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The Medical Research Council Medicinal Plants Survey in its expedition to Africa collected many specimens of the seed of Strophanthus hi8pidus P.DC. and S. sarmento8us P.DC., mostly from individual plants, in order to investigate the occurrence of sarmentogenin, a substance which forms a possible starting material for the production of cortisone. The isolation of sarmentogenin from certain of these seeds has already been reported (Callow, Meikle & Taylor, 1951). The present paper is a report on the preliminary investigation ofall the samples collected, and reveals certain interesting features about the distribution of cardiac aglycones in various Strophanthus species. As the examination of each seed sample by isolation of the constituents might take several years for completion, a method has been developed for the routine semi-micro investigation of the easily hydrolysable glycosides of the seeds. These easily hydrolysable glycosides (Reichstein, 1951), which are completely hydrolysed by refluxing for half an hour in 50% aqueous methanol which has been made 0-1N with respect to sulphuric acid, are those in which the steroidal portion of the molecule is directly attached in a glycosidic linkage to a 2deoxy sugar. Most of the best known glycosides belong to this group, which is much easier to investigate than the group in which the aglycones are directly attached to 'normal' sugars carrying a hydroxyl group at C(2), such as glucose or rhamnose, where isolation of the intact aglycone is usually difficult and has in several cases not yet been achieved. Investigation of the 'normal' glycosides in our seed samples has so far only been carried out in selected cases. The paper-chromatographic method of Zaffaroni, Burton & Keutmann (1950) (cf. Burton, Zaffaroni & Keutmann,1951)hasbeenusedbySchindler & Reichstein (1951) for separating this type of substance, but the procedure is in many ways complicated

Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s−1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).

Although myo-inositol is included in media for the successful growth of plant tissues, the actual requirement of most tissues, including soybean (Glycine max) callus in suspension culture, for myo-inositol has not been demonstrated. We have made use of deoxyglucose to reduce intracellular levels of myo-inositol. Deoxyglucose is phosphorylated to deoxyglucose 6-phosphate, which inhibits L-myo-inositol 1-phosphate synthase, an important enzyme in the synthesis of myo-inositol. Addition of deoxyglucose to the medium resulted in a decrease in the intracellular level of myo-inositol that corresponded with a decrease in cell division. Cell viability was not affected. When myo-inositol was added to cells along with deoxyglucose, cell division was restored, as were intracellular levels of myo-inositol. Addition of myo-inositol had no affect on the uptake or metabolism of deoxyglucose. From these results we propose that myo-inositol has a role in maintaining cell division in soybean callus tissue in suspension culture.

The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences.

Three cellulases and a beta-glucosidase were purified from the culture filtrate of the thermophilic fungus Thermoascus aurantiacus. The isolated enzymes were all homogeneous on polyacrylamide-disc-gel electrophoresis. Data from chromatography on Bio-Gel P-60 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated mol.wts. of 87000 (beta-glucosidase), 78000 (cellulase I), 49000 (cellulase II) and 34000 (cellulase III); the carbohydrate contents of the enzymes were 33.0, 5.5, 2.6 and 1.8% (w/w) respectively. Although the three purified cellulases were active towards filter paper, only cellulases I and III were active towards CM(carboxymethyl)-cellulose. Cellulase I was also active towards yeast glucan. The Km and catalytic-centre-activity values for the enzymes were as follows; 0.52 mumol/ml and 6.5 × 10(4) for beta-glucosidase on p-nitrophenyl beta-D-glucoside, 3.9 mg/ml and 6.3 for cellulase I on CM-cellulose, 1.2 mg/ml and 1.1 for cellulase I on yeast glucan, 35.5 mg/ml and 0.34 for cellulase II on filter paper, and 1.9 mg/ml and 33 for cellulase III on CM-cellulose.

A particulate enzyme preparation made from epicotyls of 1-week-old etiolated pea (Pisum sativum) seedlings was shown to incorporate glucuronic acid from UDP-D-[U-14C]glucuronic acid into a hemicellulosic polysaccharide. Optimum conditions for the incorporation include the presence of Mn2+ ions at between 4 and 10 mmol/litre and a pH between 5 and 6. UDP-D-xylose at 1 mmol/litre allows incorporation to continue for at least 8 h. In its absence, the reaction stops within 30 min. Analysis of the product by partial and total acid hydrolysis, followed by paper chromatography or electrophoresis, indicates that the polysaccharide produced is a glucuronoxylan.

Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)--NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.

To determine whether the prosthetic group of cytochrome c is synthesized and linked to the apoprotein in the cytosol or in connexion with the endoplasmic reticulum, we have studied the incorporation in vitro of delta-amino[(14)C]laevulinate into porphyrin compounds and cytochrome c by the cell sap from rat liver. The radioactive precursor was incorporated into a trichloroacetic acid-precipitable form partially resistant to extractions by acid solvents, suggesting the existence of a fraction covalently linked to protein. The activity was proportional to the amount of protein incubated, did not increase substantially by supplementation with the microsomal fraction and an energy source, and was very low in the pH5 fraction. Addition of increasing amounts of haemin inhibited the incorporation, as with purified delta-aminolaevulinate dehydratase. [(14)C]Protoporphyrin IX was identified by paper chromatography, together with a shoulder running as protohaem IX. The cell sap in the absence of ribosomes was also able to incorporate radioactivity into purified cytochrome c, and the addition of ribosomes significantly enhanced the activity. The precursors of haem c were synthesized in the soluble system by the known haem-synthetic pathway, as shown by the kinetics of labelling of the coproporphyrin, protoporphyrin and haem fractions, and the activities were concentrated in the precipitate obtained between 40 and 60% saturation with (NH(4))(2)SO(4). The presence of ferrochelatase was indicated by the incorporation of (55)Fe into proto- and haemato-haem identified by paper chromatography. It is concluded that the cell sap from rat liver contains the complete set of enzymes for the synthesis from delta-aminolaevulinate of haem c and its linkage to a small pool of free apoprotein c present in soluble form. This suggests that an ancillary pathway of haem synthesis occurs in the cytosol for at least the formation of the prosthetic group, which is linked post-translationally to that pool of apoprotein c synthesized by free polyribosomes.

1. Acrolein was detected in rat liver suspensions incubated in the presence of allyl alcohol and allyl formate. Acrolein was determined by condensation of the distilled aldehyde with semicarbazide, followed by spectrophotometric measurement of the semicarbazone at 257nm and identification by paper chromatography. 2. The transformation of allyl alcohol into acrolein occurred in the presence of NAD+. Inhibitors of rat liver alcohol dehydrogenase inhibited the reaction. 3. It is suggested that the hepatotoxic actions of allyl alcohol and of allyl formate on rat liver are related to their conversion into acrolein.

1. 6-Hydroxykynurenic acid (4,6-dihydroxyquinoline-2-carboxylic acid, 6-HKA) was isolated in crystalline form from both green and cured tobacco leaves. 2. A method for the determination of 6-HKA by paper chromatography and fluorimetry is described. 3. The content of 6-HKA in the flowers, stem and roots of the tobacco plant was much lower than that in the leaf. 4. The 6-HKA content increased throughout leaf development and senescence. 5. 6-HKA was detected in the leaves of plants representing 11 out of 27 families sampled. 6. 6-HKA was found to be devoid of antibacterial and antifungal activity, and was inactive in the Avena-coleoptile and cress-seed-germination tests. 7. The presence of 6-HKA is taken as evidence in plants of the tryptophan-catabolic pathway already known in mammals and micro-organisms.

1. A method for the isolation from animal tissues of UDP-glucuronic acid by one-dimensional paper chromatography is described and its concentrations in some tissues of several species of vertebrates are reported; the incorporation of [32P]-phosphate into UDP-glucuronic acid in vivo was also investigated. 2. The concentration of UDP-glucuronic acid was higher in the liver of rats, rabbits and guinea pigs than in the same tissue of some species of birds, amphibia and fishes; also, the concentration of UDP-glucuronic acid in rat liver, kidney and small intestine was several times lower than that of the same tissues of guinea pigs. 3. The rate of [32P]-phosphate incorporation into UDP-glucuronic acid was very high in rat liver and kidney and almost reached equilibrium with the radioactivity of UDP-glucose 30min after the administration of the [32P]phosphate.

1. Details of an improved method for starch-gel electrophoresis of water-soluble muscle proteins are given. 2. Methods are described for detecting enzyme activities on the starch gel after electrophoresis, by using pieces of filter paper. 3. Compositions of incubation mixtures suitable for detecting any of the enzymes of glycolysis, and certain other enzymes, are given. 4. A comparison of the various enzymes in extracts of several muscles from one rabbit was made; most differences are quantitative only. 5. A detailed comparison of the mobilities of various enzymes in extracts of muscles from a wide variety of species was made. Each species was found to have a characteristic pattern of proteins on the starch gel, and the mobilities of individual enzymes varied considerably. 6. Potential uses and extensions of the methods are discussed.

The Achilles tendon is built up mainly of collagen and it contains minute amounts of ground substances and cell materials. Opinions about its content of polysaccharides vary considerably. The ground substance of connective tissue is rich in acid mucopolysaccharides and Meyer (1957) has isolated the chondroitin sulphates from Achilles tendon. Besides the acid mucopolysaccharides the presence of other mucopolysaccharides has been suggested. First Grassmann & Schleich (1935) showed that collagen isolated from the skin and from Achilles tendon contains 0'5-0 7 % of polysaccharides. Consden (1953) and Consden, Glynn & Stanier (1953), using a combination of paper electrophoresis and paper chromatography, have shown that the connective tissues contain polysaccharides which after acid hydrolysis yield glucose, mannose, galactose and glucosamine. Bowes, Elliott & Moss (1955), and especially Moss (1955), are of the opinion that the negligible amount of hexosamine and the very small amount of hexose found in collagen may be impurities. With the aid ofthe enzyme collagemnucoproteinase isolated from the pancreas (Banga & Balo, 1956) the present authors succeeded in showing the existence of a protein fraction, collagenmucoprotein2 (mucoid2), the polysaccharide component of which, it was suggested, was not identical with the acid polysaccharides (Banga, 1958). It was thought that this substance might be responsible for the periodic acid Schiff reaction given by connective tissue. Mucoid2 is one of the important constituents of collagen fibre (Banga, 1958). It also takes part in the chemical contraction relaxation phenomenon of collagen (Balo, Szabo & Banga, unpublished work). In the present paper the isolation and analysis of mucoid2 are described.

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. Samples of thyroid and non-thyroid lymph and of thyroid and peripheral venous blood were obtained from primates and cats that had previously been given radioactive iodine. The distribution of the organic radioiodine between the protein and non-protein fractions in these samples was determined. 2. The proportion of the organic radioiodine in the form of iodoprotein was assessed by paper chromatography, acid-ethanol precipitation, hot-butanol washing, column chromatography and separate estimation of iodotyrosines after enzymic hydrolysis. 3. In thyroid lymph the relative proportion of the organic radioiodine in the form of iodoprotein was 75–98%; in blood it was much lower, probably no more than 6–7%. The absolute concentration of iodoprotein radioactivity also was considerably greater in thyroid lymph than in blood. 4. Enzymic hydrolysis of the protein of the thyroid lymph yielded a pattern of iodoamino acids that corresponded closely with that obtained after hydrolysis of protein extracted from the thyroid gland itself. 5. It can be concluded that the iodoprotein in thyroid lymph consists mainly of thyroglobulin or a closely related compound.

Methylation analysis was used to characterize the pectic polysaccharides from mustard cotyledons, a tissue with potential for rapid biological change involving the walls. The methylated sugars were identified by g.l.c. and paper chromatography after conversion of uronic acid derivatives into [3H]hexoses, and confirmed by the formation of crystalline derivatives of most of the main products, which were: 2,3-di-O-methyl-d-[6−3H]galactose, 2-O-methyl-d-[6−3H]galactose, 3,4-di-O-methylrhamnose, 3-O-methylrhamnose, 2,3,5-tri-O-methyl-l-arabinose, 2,3-di-O-methyl-l-arabinose, 2-O-methyl-l-arabinose, 2,3,4-tri-O-methyl-d-xylose and 2,3,4,6-tetra-O-methyl-d-galactose in the molar proportions 1·00:1·14:0·54:0·74:2·86:2·50:2·24:1·88:0·32. The structural units present are similar to those in wellknown polysaccharides from mature tissues, but their proportions are strikingly different. Uninterrupted and unbranched galacturonan segments can therefore contribute little cohesion to these walls, and it is suggested that this correlates with a function of the wall matrix to hydrate and permit readjustment, during germination, of structural elements or wall surfaces or both.

1. The lipids of ten Gram-positive bacteria have been isolated and the presence in each of a glycosyl diglyceride was established. 2. The glycolipid fractions were isolated and deacylated to give water-soluble glycosides which were purified by paper chromatography. Partial structures for the glycosides have been deduced from chemical and enzymic studies. 3. Nine of the glycosides were disaccharides glycosidically linked to the 1-position of glycerol: the remaining glycoside contained a trisaccharide similarly linked to glycerol.

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.

The partial amino acid sequence of two large peptides is described. These were prepared from the N-terminal half of the heavy chain of immunoglobulin G from pooled normal rabbit serum by tryptic digestion after the ∈-amino groups of the lysine residues had been blocked with S-ethyl trifluorothioacetate. These peptides are believed to account for about 145 residues of fragment C-1, the N-terminal section of rabbit immunoglobulin G heavy chain prepared by cyanogen bromide cleavage. The evidence from the present paper and the preceding paper (Cebra, Givol & Porter, 1968) suggests that it may be possible to deduce a predominant amino acid sequence for most, if not all, of this section of the molecule.

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.

The pseudouridine contents of the rRNA species of HeLa cells, mouse L-cells and Xenopus laevis cultured kidney cells were examined. Pseudouridine, like 2′-O-methylation, was found to occur relatively frequently in each of the high-molecular-weight rRNA species. However, the numerical data do not support the idea that there is a general one-to-one relationship between pseudoridine residues and 2′-O-methyl groups in vertebrate rRNA.

Degradative and n.m.r.-spectroscopic studies have been carried out on a novel mannitol teichoic acid extracted from the cell wall of bacterium N.C.T.C. 9742, for which the name Brevibacterium iodinum has been proposed. The backbone of the polymer is a poly(D-mannitol phosphate) containing 1--6 phosphodiester linkages. In most residues, pyruvic acid is acetal-linked to positions 4 and 5 of the mannitol. About half of the mannitol residues carry a beta-D-glucopyranosyl substituent at position 2. The glucosylmannitol was isolated and thoroughly characterized. At least 24 products were detected by ion-exchange chromatography and paper electrophoresis after alkaline hydrolysis of the polymer. Not all of these products could be identified. The main mechanistic pathways for depolymerization by the cleavage of phosphodiester linkages during alkaline hydrolysis involved (a) participation by the 2-hydroxy group and a cyclic phosphodiester intermediate (leading to a series of mannitol-based products) and (b) participation by the 3-hydroxy group in the cyclization of mannitol (leading to a series of products based on 1,4-anhydromannitol). The presence of glycerol phosphates in hydrolysates could be ascribed either to a linkage unit or to a separate glycerol teichoic acid. The mannitol teichoic acid was absent from the cell walls of Brevibacterium linens and Brevibacterium epidermis (one strain of each was examined).

1. Cell-wall hydrolysates from calli of all higher plants tested contained a new phenolic amino acid for which the trivial name isodityrosine is proposed. Isodityrosine was shown to be an oxidatively coupled dimer of tyrosine with the two tyrosine units linked by a diphenyl ether bridge. 2. The amount of isodityrosine in sodium dodecyl sulphate-insoluble cell-wall preparations was proportional to the amount of hydroxyproline. 3. Acidified chlorite split the diphenyl ether bridge of isodityrosine, and concomitantly solubilized the cell-wall glycoprotein. 4. Dithiothreitol inhibited isodityrosine synthesis in vivo, and suppressed in parallel the covalent binding of newly synthesized protein in the cell wall. 5. It is suggested that isodityrosine is an inter-polypeptide cross-link responsible for the insolubility of plant cell-wall glycoprotein.

About 70% of the radioactivity retained in the livers of rats dosed 48 h earlier with radioactively labelled folate was incorporated into two folate conjugates. The major derivative was purified and isolated by Sephadex G-15, DEAE-cellulose and DEAE-Sephadex ion-exchange column chromatography and paper chromatography. It was identified as 10-formylpteroylpentaglutamate by a combination of spectral, microbiological, chemical and chromatographic techniques. The minor conjugate, though less well characterized, exhibited similar properties and was assigned the structure 10-formylpteroyltetraglutamate. 10-Formylpteroylpentaglutamate (2.0nmol/g) and 10-formylpteroyltetraglutamate (0.25nmol/g) comprised about 20% of the total endogenous hepatic folate as determined by microbiological assay (Lactobacillus casei after conjugase treatment.

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. 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.

A convenient, inexpensive assay was developed for measuring relative changes in cyclic GMP in whole mouse neuroblastoma cells (clone NIE 115) based on labelling the cellular GTP pool with [8(-3)H]guanine. The time course of cell labelling and the distribution of radioactivity among possible products were studied; GTP is the only major labelled species. Radioactive cyclic GMP produced from the radioactive GTP on cell stimulation is isolated by column chromatography nad its identity has been rigorously established by paper chromatography and ion-exchange chromatography. The assay was used to study the time course of the cyclic GMP changes that occur after stimulation of neuroblastoma cells with carbamoylcholine and the dependence of the cyclic GMP changes on the carbamoylcholine concentration. The assay gives results comparable with those obtained by using a radioimmunoassay for cyclic GMP and should be applicable to other whole-cell and tissue-slice systems.

A novel o-phthalaldehyde-reactive compound was found in the h.p.l.c. chromatogram of Aplysia kurodai extract. This compound was isolated by ion-exchange chromatography and preparative high-voltage paper electrophoresis. It was shown by optical-rotatory-dispersion spectrum and optical-resolution h.p.l.c. analysis that this compound consisted of equimolar amounts of D-aspartic acid and glycine. This compound resisted cleavage in the Edman reaction. This peptide was inferred to be beta-D-aspartylglycine, and this was confirmed by synthesis. beta-D-Aspartylglycine was detected in all tissues of Aplysia kurodai, with especially high concentrations in body wall (skin and muscle) and gill.

1. Corynebacterium diphtheriae contains relatively large amounts (6.6mumoles/g. dry wt.) of a naphthaquinone whose ultraviolet-absorption spectrum is that of a typical menaquinone (vitamin K(2)), the E(1%) (1 cm.) value corresponding with that of MK-8, but on reversed-phase paper chromatograms it runs with MK-9. 2. In the presence of Adams catalyst hydrogen uptake is 2 atoms/mol. less than that calculated for MK-8. 3. Hydrogenated samples of the Corynebacterium quinone and the hydrogenation product of authentic MK-8 ran together on reversed-phase chromatograms. 4. Infrared-absorption spectra indicated close relationship with the menaquinone series, and nuclear-magnetic-resonance measurements show that one, non-terminal, double bond of the side chain has been saturated. 5. The compound is thus designated MK-8(2H), indicating a menaquinone with eight isoprene units but only seven double bonds in the side chain.

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.

1. Proteinuria was found in most sucking lambs and calves examined but was less prevalent in bottle-fed kids or bucket-fed calves. 2. This incidence of proteinuria corresponded to the presence of immune lactoglobulin in the serum. 3. The extent of proteinuria in lambs and calves rose to 20 g./l. or more, whereas in kids values of only 4 g./l. were obtained. 4. The proteins present in the urine of lambs and kids have been compared with those in the urine of calves by using boundary electrophoresis and paper electrophoresis. 5. Sedimentation analyses have been made on the proteins of lamb, kid and calf urine. Components with sedimentation coefficients 3s and less were present. The separated slow electrophoretic components were found to have sedimentation coefficients 3·3, 3·6 and 3·3s respectively. 6. The sedimentation analyses, and for calf urine the electrophoretic data, support the view that the slow-moving electrophoretic components of calf and lamb urine are degraded immune lactoglobulins. 7. There was electrophoretic evidence for other degraded proteins in some lamb urine which showed multiple zones that could not be related to serum or colostral proteins.

The oxidation of 3,5-di-tert.-butylphenyl N-methylcarbamate (Butacarb) has been studied in the flies Musca domestica and Lucilia sericata, grass grubs Costelytra zealandica and the mouse. In all species eleven oxidation products, which were formed by hydroxylation of the tert.-butyl groups and the N-methyl group, were detected.

The amino acid sequence of the light chain of the myeloma protein Dee was studied. The light chain is of the κ type and of subgroup I. The variable part contains some substitutions that are unique and also some that have been observed already in other κI chains (repeated variants). Based on these repeated variants a subdivision of the κI subgroup is proposed.

1. Glucose phosphorylation rates of about 1 mumole/g./min. have been measured at room temperature in homogenates of human placental chorionic villi, and these rates are relatively constant throughout gestation. 2. This reaction has an apparent K(m) for glucose of 3x10(-5)m both in early and term placenta. 3. Human foetal membranes, the amnion and chorion, also phosphorylate glucose at a rate about equal to that of the placenta. 4. On incubation of intact bits of villus tissue from 8-12-week or full-term placenta with labelled pyruvate, followed by paper chromatography of the tissue extract, the following distribution of label was observed: residual pyruvate, 40-60%; lactate, 30-50%; glucose, 6%; fructose, 7%; sorbitol, 0.6%. 5. The concept of the placenta acting as a foetal liver during early pregnancy is inconsistent with the observation that glucose production by this organ persists up to term.

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.

1. Maize and barley shoots have been shown to contain phylloquinone, plastoquinone, alpha-tocopherol (and gamma-tocopherol in maize), alpha-tocopherolquinone and ubiquinone-9. 2. No solanesol was detected in any tissue examined. 3. In maize shoots plastoquinone and alpha-tocopherolquinone were localized in the chloroplast; ubiquinone was in the mitochondria. 4. Etiolated (dark-grown) shoots contained smaller amounts of phylloquinone and plastoquinone; alpha-tocopherolquinone was entirely absent; ubiquinone and alpha-tocopherol concentrations were unaffected. 5. On illumination of etiolated shoots the chloroplastidic quinones phylloquinone, plastoquinone and alpha-tocopherolquinone were synthesized in step with chloroplast development. alpha-Tocopherolquinone was not formed at the immediate expense of alpha-tocopherol.

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.

1. Cysteic acid peptides from various digests of calf rennin were purified by diagonal paper electrophoresis. 2. The amino acid sequence of these peptides accounts for 38 amino acids around three unique disulphide bridges in rennin. 3. One bridge connects two acidic regions of the chain, one forms a loop of five residues and the other a loop of six residues. 4. These bridges are homologous with those of hog pepsin. 5. Tryptic peptides from the C-terminus of rennin account for 22 residues, 17 of which are homologous with the C-terminus of pepsin. 6. Altogether, sequences accounting for 94 of the 270 residues in rennin are described and the degree of homology with pepsin approximates to 70%.

Specific analyses of rat milk sugar revealed lactose and N-acetylneuraminyl-lactose. The latter showed multiple, interconvertible forms in certain paper-chromatographic systems. Mean lactose concentrations rose from 33.1μmol/ml at day 0 to 100μmol/ml at day 20 of lactation. Mean N-acetylneuraminyl-lactose concentrations rose from 7.1μmol/ml at day 0 to 25.9μmol/ml at day 4 and thereafter declined, decreasing to zero by day 20 of lactation. Similar data are given on the concentrations of these sugars in mammary tissue of rats. Neuraminyl-lactose was also detected in mouse mammary tissue.

1. A lipid–teichoic acid complex was isolated from Streptococcus faecalis N.C.I.B. 8191. The covalent nature of the linkage between teichoic acid and lipid was established. 2. The complex exhibits macromolecular properties in solution, and ultracentrifugation studies show that these are due to micelle formation. 3. From chemical studies it is concluded that the teichoic acid is a poly(glycerol phosphate) in which some of the glycerol hydroxyl groups possess kojibiosyl [2-O-α-d-glucopyranosyl-(1→2)-α-d- glucopyranosyl] substituents, together with d-alanine ester residues. 4. The lipid is 1-kojibiosyl diglyceride, already known as a membrane component of this organism, with probably a phosphatidyl substituent. The phosphatidyl kojibiosyl diglyceride is attached to the teichoic acid through a phosphodiester linkage, and the chain of the teichoic acid contains 28–35 units. 5. Although the complex represents the whole of the membrane teichoic acid in this organism, only about 12% of the membrane glycolipid is associated with teichoic acid. 6. Two phosphatidyl glycolipids, closely resembling that bearing the teichoic acid, were isolated from the lipids of the organism and were partly characterized.

The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis–Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3α-ol-20β-one (3α-hydroxypregnan-20-β-one)>oestradiol-17β 3-methyl ether>oestradiol-17β>oestriol>pregnane-3α,20β-diol>oestrone. Pregnan-3α-ol-20β-one, pregnane-3α,20β-diol and oestrone had negligible effect on glucuronidation.

1. A simple method is described for the determination of small amounts of [35S]sulphated polysaccharide with 95–100% recovery in the range from 0.3 to 150μg of polysaccharide. 2. The method is based on precipitation with cetylpyridinium chloride of polysaccharide samples applied to filter paper. It is not significantly disturbed by the presence in the sample of a large excess of inorganic35SO42-. 3. Sulphated glucosamino- and galactosaminoglycans may be determined separately by treatment of the sample with chondroitinase ABC. 4. The method is applicable to the assay of [35S]sulphated polysaccharide biosynthesis in cell cultures. A stimulation of sulphate incorporation obeying a linear dose–response curve, was demonstrated in somatomedin-incubated fibroblast and glia cell cultures. 5. The described system provides a new assay method for somatomedin. 6. The stimulatory effect of somatomedin on the synthesis of [35S]sulphated polysaccharide appeared to be general, rather than specific, for a particular type of polysaccharide.

1. Rhodesian copalwood (Guibourtia coleosperma) contains three diastereo-isomeric leuco-fisetinidins. These consist of the (−)-2,3-cis–3,4-cis (2R,3R,4R) and (−)-2,3-cis–3,4-trans (2R,3R,4S) 3′,4′,7-trihydroxyflavan-3,4-diols, and the third was shown to be a 2,3-trans–3,4-cis isomer by means of paper ionophoresis. 2. There occurrence in similar proportions as tannin precursors also in the tropical hardwoods G. tessmannii and G. demeusii implies a close taxonomic relationship between these, and with G. coleosperma. 3. Epimerization of the natural (−)-3′,4′,7- trihydroxy-2,3-trans-flavan-3,4-trans-diol affords a mixture from which the (−)-2,3-cis–3,4-cis isomer was separated readily, but the (−)-2,3-trans–3,4-cis isomer was obtained with difficulty. These were formed by epimerization of the (−)-2,3-trans–3,4-trans isomer at C-2 and C-4, and at C-4, respectively.

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma α1-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1→4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2→2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl→N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas–liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.

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.