trypsin, ehymotrypsin and pepsin is reported. The data obtained permitted the assembly of a unique sequence of amino acids for the poly-peptide chain of the protein. Various corrections and one addition to the sequence previously communicated are made. [ABSTRACT FROM AUTHOR]

After the recent investigation on the biochemical properties of the 2-keto-3-deoxy-gluconokinase and 2-keto-3-deoxy-6-phospho-gluconate aldolase in Escherichia coli K12, we report in this paper the induction mechanism of these two enzymes and the genetic control of a new enzymatic activity: the 2-keto-3-deoxy-gluconate transport system. The D-glucuronate and D-galacturonate raise the high basal levels of the kinase and aldolase on glycerol by a factor of 5 and 3-4, respectively, the kinase and aldolase being enzymes of the common degradative pathway of D-glucuronate and D-galacturonate. The differential rates of synthesis of these two enzymes are constant from the addition of the galacturonate. Under a variety of conditions (different inducers and substrates of growth) kinase and aldolase have been found to be synthesized non coordinately. The strongest decoordination is carried out by gluconate: this compound which is a good inducer of the aldolase, not only is unable to induce the kinase but also represses it. Moreover, the aldolase seems to be less sensitive to the catabolic repression than the kinase. By means of appropriate negative mutants we have established that the two hexuronates and two keto-uronates do not induce directly kinase and aldolase, but by sequential conversion into 2-keto-3-deoxy-gluconate. Furthermore, the fact that the hexuronates induce the kinase in aldolase-negative mutants (kdg A) and besides, "over" induce the aldolase in kinase negative mutants (kdg K) strongly suggests that, 2-keto-3-deoxy-gluconate is a true inducer for both enzymes. In addition, the gluconate which still induces the aldolase in an edd strain suggests that, this compound and/or the 6-phospho-gluconate are/is also inducer(s) of this enzyme. Moreover, we have isolated spontaneous mutants able to utilize the 2-keto-3-deoxy-gluconate as a unique carbon source. Two classes have been identified. The mutants of these classes which arise at high frequency (10-5 to 10-6) are either simultaneously constitutive for a specific 2-keto3-deoxy-gluconate transport system, kinase and aldolase (class kdg Rc) or only constitutive for the transport system (class kdg Pc). The 2-leto-3-deoxy-gluconate as exogenous substrate, which cannot induce its transport system in the wild type, behaves like a non-inducer substrate. So the strains kdg Rc and kdg Pc are respectively i- and oc lac-like mutants. These findings and the fact that we have found thermosensitive mutants mapping in the kdg R locus (34.5 min) and which are simultaneously derepressed for the permease, kinase and aldolase at 42 °C but not at 28 °C, strongly suggest that the synthesis of the three sequential enzymes degrading the 2-keto-3-deoxy-gluconate is negatively controlled by a common regulator gene product. The decoordinated syntheses of these three enzymes are in agreement with the scattering of the corresponding operons on the chromosome: transport system operon (76.5 min), kinase operon (69 min), aldolase operon (34 min). [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]