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The electrophoretic behaviour of oligodeoxynucleotides on ion-exchange papers was studied systematically with particular attention to applications in sequence analysis. Complex mixtures of larger oligonucleotides were fractionated by electrophoresis on cellulose acetate followed by t.l.c. on polyethyleneimine-cellulose. The behaviour of the oligonucleotides on polyethyleneimine-cellulose and on the two-dimensional system can provide a useful guide to their sequence. Detailed results from some of these experiments have been deposited as Supplementary Publication SUP 50031 (13 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

1. The peptides obtained by tryptic digestion of S-[14C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.

1. Triose phosphate isomerase was prepared by chromatography on DEAE-cellulose of an (NH4)2SO4 fraction of an extract of homogenized chicken breast muscle. The product is homogeneous on gel electrophoresis and is suitable for growing crystals for X-ray work. The specific activity is 10000 units/mg and the value for E0.1%280 is 1.20. 2. Comparison between the sum of the amino acid compositions of the tryptic peptides of the protein and the amino acid composition obtained on total hydrolysis of the protein indicates that the relative subunit mass is about 27000. 3. These data, together with the results of the examination of the amino acid compositions of a number of minor peptides, the number of peptides in the tryptic digest and the complete amino acid sequences of the tryptic peptides (the determination of which is described here), give no indication that the subunits are dissimilar. 4. A tentative amino acid sequence is presented for the protein, in which the ordering of the tryptic peptides is derived by homology with the sequence of the rabbit muscle enzyme (Corran & Waley, 1973). 5. An appendix describes the use that was made of mass spectrometry in the determination of some of the sequences. Mass-spectrometric data have been obtained for 35 residues, that is about 15% of the total sequence of the protein. 6. An extended version of the present paper has been deposited as Supplementary Publication SUP 50025 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

1. UDP-glucose dehydrogenase (EC 1.1.1.22) was extracted from epiphysial-plate cartilage of newborn pigs and from whole bovine corneas. 2. Formation of UDP-glucuronic acid was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c.: in these conditions a radioactive glucuronic acid spot also appears. 3. UDP-xylose prevented the formation in the incubation mixture of both UDP-glucuronic acid and free glucuronic acid. 4. In both tissues the dependence of the enzyme activity on pH and the Km values for UDP-glucose and NAD+ were determined. 5. Inhibition by UDP-xylose with respect to UDP-glucose was investigated. The plots of 1/v versus 1/[UDP-glucose], and of percentage inhibition versus UDP-xylose concentration and the Hill coefficient showed that a co-operative effect existed between UDP-xylose-binding sites. 6. The physiological meaning of the different affinities of cartilage and cornea enzymes for UDP-xylose is discussed and related to the different glycosaminoglycan contents of the two connective tissues studied.

1. This paper deals with the identification of three O-methyl sugars in lipopolysaccharides isolated from strains of the Gram-negative photosynthetic family Rhodospirillaceae. In addition to the previously described 3-O-methyl-l-xylose, a second O-methyl sugar was encountered in the lipopolysaccharide of Rhodopseudomonas viridis F, namely 3-O-methyl-d-mannose. The lipopolysaccharides of two strains of Rhodopseudomonas palustris (strain 1e5 and 8/1) contain two O-methylsugars, 4-O-methyl-d-xylose and 3-O-methyl-6-deoxy-d-talose (d-acovenose). 4-O-Methyl-d-xylose, but not 3-O-methyl-6-deoxy-d-talose, could be identified in the lipopolysaccharides of the strains K/1 and 2/2 of the same species. 2. The O-methyl sugars described in this communication were isolated by paper chromatography and identified by g.l.c., paper chromatography, high-voltage electrophoresis and mass spectrometry. Besides the genuine sugars, their alditol acetates and their demethylated (parental) forms were investigated. Optical rotation measurements and, in one case, enzymic reactions were used to establish the optical configuration of the sugars under investigation.

1. Microsomal and soluble RNA of rat liver have been studied by column and paper chromatography after administration of [Me−14C]methionine; evidence was obtained for the occurrence of 7-methylguanine, the methyl group being derived from methionine. 2. No evidence was obtained for the occurrence of 7-methylguanine in DNA.

1. A highly active and electrophoretically homogeneous dipeptidase was purified from the soluble extracts of monkey small-intestinal mucosa. 2. By gel-filtration studies the molecular weight of the enzyme was found to be 107000. It is composed of two identical, subunits of molecular weight 54000. 3. A paper-chromatographic method of dipeptidase assay was developed to overcome some of the difficulties encountered in the generally used spectrophotometric procedure. By using this method, the Km and k0 values of a few substrates were determined. 4. The substrate specificity of the enzyme was investigated in great detail with substrates of a wide range of possible structural types. The enzyme hydrolyses a very large proportion of the range of dipeptides tested. This enzyme, which exhibits such a wide range of action, has been termed the ‘master’ dipeptidase of the intestine. Glycylglycine, glycyl-l-proline, glycyl-l-histidine, l-prolylglycine and some of the arginine- and aspartic acid-containing dipeptides were not substrates and are possibly hydrolysed by other peptidases. These results therefore suggest that in the intestine the number of dipeptidases is rather limited. 5. In the light of these findings, the implications on the role of dipeptidases in intestinal peptide transport are discussed.

1. Human skin epithelium and human skin were found to absorb both free bilirubin and serum-bound bilirubin from an aqueous buffered medium. The serum-bound bilirubin thus absorbed was readily released when human skin epithelium or human skin were transferred to media containing no bilirubin. 2. The K(m) values for serum-bound bilirubin were 1.8x10(-3)m and 2.2x10(-3)m respectively for human skin epithelium and human skin; corresponding K(m) values for free bilirubin were 3.0x10(-4)m and 5x10(-4)m. The V(max.) for bound and free bilirubin was of the same magnitude, the apparent V(max.) being 1.0 and 1.66mumol/g of tissue for human skin epithelium and human skin respectively. 3. When human skin that had acquired a yellow tinge by absorbing bilirubin was incubated in a buffered medium and exposed to a mercury-vapour light, the yellow colour disappeared and decomposition products of bilirubin accumulated in the medium. 4. Experiments with [(3)H]bilirubin indicated that the pigment absorbed by skin was photo-oxidized to products that were soluble in water and the quantity and number of such products increased with the time of exposure of human skin to the light-source. Under similar conditions [(3)H]bilirubin alone in buffered medium was also oxidized and gave products which by paper chromatography appeared to be different from those released by human skin that had absorbed bilirubin. 5. The results suggest that by virtue of its large surface area human skin can act as a matrix for the degradative action of light on bilirubin.

It has been previously observed that rabbit erythrocyte cell surface galactosyltransferase appears to play a role in concanavalin A agglutination of these erythrocytes (Podolsky et al., 1974). Further, a correlation between the occurrence or level of cell surface galactosyltransferase and concanavalin A agglutinability of other cell types has also been observed. The mechanism by which rabbit erythrocyte galactosyltransferase participates in concanavalin A agglutination has now been further defined. The enzyme was solubilized and purified. Characterization of the enzyme properties has shown them to be similar to those reported for other purified galactosyltransferases. Amino acid and carbohydrate analysis showed a high asparagine content and the presence of D-mannose. Specific α-mannosidase treatment of the enzyme showed that some of these D-mannose residues were terminal sugars. The purified enzyme also conferred concanavalin A agglutinability to non-agglutinable human erythrocytes. However, the ability to confer concanavalin A agglutinability was unrelated to the enzyme activity per se (as measured with fetuin acceptor) but appeared to be entirely dependent on the presence of terminal α-linked D-mannosyl residues in the enzyme structure. These findings suggest that the presence of terminal α-mannosidyl residues on cell surface glycoproteins such as galactosyltransferase may be the determining factor in agglutination of cells by concanavalin A.

1. 18-Hydroxyoestrone was reduced by NaBH(4) in methanol, giving 18-hydroxyoestradiol-17alpha and 18-hydroxyoestradiol-17beta in the ratio 3:7. 2. Treatment of 18-hydroxyoestrone with a strong alkali yielded 18-noroestrone; however, the 18-hydroxyoestradiols did not undergo transformation to their respective 18-nor derivatives. 3. All the 18-hydroxylated oestrogens were stable under acid conditions. They formed Kober chromogens: the chromogenicity of 18-hydroxyoestrone was only one-third that of the 18-hydroxyoestradiols and oestriol. 4. Paper-, thin-layer- and gas-liquid-chromatographic systems for the characterization of these compounds are described. 5. An examination of the mass spectra revealed peaks characteristic of the substituted carbon atoms. Definite assignment of the 17alpha- and 17beta-hydroxyl groups of the epimeric 18-hydroxyoestrogens was possible by characteristic fragmentation of the free steroids. Further, the configuration of 18-hydroxyoestradiol-17beta was confirmed by the formation of the dimethylsildioxy derivative of the 3-methylether of the steroid. 6. Both rat and rabbit liver slices reduced 18-hydroxyoestrone to 18-hydroxyoestradiol-17beta and some other labile, polar metabolites with properties similar to 2-hydroxylated oestrogens. No formation of 18-hydroxyoestradiol-17alpha in vitro was observed. 7. The results are discussed with respect to the possible influence of the 18-hydroxyl group on reactions at C-17, as well as the reactions of 18-hydroxylated oestrogens with strong acid (Kober reactions) and alkali.

1. Sulphate-dependent PP(i)-ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP(2-) and MgP(2)O(7) (2-); ATP sulphurylase activity was not correlated with the concentration of free Mg(2+). 2. Sulphate-dependent PP(i)-ATP exchange was independent of PP(i) concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PP(i)-ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [(32)P]ATP from [(32)P]PP(i) and adenosine 5'-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PP(i) and adenosine 5'-sulphatophosphate. 5. The synthesis of ATP from PP(i) and adenosine 5'-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PP(i) and adenosine 5'-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5'-sulphatophosphate and non-competitive with respect to PP(i). It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP(2-) was the first product to react with the enzyme and MgP(2)O(7) (2-) was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5'-sulphatophosphate could not be demonstrated.

1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.

The amino acid sequence of the cytochrome c′ from Alcaligenes sp. N.C.I.B. 11015 (Iwasaki's ‘Pseudomonas denitrificans’) has been determined. This organism is the only non-photosynthetic bacterium in which the protein has been found. The protein consists of a single polypeptide chain of 127 residues, with a single haem covalently attached to two cysteines. Unlike normal cytochromes c, the haem attachment site is very close to the C-terminus. The amino acid sequence around the haem attachment site is very similar to that of Chromatium vinosum D cytochrome c′. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50022 at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

Lipid A fractions from Pseudomonas aeruginosa and Pseudomonas alcaligenes have similar compositions and structural features. By means of hydrazinolysis of the parent lipopolysaccharides and partial hydrolysis of the deacylation products, it was established that both lipids are derived from the β-(1→6)-linked disaccharide of glucosamine. Phosphorylated derivatives of the disaccharide from Ps. aeruginosa were also characterized. The lipids differ mainly in the absence of hexadecanoic acid and 2-hydroxydodecanoic acid from the lipid from Ps. alcaligenes. Evidence that in Ps. aeruginosa these acids are ester-linked to residues of 3-hydroxyalkanoic acids (including 3-hydroxydecanoic acid) was obtained. Heterogeneity of lipid A fractions was indicated by t.l.c., and by gel filtration of de-O-acylation products from mild alkaline methanolysis of the lipids.

A heparan sulphate sulphotransferase was partially purified from an ox lung homogenate by (NH(4))(2)SO(4) precipitation. Various glycosaminoglycans were assayed as sulphate acceptors with this enzyme. The highest acceptor activity was obtained with desulphated heparin and heparan sulphate, which indicates that sulphate transfer may be to free amino groups of the substrate. Some heparan sulphate was (35)S-labelled by incubation with the enzyme and re-isolated. On treatment of this heparan [(35)S]sulphate with nitrous acid and separation of the degradation products on Sephadex G-15, a major peak of radioactivity was obtained, and identified as [(35)S]sulphate by high-voltage electrophoresis at pH5.3. The [(35)S]sulphate is believed to be derived from N-[(35)S]sulphated groups of heparan [(35)S]-sulphate. That the ox lung preparation contained an N-sulphotransferase was confirmed by the isolation of 2-deoxy-2-[(35)S]sulphoamino-d-glucose as the major product from the flavobacterial degradation of heparan [(35)S]sulphate.

Haemoglobin mRNA isolated from EDTA-treated polyribosomes has an apparent molecular weight of 120000–180000 estimated by condensation with 3H-labelled isoniazid after periodate oxidation. Analysis of the ribonuclease digests of isoniazid-labelled RNA by paper electrophoresis and column chromatography enables the amount of contaminating 18S, 7S, 5S and 4S RNA to be estimated, and a corrected molecular weight of globin mRNA as the acid is 161000 or 500 nucleotides in length. This molecule contains two groups of 3′-terminal sequences in equal yield; G-Y-A6 and G-Y-A7 in the ratio 3:2, and G-N9–16-Y-A2 and G-N9–16-Y-N3 in the ratio 3:2. The significance of these sequences is discussed in relation to the poly(A) content of globin mRNA, the specificity of the sequences, and possible function in processing and biosynthesis of mRNA.

The complete amino acid sequences of wool protein SCMKB-IIIA3 (131 residues) and a minor component SCMKB-IIIA3A (130 residues) have been determined. The proteins are mutually homologous and have free threonine as the N-terminal residue and carboxymethylcysteine as the C-terminus. The peptides used for the sequence work were obtained by trypsin, thermolysin, pepsin and chymotrypsin digestions and were fractionated by chromatography on DEAE-cellulose, gel filtration on Sephadex G-25 and G-50, paper chromatography and electrophoresis. The Edman degradation method (employing both the Beckman Sequencer and a non-automatic procedure) was used to obtain the sequences of the peptides.

Quantities of the order of 10mg of three alkali-stable trinucleotides were prepared from yeast ribonucleic acid. Analyses showed that they were of the type N1m-N2m-N3p, where m signifies 2′-O-methylation of the pentose residue. One, Am-Um-Gp, is newly described.

The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody to type III pneumococci was determined. This L chain, designated BS-5, exhibits a greater degree of homology with the basic sequence of human κ chains of subgroup I (72%) than with subgroups II and III. L-chain BS-5 differs from another L chain (BS-1), also derived from an antibody to type III pneumococci (Jaton, 1974), by eight amino acid residues, even though the chains are identical within the N-terminal 30 residues. Six of these eight substitutions are located within the three hypervariable sections of the variable half: Asn/Ser in position 31, Glu/Ala in position 55, Asx/Thr, Thr/Gly, Thr/Gly and Val/Tyr in positions 92, 94, 96 and 97 respectively. The two anti-pneumococcal L chains BS-1 and BS-5 are much more similar to each other than to an anti-azobenzoate L chain (Appella et al., 1973), from which they differ by 30 and 29 residues respectively. Of these interchanges 13–15 are confined to the three hypervariable sections, and 11 occur within the N-terminal 27 positions. The three chains have an identical sequence from residue 98 to residue 139, except for a possible inversion of two residues in positions 130–131 of the anti-azobenzoate chain.

The effect of introducing methyl groups into DNA substrates was studied by using the spleen exonuclease (EC 3.1.4.1), an enzyme which hydrolyses oligonucleotides in a sequential manner by splitting off 3′-phosphomononucleotides starting from the 5′-hydroxyl terminus. Analyses of oligodeoxyribonucleotide 3′-phosphate substrates after reaction in vitro with dimethyl sulphate demonstrated that the resultant methylation pattern differed from the previously found for native DNA, particularly with respect to the relative amounts of 1- and 3-methyladenine produced. Although after treatment with increasing amounts of dimethyl sulphate the substrate became progressively resistant to degradation by the exonuclease, the methylation products themselves were only partially responsible for the observed inhibition of enzyme activity. The incomplete degradation encountered was apparently due to the presence of apurinic sites, which arose as secondary lesions after the spontaneous release of the labile alkyl purines from the methylated substrate. Inhibition of enzyme activity appeared to be competitive, being characterized by constant values for apparent Vmax, and increased values for apparent Km. the interpretation of this, however, is complicated by the complex nature of the substrate, and these aspects are considered in some detail.

Membranes of Micrococcus lysodeikticus, Micrococcus flavus and Micrococcus sodonensis contain acidic lipomannans. Lipoteichoic acids could not be detected in these organisms, and the suggestion that they are substituted for by the lipomannans is strengthened by the chemical and physical resemblances between the two polymers. The mannans contain glycerol, ester-linked fatty acids and mono-esterified succinic acid residues, giving them both hydrophobic and charged properties. The M. lysodeikticus mannan has a chain of about 60 hexose units with two branch points, and is joined at its reducing end to the 1-position of a glycerol moiety bearing two fatty acid residues. Succinic acid on the mannan enables it to bind Mg2+ efficiently, and the polymer is firmly associated with the cytoplasmic membrane, probably by intercalation of its fatty acids with those of the membrane lipids.

1. 2-Deoxy-2-fluoro-d-galactose, 3-deoxy-3-fluoro-d-galactose, 4-deoxy-4-fluoro-d-galactose, 6-deoxy-6-fluoro-d-galactose and 2-deoxy-d-lyxo-hexose are substrates for yeast galactokinase. 2. The variation in Km values for the d-hexose derivatives was not associated with a variation in the value of Km for MgATP2- indicating that the binding of MgATP2- is not modified by the binding of the sugar substrate. 3. Donated H bonds from OH-3, OH-4 and OH-6 and an accepted H bond to OH-2 of the d-hexose are important for the binding of the sugar substrate to galactokinase. 4. Yeast galactokinase exhibits similar kinetics to the galactokinase from Escherichia coli and operates by a similar random sequential mechanism. 5. 4-Deoxy-4-fluoro-d-glucose was neither a substrate for nor an inhibitor of yeast galactokinase.

Phosphate-activated glutaminase (EC 3.5.1.2; l-glutamine amidohydrolase) purified from pig kidney and brain is activated by CoA and short-chain acyl-CoA derivatives. Acetyl-CoA is the most powerful activator (KA about 0.2mm). Acetyl-CoA is maximally effective in the absence of other activating anions such as phosphate and citrate, and at low glutamine concentrations. The negative co-operative substrate activation observed at pH7 becomes more pronounced in the presence of acetyl-CoA. Similarly to phosphate, acetyl-CoA produces at high protein concentrations a different type of activation, which is time-dependent, depends on protein concentration and is accompanied by an increase in the sedimentation coefficient. Acetyl-CoA, phosphate and citrate appear to have binding sites in common. No significant difference was observed between kidney and brain phosphate-activated glutaminase.

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic-enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their beta-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its beta-lactam ring. In Tris-NaCl-MgCl(2) buffer at pH7.7 and 37 degrees C, the rate constants for the dissociation of the antibiotic-enzyme complexes were 2.8x10(-6), 1.5x10(-6) and 0.63x10(-6)s(-1) (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [(14)C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a beta-lactam-antibiotic-destroying enzyme but did not function as a beta-lactamase. Incubation at 37 degrees C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [(14)C]benzylpenicillin-enzyme complex. The rate constants were 1.6x10(-5)s(-1) and 0.8x10(-4)s(-1) respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.

1. C-teichoic acid (C-substance) from the walls of Diplococcus pneumoniae contained free amino groups accessible to attack by nitrous acid. Treatment with nitrous acid, followed by reduction with borohydride and hydrolysis with acid, gave ribitol, glucitol and their respective phosphates. 2. Hydrolysis of the polymer with alkali followed by treatment of products with nitrous acid yielded glucose. 3. When alkali hydrolysis was followed by treatment with a phosphomonoesterase, nitrous acid degradation of C-substance yielded glucose and a disaccharide identified as 2-O-(N-acetylgalactosaminyl)-d-ribitol. 4. A partial structure for C-teichoic acid was deduced in which the order of the constituent residues and the position of phosphodiester linkages were established.

Methods of synthesizing a series of chemically-defined AMP, ADP, ATP, adenylyl imidodiphosphate and pyrophosphate derivatives suitable for affinity chromatography are extensively described. Each derivative has a single primary amino group at the end of a hexamethylene ‘spacer’ chain for attachment to CNBr-activated agarose. The synthesis of the derivative where the ‘spacer’ arm is attached directly to the 8 position of the adenine ring to produce 8-(6-aminohexyl)amino-AMP involves the direct bromination of AMP in the 8 position followed by displacement of the halogen by 1,6-diaminohexane. This monophosphate derivative can then be converted into the corresponding di- or triphosphate forms by direct phosphate condensation with carbonyl di-imidazole. A second series of adenosine phosphate derivatives with the phosphate moieties unsubstituted has been similarly prepared from N6-(6-aminohexyl)-AMP (Guilford et al., 1972). A third type of ligand has been synthesized by condensing the phosphoryl imidazolide of AMP with 6-aminohex-1-yl phosphate. This compound, P1-(6-aminohex-1-yl) P2-(5′-adenosyl) pyrophosphate, has an unsubstituted adenine ring. The synthesis of a fourth type of ligand, 6-aminohex-1-yl pyrophosphate, was done by heating 6-aminohexan-1-ol with crystalline pyrophosphoric acid under reduced pressure. The structures of the synthesized compounds were confirmed by chemical, electrophoretic and chromatographic methods and by u.v. spectrometry. The general applicability of the synthetic methods used is discussed in relation to the preparation of other affinity adsorbents. Examples are given where these derivatives have been successful in reversibly binding dehydrogenases, kinases and myosin and its proteolytic subfragments. The partial purification of rat liver glucokinase on an ADP derivative is shown.

The identity of a novel form of sialyl-lactose found in milk of the echidna (Tachyglossus aculeatus) was investigated. The sialyl-lactose yielded equimolar amounts of N-acetylneuraminic acid and lactose during mild acid hydrolysis but was resistant to the action of a bacterial neuraminidase. A viral neuraminidase hydrolysed it to lactose plus a form of sialic acid that reacted positively with thiobarbituric acid reagent but whose chromatographic mobility was greater than that of N-acetylneuraminic acid. Treatment with alkali converted the sialyl-lactose into a substance with the same chromatographic mobility as N-acetylneuraminyl-(2→3)-lactose and made it susceptible to the action of bacterial neuraminidase. The sialyl-lactose contained one mol of ester (identified as acetyl), and released one mol of formaldehyde during periodate oxidation, per mol of sialic acid. It did not contain N-glycollylneuraminic acid. These results indicate that the sialyl-lactose is N-acetyl-4-O-acetylneuraminyl-(2→3)-lactose. Echidna milk contained, in addition, a small amount of N-acetylneuraminyl-(2→3)-lactose.

1. Phenoxymethylpenicillin sulphoxide 4-methoxybenzyl ester was labelled with 3H in its 2-β-methyl group. Its specific radioactivity was 362 mCi/mmol. 2. Removal of the side chain of this compound yielded the corresponding ester of 6-aminopenicillanic acid sulphoxide and coupling of the latter with the appropriate protected α-aminoadipic acid gave 4-methoxybenzyloxycarbonylisopenicillin N sulphoxide di-4-methoxybenzyl ester or the corresponding derivative of penicillin N. 3. Removal of the protective groups by hydrogenolysis and reduction of the sulphoxide group yielded 3H-labelled isopenicillin N or penicillin N. 4. 3H-labelled phenoxymethylpenicillin sulphoxide was obtained by hydrogenolysis from its 4-methoxybenzyl ester. Reduction of its sulphoxide group and subsequent removal of the side chain gave 3H-labelled 6-aminopenicillanic acid.

Studies of crude extracts of pea seeds (Pisum sativum, var. Green feast) revealed the presence of three enzymes that hydrolyse the amide bond of aminoacyl β-naphthylamides. They differ in their specificity towards the aminoacyl moiety; one is proline-specific, whereas the other two hydrolyse the β-naphthylamides of primary amino acids. Of the latter, one is highly specific for hydrophobic aminoacyl residues whereas the other has a broader, somewhat complementary specificity, showing preferential hydrolysis of non-hydrophobic aminoacyl residues. These latter two aminoacyl-β-naphthylamidases have been separated and partly characterized with regard to substrate specificity and antagonism by inhibitors. Both are true aminopeptidases, requiring the presence of a free amino group and hydrolysing the amide bonds of amino acid amides, dipeptides and oligopeptides consecutively from the N-terminal end.

The structure of dermatan [35S]sulphate-chondroitin [35S]sulphate copolymers synthesized and secreted by fibroblasts in culture was studied. 35S-labelled glycosaminoglycans were isolated from the medium, a trypsin digest of the cells and the cell residue after 72h of 35SO42-incorporation. The galactosaminoglycan component (dermatan sulphatechondroitin sulphate copolymers) was isolated and subjected to various degradation procedures including digestion with testicular hyaluronidase, chondroitinase-AC and-ABC and periodate oxidation followed by alkaline elimination. The galactosaminoglycans from the various sources displayed significant structural differences with regard to the distribution of various repeating units, i.e. IdUA-GalNAc-SO4 (L-iduronic acid-N-acetyl-galactosamine sulphate), GlcUA-GalNAc-SO4 (D-glucuronic acid-N-acetylgalactosamine-sulphate) and IdUA(-SO4)-GalNAc (L-iduronosulphate-N-acetylgalactosamine). The galactosaminoglycans of the cell residue contained larger amounts of IdUA-GalNAc-SO4 than did those isolated from the medium or those released by trypsin. In contrast, the glycans from the latter 2 sources contained large proportions of periodate-resistant repeat periods [GlcUA-GalNAc-SO4 and IdUA(-SO4)-GalNAc]. Periods containing L-iduronic acid sulphate were particularly prominent in copolymers found in the medium. Kinetic studies indicated that the 35S-labelled glycosaminoglycan of the cell residue accumulated radioactivity more slowly than did the glycans of other fractions, indicating that the material remaining with the cells was not exclusively a precursor of the secreted polymers. The presence of copolymers rich in glucuronic acid or iduronic acid sulphate residues in the soluble fractions may be the result of selective secretion from the cells. Alternatively, extracellular, polymer-level modifications such as C-5 inversion of L-iduronic acid to D-glucuronic acid, or sulphate rearrangements, would yield similar results.

1. The preparative Edman degradation of desmosine-containing peptides permitted the isolation of peptides C-terminal to the desmosine cross-links in bovine, porcine and human aortic elastin as well as bovine ligamentum nuchae elastin. This identifies the lysines in the tropoelastin which give rise to the desmosine cross-links. 2. The sequences from bovine aortic elastin were identical with those obtained from bovine ligamentum nuchae elastin but differed from those obtained from the other species. The most striking difference involves the occurrence of phenylalanine in bovine elastin and tyrosine in porcine and human elastin C-terminal to the desmosine cross-links. 3. The sequences of the C-terminal peptides were found to fall into two distinct classes, one starting with hydrophobic residues, the other starting with alanine. It is proposed that thehydrophobic residue prevents the enzymic oxidative deamination of the adjacent lysine e-amino group and this then contributes the nitrogen to the pyridinium ring of the cross-links.

A number of parameters were used to correlate a change in control or expression of tRNA methylase activity and the transformation by adenovirus-12 in vitro. The earliest change observed corresponding to visible morphological changes was a decrease in the tRNA methylase inhibitor(s).

The amino acid sequence of plastocyanin from potato was determined. It consists of a single polypeptide chain of 99 residues, of molecular weight 10332. The sequence was determined by using a Beckman 890c sequencer and by dansyl–Edman analysis of peptides derived from purified CNBr fragments. The sequence shows considerable similarity with that of Chlorella fusca, and also with the C-terminal region of bacterial azurins.

The amino acid sequence of plastocyanin from broad bean was determined. It consists of a single polypeptide chain of 99 residues. The sequence was determined by using a Beckman 890C sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic cleavage of purified cyanogen bromide fragments. Some parts of the sequence depend on the results of Edman degradation of peptides for which amino acid analyses were not obtained. The evidence for one overlap is not strong.

1. Particulate enzyme preparations obtained from Bacillus stearothermophilus B65 by digestion with lysozyme were shown to catalyse teichoic acid synthesis. With CDP-glycerol as sole substrate the preparations synthesized 1,3-poly(glycerol phosphate). It was characterized by alkaline hydrolysis, by glucosylation to the alkali-stable 2-glucosyl-1,3-poly(glycerol phosphate) with excess of UDP-glucose and a Bacillus subtilis Marburg enzyme system, by degradation of this latter product with 60%HF and periodate oxidation of the resulting glucosylglycerol. The specificity of the B. subtilis system previously reported (Glaser & Burger, 1964), was confirmed in the present work. 2. Pulse-labelling experiments, followed by periodate oxidation of the product and isolation of formaldehyde from the glycerol terminus of the polymer, showed that the B. stearothermophilus enzyme system transferred glycerol phosphate units to the glycerol end of the chain. The transfer reaction was irreversible. It was not determined if these poly(glycerol phosphate) chains were synthesized de novo, but it was shown that the newly synthesized oligomers were bound to much larger molecules. 3. When the B. stearothermophilus enzyme system was supplied with both CDP-glycerol and UDP-glucose, 1-glucosyl-2,3-poly(glycerol phosphate) was synthesized in addition to the 1,3-isomer. The former polymer was characterized by acid and alkaline hydrolysis, degradation with HF and periodate oxidation of the resulting glucosylglycerol, and periodate oxidation of the intact polymer followed by mild acid hydrolysis. This latter procedure removed the glucose substituents without disrupting the poly(glycerol phosphate) chain. 4. The poly(glycerol phosphate) isomers were distinguished by glucosylation with the B. subtilis enzymes and alkaline hydrolysis, the 2,3-isomer remaining alkali-labile. The proportion of 2,3-poly(glycerol phosphate) in the product increased with increasing amounts of UDP-glucose in the incubation mixture, but the total glycerol phosphate incorporated into products remained constant. It is suggested that the synthetic pathways of the two poly(glycerol phosphate) species may share a rate-limiting step.

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with 32P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([14C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([14C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of 32P-labelled phosphatidylcholine or 32P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and Pi in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and Pi. The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.

1. Measurable incorporation of radioactive carbon from [U-14C]pyruvate, [U-14C]-glutamate and [14C]bicarbonate into the glycogen synthesized by brain slices in vitro was demonstrated. 2. The fructose diphosphatase activity of guinea-pig brain was determined and found to be about 0.03 μmol of substrate degraded/min per g of fresh tissue. 3. The specific radioactivity of the glucose carbon from glycogen relative to that of the precursor added to the incubation medium gave approximate values of 0.195 for glucose, 0.006 for pyruvate, 0.039 for glutamate and 0.001 for bicarbonate.

The synthesis of peptidoglycan by an autolysin-deficient β-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with β-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.

Dopamine 3-O-sulphate (3-O-hydrosulphato-4-hydroxyphenethylamine) was isolated from newly ecdysed cockroaches, Periplaneta americana (L.), and its structure established by chemical and physical techniques and by synthesis. Relatively high concentrations (about 1μmol/g wet. wt.) of dopamine 3-O-sulphate exist in the newly ecdysed insect, and these concentrations decrease sharply as sclerotization of the cuticle proceeds. At least 40% of the radioactivity of 14C-labelled dopamine 3-O-sulphate injected into newly ecdysed nymphs was recovered in the sclerotized cuticle 7–12 days after the injection. However, less than 1% of the radioactivity of injected dopamine 3-O-[35S]sulphate was recovered, and this value was not appreciably different from that for the incorporation of Na235SO4. Apparently, little or none of the sulphate moiety of dopamine 3-O-sulphate is incorporated directly into the cuticle as the intact sulphate ester. These observations are discussed in relation to current concepts of cuticular sclerotization in insects.

1. 3-Hydroxypropylmercapturic acid, i.e. N-acetyl-S-(3-hydroxypropyl)-l-cysteine, was isolated, as its dicyclohexylammonium salt, from the urine of rats after the subcutaneous injection of each of the following compounds: allyl alcohol, allyl formate, allyl propionate, allyl nitrate, acrolein and S-(3-hydroxypropyl)-l-cysteine. 2. Allylmercapturic acid, i.e. N-acetyl-S-allyl-l-cysteine, was isolated from the urine of rats after the subcutaneous injection of each of the following compounds: triallyl phosphate, sodium allyl sulphate and allyl nitrate. The sulphoxide of allylmercapturic acid was detected in the urine excreted by these rats. 3. 3-Hydroxypropylmercapturic acid was identified by g.l.c. as a metabolite of allyl acetate, allyl stearate, allyl benzoate, diallyl phthalate, allyl nitrite, triallyl phosphate and sodium allyl sulphate. 4. S-(3-Hydroxypropyl)-l-cysteine was detected in the bile of a rat dosed with allyl acetate.

A sequence of 33 nucleotides from the coliphage R17 RNA genome was determined. It constitutes the main component of a mixture of fragments that migrate together on electrophoresis in a separation according to molecular weight. Fragments of comparable chain length from 3′ end of RNA from coliphage R17, from a region preceding and overlapping the coat-protein cistron ribosome binding site and from the beginning of the A-protein cistron, were also found and characterized. ‘Hairpin’-like secondary structures are proposed for the longer fragments, one of which appears to have a tetranucleotide excised in the loop region.

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a β-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN′-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The Km value for hydrolysis of p-nitrophenyl β-2-acetamido-2-deoxy-d-glucopyranoside at 37°C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.

With a particulate enzyme preparation from Phaseolus aureus hypocotyls, UDP-α-d-[U-14C]galactose served as a precursor for a number of products. One of these products was characterized as a β-(1→4)-linked galactan. The ADP-, GDP-, TDP- and CDP- derivatives of α-d-galactose did not serve as biosynthetic precursors for any products insoluble in 70% ethanol, nor as substrates for a sugar nucleotide 4-epimerase which is present in the particulate enzyme preparation. The 14C-labelled β-(1→4)-galactan is alkali-insoluble and was characterized by analysis of partial acetolysis products. The labelling pattern of the [14C]oligosaccharides derived from acetolysis indicates that (1) only slightly more than two [14C]galactose moieties are added to the growing polysaccharide chain on average, and (2) these additions take place at the reducing end of the polysaccharide chain. The radioactive β-(1→4)-linked galactan chain represented 8.5% of the radioactivity initially added, and 20% of the water- and butanol-insoluble products derived from UDP-α-d-[14C]galactose. Total hydrolysis of the alkali-insoluble fraction of Phaseolus aureus hypocotyl yielded d-glucose and d-mannose in a 5:1 ratio but no detectable quantities of d-galactose. A trace quantity of a radioactive disaccharide, identified as (1→3)-linked galactobiose, was isolated from the partial acetolysate of the alkali-insoluble [14C]polysaccharide material. Also isolated from this partial acetolysate was a C-1 derivative of [14C]galactose, which could not be identified. An alkali-soluble galactose-containing polysaccharide was also synthesized in this enzymic reaction, and represented 20% of the water- and butanol-insoluble products derived from UDP-α-d-[14C]galactose. The spectrum of radioactive oligosaccharides produced by partial acetolysis of this alkali-soluble polysaccharide material was different from that obtained from the alkali-insoluble polysaccharide, indicating a different structure.

1. The trehalase found in an extract prepared from a yeast strain that cannot ferment trehalose was studied and characterized. The enzyme is highly specific for trehalose with Km 1·02×10−2m, and an optimum pH of 6·9. 2. It is inhibited by glucose and by trehalose 6-phosphate, and does not facilitate any significant transglucosylations. 3. pK values 7·7 and 5·8 were detected for the groups associated with binding of the non-ionized substrate to the enzyme. 4. The trehalase was found to be highly labile and was inhibited by thiol-binding reagents. 5. The possible role of this enzyme in the trehalose-dissimilation patterns in the yeast cell was evaluated.

1. The type-specific substance from Pneumococcus type 11A(43) is a polymer containing d-glucose, d-galactose, glycerol, phosphate and O-acetyl in the approximate molecular proportions 2:2:1:1:2. 2. Removal of the O-acetyl groups with ammonia gave a compound no longer active towards type 11A antiserum. 3. Treatment of S.11A with sodium borohydride, followed by hydrolysis with alkali yielded a phosphorus-free polysaccharide, whose structure was studied by methylation and by degradation with periodate. 4. Examination of S.11A and its de-O-acetyl derivative by periodate oxidation led to the partial structure (XI) for the type-specific substance, which thus has several features in common with S.18.

1. Arvin, a commercial preparation of the coagulant activity from the venom of Agkistrodon rhodostoma, is shown to contain a non-coagulant caseinolytic fraction. 2. A method is described for the purification of the coagulant enzyme free from any detectable contaminating protein. 3. The coagulant enzyme is identified as a glycoprotein which probably consists of a single polypeptide chain containing approx. 29% by weight of carbohydrate. Amino acid and carbohydrate analyses are reported and the N- and C-terminal amino acid residues identified. 4. Electrophoresis on polyacrylamide gel reveals the polymorphic nature of the glycoprotein. Five forms of the enzyme are observed. 5. The coagulant action is correlated with an arginine esterase activity and kinetic properties are studied with both arginine and lysine esters as substrates. The inhibitory nature of guanidine and arginine toward the esterase activity is reported.

The Cuvierian tubules of Holothuria forskali Della Chiaje, a sea cucumber found in the Adriatic Sea, were investigated with regard to their carbohydrate moieties. From a Pronase digest of these tubules three types of carbohydrate units were isolated and characterized. 1. A high-molecular-weight glycopeptide fraction was shown to contain sulphated polyfucose, galactosamine, a uronic acid and a previously unknown neuraminic acid derivative. The sulphate was shown by i.r. analysis to be present as an O-ester. The carbohydrate unit was linked O-glycosidically to threonine and serine residues in the polypeptide chain. The hitherto unknown neuraminic acid derivative (Hf-neuraminic acid) was resistant to enzymic cleavage by neuraminidase, even after mild alkaline hydrolysis for the removal of O-acyl residues. However, the glycosidic linkage of this compound to the other part of the carbohydrate moiety was readily cleaved by mild acid hydrolysis. Its chromatographic properties distinguished Hf-neuraminic acid from other known neuraminic acid derivatives (N-acetyl-, NO-diacetyl-, NOO-triacetyl- and N-glycollyl-neuraminic acid). Further, this acidic sugar was shown to possess neuraminic acid as its basic structure. Thus, an as yet unknown substituent lends the distinct properties to Hf-neuraminic acid. 2. The carbohydrate composition of a second glycopeptide fraction consisting of a derivative of neuraminic acid, galactose, mannose and glucosamine was similar to that of the well-known carbohydrate groups of the globular glycoproteins. 3. The third fraction contained two glycopeptides containing the disaccharide, glucosylgalactose, which was shown to be linked to the hydroxyl group of hydroxylysine residues of a collagen-like protein. Approximately half of these residues were glycosylated. In addition to these glycopeptides, a small amount of a third glycopeptide that carried only a galactosyl residue was detected. The amino acid sequence of the two major compounds were found to be Gly-Ala-Hyl*-Gly-Ser and Gly-Pro-Hyl*-Gly-Asp, where Hyl* represents a glycosylated amino acid residue.

Amino acid analysis and terminal-group analysis of tryptic and chymotryptic peptides from sheep haemoglobin A have enabled a partial amino acid sequence to be worked out. By comparing this partial sequence with the known amino acid sequences of human haemoglobins A and F as well as horse slow haemoglobin the most probable sequence of sheep haemoglobin has been deduced.

Tryptophan 5-hydroxylase was partially purified from rat small intestine and characterized. The enzyme activity was mainly localized in the distal one-fourth of the small intestine. The enzyme required Fe2+, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine and oxygen for full activity. The pH optimum of the reaction was 8.0. The hydroxylation rate of d-tryptophan by the enzyme was one-third that of l-tryptophan. l-Phenylalanine and l-tyrosine could not serve as substrates. The physiological significance of the enzyme is discussed.