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A method for the quantitative determination of plasma concentrations of ampicillin and cloxacillin in mixture is described. The antibiotics were separated by high voltage paper electrophoresis. The high affinity of cloxacillin for albumin seriously interfered with the electrophoretic separation of the antibiotics and the quantitation of cloxacillin. Therefore, prior to electrophoresis, the plasma proteins were digested with trypsin to release cloxacillin. Subsequent to the electrophoretic separation the antibiotics were developed micro-biologically by agar diffusion using the separated spots on the paper as diffusion centres. The inhibition zones were clearly visible after staining and could be quantitated. The method was evaluated statistically and showed good agreement with the paper disc diffusion method.

Aeromonas liquefaciens and Aeromonas salmonicida were both found to produce considerable amounts of extracellular proteinases, when grown on semi-solid skim milk agar at 30° C. The proteinase A, produced by Ae. liquefaciens only, and the proteinase B, produced also by Ae. salmonicida were purified approximately 100 times by precipitation twice with (NH4)2SO4, batchwise treatment with DEAE-cellulose and, finally, gel filtration on Sepha-dex G-100. The pH-optimum was estimated to be 7.9 for proteinase A and 9.0 for proteinase B. Differences between the proteinases were also found with regard to thermoresistance, and to their behaviour in the presence of some naturally occurring proteinase inhibitors. The addition of iron and cobalt ions increased the activity of both proteinases. Molecular weights were estimated to be 22 100 for the proteinase A and 43 600 for proteinase B, on the basis of gel filtration on Sephadex G-100.

Urinary metabolic studies by means of high-voltage paper electrophoresis (HVPE) and some qualitative chemical tests were performed in 40 patients in a family with hereditary macular degeneration and in 40 nonaffected members of the same family. No biochemical abnormalities were observed in the examined urine samples. The results are discussed with regard to earlier published reports of aminoaciduria in hereditary macular degeneration.

Three Danish pedigrees have been studied with a view to reduced thyroxine-binding globulin (TBG). The study confirmed that this defect is transmitted by X-chromosome-linked dominant inheritance, there being no man-to-man transmission, but also that it may occur in two types: deficient TBG and low, non-zero TBG. Definite changes of TBG and thyroid function tests were found in the hemizygous men, whereas the heterozygous women showed a marked variation in the range between the values for hemizygous men and normal persons. Determination of total thyroxine appears to afford more guidance than the protein-bound iodine suggested by others. Two of four women on oral contraception exhibited such deviating TBG values that they are assumed to be TBG heterozygous. In all persons free thyroxine proved normal or slightly reduced but all the persons tested were euthyroid. However, the propositus of one family had previously been treated for Graves' disease.

Serum γ-globulin levels have been determined by paper electrophoresis in 54 patients with cirrhosis of the liver before and after establishment of a portocaval shunt. As a control group 23 patients operated on for cancer of the lung were studied in the same way. A significant average increase from 18.6 to 23.0 g/1 in the cirrhotics was found, whereas no increase in the control group was observed. Serum immunoglobulins and antibodies to E. coli 0 group antigens were studied in 31 patients with cirrhosis and a portocaval shunt and 128 patients with cirrhosis without portocaval shunt. Significantly higher levels of IgG and E. coli antibodies were found in the patients with a portocaval shunt. It is proposed that these observations can be explained by the change in liver circulation brought about by the operation. If the cirrhotic liver has retained some of the ability of the normal liver to inhibit immunogens as previously proposed by us, the observed changes in immunoglobulins and E. coli antibodies should be expected.

In a patient with chronic lymphocytic leukaemia, two M components of the IgGkappa and IgGlambda classes were demonstrated in the serum at the time of diagnosis. The patient was first followed without treatment; after 18 months multiple myeloma developed. At that time, immunofluorescence study of lymphocytes of the peripheral blood showed mainly membrane-bound immunoglobulins (S-Ig) of the IgGkappa class. The bone marrow disclosed a definite predominance of plasma cells with cytoplasmic IgGlambda, suggesting that the two B cell-derived diseases had arisen from two different cell clones. During the development of multiple myeloma, the serum concentration of the M component IgGlambda increased. Concurrently, the M component of IgGkappa gradually disappeared from the serum, and the concentrations of the normal immunoglobulins IgA and IgM declined. Following cytostatic treatment, the concentration of the myeloma-derived M component IgGlambda was halved and simultaneously the M component IgGkappa reappeared in the serum. To our knowledge, this case is the second reported of chronic lymphocytic leukaemia and multiple myeloma indicating development of the two diseases from different cell clones, and the first reported cases with myeloma-induced suppression of M-component secretion from malignant cells.

Nucleolar and nuclear size of bone marrow plasma cells has been studied in 26 cases of myeloma, 19 of benign essential monoclonal gammapathy (BEMG) and 9 without an M-component. Bone marrow smears were Feulgen-stained to visualize nucleoli and nuclei. Nucleolar area, nuclear area and the ratio nucleolar area/nuclear area were calculated. The myeloma group differed from the other two groups in having plasma cells with larger nucleoli, larger nuclei and an increased ratio nucleolar area/nuclear area. No difference was found between the BEMG group and the cases without an M-component. In some cases--in which the absence of osteolytic lesions at X-ray examination, a low plasma cell percentage in bone marrow smears or a low M-component concentration initially made the diagnosis of myeloma uncertain--the observation of enlarged nucleoli could help to establish the diagnosis. Three cases were observed in which the onset of myeloma was preceded by a long period of essentially unchanged M-component concentration. In these cases a sudden increase of M-component concentration was accompanied by an increase of mean nucleolar size of bone marrow plasma cells.

A series of 71 consecutive patients with myelomatosis formed the basis of determining survival in relation to sex, type of M-component, and its initial magnitude. For the entire material the median survival was 29 months. It was essentially shorter for IgA (15 months) than for IgG myelomatosis (37 months), whereas the survival was the same for both types among patients who were still alive 5–6 years after diagnosis. The magnitude of the M-component at diagnosis appears to play no prognostic role. 49 patients were treated with cytostatics, and 21 also with prednisone, for a minimum of 2 months. 21 patients responded within 2 months by a decrease in the serum concentration of the M-component exceeding 0.6 g/100 ml. On the other hand, 28 patients showed no significant change in the magnitude of the M-component during the first 2 months' medication. However, the latter patients had a median survival of 62 months as compared to only 13 months for the quickly responding patients. Thus, the quick responders had a significantly poorer prognosis.

This is the first case, hitherto described, of benign essential gammopathy, presenting symptoms and histological evidence of peripheral neuropathy. Furthermore, in no previous case, has a specific localization of immunoglobulin (type IgM) to the areas of nerves involved by the lymphocytes been demonstrated. After six months' treatment with chlorambucil the neurological symptoms disappeared and in a biopsy from the right n saphenus no endoneural lymphocytes or local IgM could be found.

An immunological study has been made of 12 patients with myelofibrosis. The study included determinations of serum protein and immunoglobulin concentrations, serial tuberculin skin tests, and in vitro stimulation of peripheral lymphocytes with phyto-haemagglutinin (PHA). In two patients, very low gammaglobulin values were found (low IgG and IgA concentrations; low IgM in one of them). Four other patients had subnormal IgA values. Depressed delayed-type hypersensitivity reactions were observable in four of eight patients tested. The immuno-deficiency noted was probably secondary to the bone marrow damage. Nevertheless, a disturbed immune response may contribute to the development of bone marrow fibrosis and myeloid metaplasia.

The transfer of selenite via the erythrocytes to the plasma was investigated using Se 75-bodium selenite and cow blood. The selenite was initially fluxed into the erythrocytes by a cyanide sensitive mechanism. During the uptake period (within 1 min.) the erythrocytes consumed oxygen and it can be concluded that selenium is reduced and catalyzes the electron flow to the oxygen. The lack of electrons thus produced is balanced by the oxygenation of GSH. The release is dependent on GSH and it is possible that selenium is ejected from the erythrocytes as a glutathione complex which is fixed to the SH of plasma albumin.

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).

The peptide [15, 16-D-lysine, 17, 18-D-arginine]-adrenocorticotropin-(1-19) and an all-D-retropeptide related to the amino terminal octadecapeptide of adrenocorticotropin have been synthesized by the solid-phase method. The nonadecapeptide was shown to possess 10-15% of the steroidogenic activity and 3% of the lipolytic activity of adrenocorticotropin-(1-19). The all-D-retropeptide showed no activity and exhibited no inhibitory activity in steroidogenesis and lipolysis.

Digestion of a commercial mixture of the peptides polymyxin B1 and B2 with subtilopeptidase A yielded a peptide whose amino acid composition corresponded to the ring fragment common to both antibiotics. However, in contrast to the earlier chemical studies of Suzuki and collaborators, our high resolution nmr studies indicated that the ring had been split. Edman degradation, hydrazinolysis and exopeptidase digestion showed the rupture to have occurred between positions 8 and 9.

Ten new nonapeptide and decapeptide analogues of bradykinin were prepared by solid phase synthesis. Analogues of proline (3,4-dehydroproline; azetidine-2-carboxylic acid; cis,trans-4-methylproline; thiazolidine-4-carboxylic acid and trans-4-hydroxyproline) were successfully incorporated into the peptides in place of proline at position 3. In vivo and in vitro pharmacological studies revealed that the analogues were in general less active than bradykinin. Several structure-activity relationships were postulated from rat blood pressure, rat uterus and guinea pig ileum studies.

A nonapeptide, Arg-Glu-Leu-Glu-Asp-Gly-Thr-Pro-Arg, corresponding to the 123–131 sequence of bovine pituitary growth hormone, was prepared by a modified solid phase synthesis. To avoid an acid- or base-catalysed rearrangement at the Asp-Gly bond, which gives rise to an aspartimide-containing peptide, a new combination of protecting groups was introduced. The 2-(4-biphenylyl)-2-propyloxycarbonyl group was used for Nα-protection, a t-butyl ester for the β-carboxyl of aspartic acid, and a p-alkoxybenzyl alcohol ester for the anchoring bond to the resin support. The nonapeptide showed growth hormone activity in the hypophysectomized rat tibia epiphyseal width test, but was inactive in the hypophysectomized rat body weight gain test.

The binding of pyridoxal 5′-phosphate (PLP) to bovine serum albumin (BSA), and to large BSA fragments obtained after proteolytic hydrolysis, was investigated in order to study the structure of these fragments in relation to the albumin structure itself, and to get information about the PLP binding sites on albumin. From absorbance and circular dichroism spectra, combined with peptide mapping of the tryptic digests of the reduced PLP-protein complexes, it could be concluded that the primary binding site is localized within the NH2-terminal part of the albumin molecule. The COOH-terminal part contains one or more secondary sites. It appeared that in albumin and in the largest NH2-terminal fragment, the environment of the primary binding site is rather apolar in character. However, in the smallest NH2-terminal fragment this site is more exposed to the solvent. This suggests that the part of the peptide chain which is not common in both fragments has a stabilizing effect on the structure around the primary binding site.

Fluorescamine was shown to be an excellent terminating agent for blocking unreacted amino groups during solid phase peptide synthesis. A comparison of the termination efficiency of fluorescamine versus that of acetylation revealed that the former method gave superior products as assessed by peptide analysis, dansyl-amino end group determination and biological assay. In addition, fluorescamine terminated fragments were converted to non-fluorescent spirolactones during the deprotection stage. These spirolactones were stable to subsequent solid phase reaction conditions and were readily removed from the target peptide.

The solid-phase syntheses of [Sar2]-, [Ala2]-, [D-Leu2]-, [D-Lys2]-beta-endorphins and [Pro5]-, [Leu5]-, [D-Leu5]-, [D-Ala2, D-Leu5]-beta-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu2 analog to be 48% as potent as betah-endorphin while the Ala2, D-Lys2, Leu5, and [D-Ala2, D-Leu5] analogs were 8 to 17% as active. The Sar2, D-Leu5, and Pro5 analogs were less than 1% as potent.

A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl glycine in the dry state. Fast atom-bombardment (FAB) mass spectrometry and Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) showed that the product is a cyclic imide. Cyclic imide formation also was shown to occur with the tripeptides Gly-Asp-Gly and Gly-Asn-Gly. The cyclization of aspartyl peptides depends upon protonation of the beta-carboxyl group and can be promoted by acidification or by heating the NH4+ salt in the dry state.

The neurohypophyseal hormones of three species of Australian marsupials belonging to the family Macropodidae (Macropus rufus, Macropus giganteus, Macropus robustus). have been investigated. In these species three active peptides have been disclosed, one with oxytocic activity and two with pressor activity. The three hormones are present in all the individual glands which have been examined. Purification has been undertaken by ion-exchange chromatography and paper chromatoelectrophoresis. The more abundant of the pressor hormones (about 80% of the total pressor activity) has been identified as lysine-vasopressin by its biological properties, its chromatographic behaviour and its amino acid composition. The second pressor principle, more basic, has not yet been chemically characterized but its pharmacological properties and its chromatographic behaviour suggest that it is closely related or identical to arginine-vasopressin. Dimorphism of vasopressins seems peculiar to Metatheria since it has not been observed in Protheria and very rarely in Eutheria.

A method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences. The main point of modification is that before every coupling cycle the resin is divided into equal parts and each portion is coupled with a different amino acid. Then the portions are mixed and before the next coupling cycle the resin is again distributed into equal portions. The method is illustrated by the synthesis of a mixture of 27 tetrapeptides and that of 180 pentapeptides.

A single insulin component has been isolated by ion-exchange chromatography on CM-cellulose from a commercially available mixture of tuna, bonito, and swordfish insulin. The biological activity of this component is in the range of 17 i.u./mg as determined with the rat adipose tissue assay. This fish insulin ha s been reduced and aminoethylated. The two chain derivatives were separated by column chromatography on IRC-50. The A and B chain derivatives contain 21 and 30 amino acids, respectively. The two chain derivatives were digested with trypsin and the resulting peptides separated by high voltage paper electrophoresis, paper chromatography, and, in some cases, by ion-exchange chromatography and gel filtration. Certain tryptic peptides were further digested with chymotrypsin. The amino-acid sequences of the resulting peptides were determined by a modified “dansyl”-Edman technique and by the use of aminopeptidase M, carboxypeptidase A and B, and citrus carboxypeptidase. The complete amino-acid sequence of chains A and B is given.

In order to find the conditions under which Tc-99m-methylenediphosphonate (Tc-99m-MDP) and Tc-99m(V)-dimercaptosuccinate (Tc-99m(V)-DMSA) may become tumour-seeking agents, leaving healthy organs free from the radionuclide uptake, the solution chemistry of these radiopharmaceuticals was studied by paper chromatography and paper electrophoresis in distilled water, in physiological saline, in NAHCO3, and ascorbic acid solutions. Both radiopharmaceuticals are anionic in the radiopharmaceutical solution but get easily hydrolysed to form cationic Tc-99m species which concentrates in healthy bone and in some bone metastases. Tc-99m (V)-DMSA being more stable remains long in the blood pool giving undesirable presence of the radionuclide in lung, heart and kidneys, in addition to its reduced uptake in bone metastases and in some primaries. We are trying to eliminate these drawbacks of healthy organ uptake of Tc-99-m(V)-DMSA not only to get a clean scintigraphic image of the tumour with this radiopharmaceutical but to extend its formulation, thus obtained, to prepare radiopharmaceutical with Re-188, which is the higher homologue of Tc-99m, for systemic therapy of cancer. Essentially similar solution chemistry of both radiopharmaceuticals suggests that like Tc-99m-MDP, technetium-99m-dimercaptosuccinate is also a complex of tetravalent Tc-99m and not of pentavalent Tc-99m as so far supposed to be.

Exposure of the skin to sunlight results in an increase of the content of epidermal trans-urocanic acid, a key metabolite of L-histidine, and also in occurrence of the isomerization of trans-urocanic acid to the cis isomer. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis-urocanic acid by incubation with rat liver extract that contained glutathione S-transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with gamma -glutamyl transpeptidase resulted in the formation of N-[S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteinyl]glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high-voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis-urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.

A new method has been developed for the isolation and rapid identification of anthocyanins from two floricultural crops based on the use of high-voltage paper electrophoresis with bisulphite buffer. Using this method, anthocyanin pigments were successfully purified as their negatively charged bisulphite-addition compounds from crude extracts of plant tissue. In conjunction with liquid chromatography–electrospray mass spectrometry, the method enabled the anthocyanins from the flowers of two Banksia species and the leaves of two Acacia species to be identified. The Banksia flowers contained both cyanidin and peonidin-based pigments, while the Acacia leaves contained cyanidin and delphinidin derivatives. Copyright © 2003 John Wiley & Sons, Ltd.

Paralytic shellfish poisoning (PSP) is caused by mixtures of saxitoxin analogs of which more than eighteen are known. Reliable and sensitive analytical methods and assays for these toxins are essential to protect the consumer and the shellfish industry, but research has been restricted by a shortage of the pure compounds. Only saxitoxin has so far been generally available as a PSP toxin standard, yet sulfated analogs usually occur in higher concentrations than saxitoxin in toxic marine algae and shellfish. Methods are described for the purification of some of the common PSP toxins, in quantities sufficient for the preparation of PSP standards from the dinoflagellate Alexandrium excavatum, the giant sea scallop (Placopecten megallanicus) hepatopancreas, and the cyanobacterium Aphanizomenon flos-aquae. Purity was monitored by high performance liquid chromatography with fluorescence detection (HPLC-FD), ionspray mass spectrometry (ISP-MS), capillary electrophoresis (CE), and proton NMR spectroscopy.

N-Linked sugar chains of normal mammary gland, mammary carcinomas (primary lesion), and axillary lymph node metastases of mammary carcinomas were released from their membrane preparations by hydrazinolysis and their structures were analyzed. Fractionation using a Datura stramonium agglutinin (DSA)-Sepharose column revealed that the metastasized carcinomas contain more than twice as much DSA-binding oligosaccharides as the normal gland, and the primary carcinomas contain an intermediate amount. These oligosaccharides were elucidated to have tri- and tetraantennary structures containing the GlcNAc beta 1--6(GlcNAc beta 1--2)Man group with and without N-acetyllactosamine repeating units. Lectin blot analysis of membrane glycoproteins and histochemical staining of tissues using biotinylated DSA indicated that these glycosylation changes predominantly occur in a limited number of glycoproteins with apparent molecular weights of 90, 160, and 210 kilodaltons, and mammary carcinomas are distinguishable from normal gland by their intense intracytoplasmic staining.

Methylarginines in free form were identified in bovine brain. Three compounds were isolated from the basic aliphatic amino acid fraction of bovine brain with several ion-exchange chromatographies. They showed the same Rf values in paper and thin-layer chromatographies as those of authentic NG-monomethylarginine, NG,NG-dimethylarginine, and NG,N'G-dimethylarginine. The migration distance of the isolated compounds in high-voltage paper electrophoresis and the retention times in ion-exchange HPLC were also identical to those of the above authentic methylarginines. We concluded that these three compounds are the methyl derivatives of arginine described above. The amount of these three compounds isolated from 1,090 g of bovine brain was 0.3 mumol of NG-monomethylarginine, 0.1 mumol of NG,NG-dimethylarginine, and 0.5 mumol of NG,N'G-dimethylarginine. The occurrence of these free methylarginines may have an important role in regulating the signal transduction through the nitric oxide system.

Structures of the N-linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (sCD4) have been investigated by enzymic microsequencing. The glycoprotein has two N-glycosylation sites, Asn271 and Asn300, at both of which evidence for the presence of complex type biantennary sialo-oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S.A., Hemling, M.E., Folena-Wasserman, G., Sweet, R.W., Anumula, K., Barr, J.R., Huddleston, M.J. & Taylor, P. (1989) J. Biol. Chem. 264, 21,286-21,295]. Among oligosaccharides released from sCD4 by hydrazinolysis and labelled with NaB3H4, neutral (12.8%) and acidic (87.2%) oligosaccharides were detected by paper electrophoresis. The latter were rendered neutral following sialidase treatment indicating that acidity was due exclusively to the presence of sialic acid residues. By enzymic microsequencing of the sialidase-treated oligosaccharides (fractionated on affinity columns of Ricinis communis agglutinin 120 and concanavalin A) in conjunction with methylation data from the earlier study, 14 sequences were identified. These accounted for over 80% of the sialidase-treated oligosaccharides of sCD4 as follows: [formula: see text] where +/- indicates residues present on only a proportion of chains. The spectrum of oligosaccharide structures released from each glycosylation site was assessed as being similar to that of total oligosaccharides on the basis of their chromatographic profiles on the lectin columns and on Bio-Gel P-4.

N-Linked sugar chains of normal human esophageal epithelium and esophageal squamous carcinoma were quantitatively released as oligosaccharides from their membrane preparations by hydrazinolysis. After being fractionated by serial lectin column chromatography using concanavalin A-Sepharose and Datura stramonium agglutinin-Sepharose, their structures were elucidated by exoglycosidase digestion in combination with methylation analysis. Both normal epithelium and esophageal carcinoma contained bi-, tri- and tetraantennary oligosaccharides as well as high mannose-type oligosaccharides. Interestingly, carcinoma had about 1.6 times larger amounts of tri- and tetraantennary oligosaccharides with the GlcNAc beta 1----4Man alpha 1----and/or the GlcNAc beta 1----6Man alpha 1----linkages than normal epithelium. Tri- and tetraantennary oligosaccharides with N-acetyllactosamine repeating units (the Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc group) were also increased in carcinoma. These data indicated that the altered glycosylation of proteins previously found in transformed rodent cells also occurs widely in human esophageal carcinoma.

Using the active-site-directed irreversible inhibitor bromohydroxy acetone phosphate (BHAP) Coulson et al. [l] isolated a hexapeptide from the active centre of chicken triose phosphate isomerase to which the inhibitor was bound covalently. Waley et al. [2] isolated a peptide of homologous sequence from the rabbit enzyme. Later de la Mare et al. [3] elucidated the residue to which the inhibitor moiety became attached and that to which it subsequently migrated. We have isolated from the chicken enzyme the tryptic peptide of fifteen residues that include the former hexapeptide sequence and have established its sequence by a combination of conventional and massspectrometric techniques. The sequence is identical to that derived for rabbit enzyme by Corran and Waley [4], but differs from those put forward, also for the rabbit enzyme, by Hartman [S, 61. Preliminary results bbtained by conventional techniques were reported by Offord et al. [7].

The disulphide linkages of the 16 half-cystine residues in the sweet-tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high-performance liquid chromatography and peptide mapping by paper high-voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9-204, 56-66, 71-77, 121-193, 126-177, 134-149, 145-158 and 159-164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys-145 and Cys-158.

The incorporation of radioactivity in vitro from UDP-[14C]glucose into endogenous acceptors in a crude enzymatic preparation from Acetobacter xylinum is described. A fraction of the total incorporated radioactivity could be extracted with organic solvents, like butanol. The remainder was mostly solubilized by hot alkali, leaving an insoluble fraction. No incorporation was observed in the absence of magnesium ions. Mn2+ could only partially replace Mg2+, and had an inhibitory effect when both ions were present. Analysis of the butanol-soluble fraction permitted the isolation of at least three components characterized as: lipid-diphosphate α-glucose, lipid-diphosphate α-cellobiose and lipid-monophosphate β-galactose. In the presence of [β-32P]UDP-glucose the system incorporated radioactivity only into the diphosphate derivatives, indicating the transfer of a α-glucose 1-phosphate group from UDP-glucose. Experiments with doubly labelled substrate confirmed these results. The role these compounds might play in polysaccharide biosynthesis is discussed.

It was previously shown that in a radiochemical assay using anion exchange resin the capacity to synthesize acetylcholine in salivary glands was markedly overestimated unless suitable controls were introduced. By the use of paper electrophoresis of the incubation mixture it is shown that the labelled acetyl group is not only transferred from acetyl-Coenzyme A to choline but also to carnitine in both salivary glands and urinary bladders. The two enzymes responsible for these processes, choline acetyltransferase and carnitine acetyltransferase, are shown to behave differently following denervation of the glands and bladders. Further, the adequacy of the introduced controls is demonstrated.

In human tissues the pyruvate kinase reaction (ATP: pyruvate phosphotransferase; E C 2.7.1.40) is catalysed by at least three isozymes [l-3] which can be distinguised by their kinetic, electrophoretic and immunological properties: M1 present in muscle, L the major form in liver and Mz found in liver, kidney and adipose tissue. The isozyme present in red cells (R) is kinetically similar to the L isozyme but can be distinguished from it by electrophoresis [2-41. The R isozyme can be converted into a form which is electrophoretically identical to the L isozyme either by treating with a liver homogenate or repeated freezing and thawing [4 ] . The molecular structures and interrelationships of the isozymes are unknown. We have examined the human M1 and R isozymes by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and two dimensional fingerprinting of their cyanogen bromide peptides. The evidence is consistent with identical subunits in the M1 tetramer and two pairs of non-identical subunits in the R tetramer. One of the R subunits may be homologous with the M1 subunit but is probably not identical to it.

The O antigen polysaccharide of Salmonella montevideo was isolated from a core-defective mutant by the phenol/water procedure, and was suspected to contain phosphomonoester and cyclic phosphodiester at its reducing end in anology to the O hapten from Salmonella typhimurium (Kent and Osborn, 1968). Therefore, it was chromatographed on a DEAE-cellulose column. Whereas one part eluted with water the other part of the polysaccharide could only be eluted with buffer. Both fractions were further purified on Sephadex G100 and contained mannose, glucose, N-acetylglucosamine and phosphate in a molar ratio of 4:1:1: < 0.1. In order to specifically label the reducing end phosphate was removed enzymatically, or the presumed cyclic diester was cleaved by mild hydrolysis, and the fractions were reduced with sodium boro[3H]hydride. Both fractions yield mainly [3H]glycerol after hydrolysis and paper chromatography. In addition, [3H]mannitol and [3H]monohydroxyacetone could be identified by paper chromatography and were concluded to be the result of phosphate migration and β-elimination reactions taking place during the isolation procedure and the various treatments prior to sodium boro[3H]-hydride reduction. These findings in addition to periodate oxidation studies indicated that the O antigen polysaccharide of Salmonella montevideo had glyceraldehyde phosphate at its reducing end. From the incorporation of 3H into the polysaccharide the O antigen was calculated to consist of about 19 repeating units of 6 sugar residues each.

Three rat alpha 1-fetoprotein fractions were obtained by chromatography on concanavalin-A--Sepharose: one non-reactive, one weakly reactive and one reactive to concanavalin A. The non-reactive and reactive variants were found to vary in the structure of their carbohydrate chains while the conformation of the weakly reactive form may modulate the accessibility of these chains to the lectin. N-Glycosidically linked glycans from unfractionated alpha 1-fetoprotein were isolated and chemically characterized. Particular attention was paid to develop sensitive methods based upon hydrazinolysis, quantitative re-N-acetylation of glycans with [14C]acetic anhydride and thin-layer chromatography of labeled compounds. With the aid of these methods two main kinds of glycans (1a and 2a) were obtained and fractionated on concanavalin-A--Sepharose into non-reactive (1a) and reactive (2a) molecules. Moreover it was demonstrated that each alpha 1-fetoprotein variant contained either two glycans 1a or two glycans 2a, not randomly, but a pair of the identical carbohydrate chains at the two glycosylation sites.

The isolation and amino-acid and carbohydrate composition of three tryptic glycopeptides, containing 16,17 and 20 amino-acid residues, respectively, from the neuraminidase-treated major form of human ceruloplasmin have been described earlier. It has also been suggested that the peptides possibly represent all of the carbohydrate-binding sites in the protein. The present paper describes the amino-acid sequences of these peptides as obtained by stepwise Edman degradation and carboxypeptidase digestions of the whole peptides and their fragments after cleavage by chymotrypsin, pepsin and thermolysin. The results confirm that the carbohydrate in all cases is attached via an asparagine (or aspartic acid) residue, that is followed by an X-Thr/Ser sequence. No sequence homologies are found when the peptides are compared to each other or to other glycopeptides of known sequence.

The presence and identity of 5'-terminal cap structures in rat brain polysomal mRNA were investigated by radiolabeling the mRNA by periodate oxidation and [3H]sodium borohydride reduction or by beta-elimination of 5'-terminal nucleoside and incorporation of 32P in the presence of polynucleotide kinase. The labeled mRNAs were digested with nucleases and the cap structures were isolated and identified by chromatographic and electrophoretic procedures The results showed that rat brain mRNAs contained cap 1 and cap 2 structures and no caps of the zero type. The proportion of cap 2 was higher than that of cap 1. Both caps had 7-methylguanosine (m7G) as the 5'-terminal nucleoside, which was linked to the next nucleoside by an inverted triphosphate bridge, as in other eukaryotic mRNAs. The most prominent nucleoside in the 5'-penultimate position was 6-methyl-2'-O-methyladenosine [m6A(m)] followed by 2'-O-methyladenosine [A(m)], which together contributed to nearly 70% of both cap 1 and cap 2 structures. 2'-O-Methylguanosine [G(m)] accounted for approximately 18%, the rest being made up of 2'-O-methylcytidine [C(m)] and 2'-O-methyluridine [U(m)].

A cyclic choline analogue, 3-hydroxy-N,N-dimethylpiperidinium, has been shown to be transported into a crude preparation of synaptosomes by high and low affinity transport mechanisms. Under conditions favouring the high affinity system the synaptosomes metabolized approximately 50% of the accumulated analogue to 3-acetoxy-N,N-dimethylpiperidinium, which was detected by paper electrophoresis. The phrenic nerve endplate region of a mouse hemidiaphragm accumulated 3-hydroxy-N,N-dimethylpiperidinium on nervous stimulation. This tissue metabolized approximately 60% of the accumulated analogue to the acetylated derivative which was released on nervous stimulation into the bathing medium. Compared to acetylcholine, 3-acetoxy-N,N-dimethylpiperidinium was shown to be 57 times less potent an agonist at the nicotinic receptors of the frog rectus abdominis muscle and 162 times less potent an agonist at the muscarinic receptors of the guinea-pig ileum. It is concluded that 3-hydroxy-N,N-dimethylpiperidinium is a precursor of a false cholinergic transmitter.

SYNOPSIS The amino acid composition of the acid soluble fraction of Euglena gracilis was determined from cells grown in 4 different culture media. Glutamic acid is the major free amino acid. Hydrolysis of this fraction increases the amount of free amino groups, the major amino acids found are then glutamic acid, aspartic acid, glycine and arginine. The pattern of amino acid distribution is similar in all 4 culture media. L-arginyl-L-glutamine was isolated and identified in extracts from all 4 culture conditions. It was shown to be a metabolic intermediate by radioactivity chase experiments.

A partial sequence of the manganese-containing superoxide dismutase from Bacillus stearothermophilus has been determined by mass spectrometry. A new fragmentation reaction at N-terminal proline is described and an 'in-chain' fragmentation, also occurring at proline, has been observed. There is some evidence that the latter cleavage may also occur at other residues. Some differences from the classically determined sequence are reported, and the validity of these observations is discussed.

Incubation of calf lens cortex homogenate with [14C]putrescine or dansylcadaverine, followed by two-dimensional gel electrophoresis and fluorography, enabled the identification of three different beta-crystallin chains as the endogenous substrates of Ca2+-dependent lens transglutaminase (R-glutaminyl-peptide:amine-gamma-glutamylyltransferase, EC 2.3.2.13). One of these is beta Bp, the predominant subunit of beta-crystallin, of which the amino acid sequence is known. The site of amine-labeling in beta Bp could be located, by limited proteolysis, in the N-terminal domain of this chain. Tryptic digestion of the N-terminal domain and subdigestion with elastase of the N-terminal tryptic peptide identified glutamine-7 as the single residue to which the amines are bound. This is the first example of an endogenous substrate of intracellular transglutaminase in which the site of the acyl-donor glutamine residue has been established. Tryptic digestion of the putrescine-labeled beta-crystallin aggregate, followed by high-voltage paper electrophoresis, provided a preliminary characterization of the labeled peptides originating from the other two labeled beta subunits.

High-affinity penicillin binding sites from which the antibiotic could not be removed by washings at 4°C in 0.017 M K2HPO4 or 0.05 M Tris-HCl pH 7.5, were shown to occur in the isolated membranes of Streptomyces R61. These sites caused the attachment of 25 picomoles of [14C]benzylpenicillin per milligram membrane protein. Penicillins and cephalosporins competed for the same binding sites. The antibiotic concentrations which excluded [14C]benzylpenicillin from 50% of the binding sites were those which inhibited by 50% the membrane-bound transpeptidase. The same rate constant (about 1 × 10−4 s−1) for the dissociation of the benzylpenicillin membrane complex at 37°C and in 0.017 M K2HPO4, was calculated either from the release of the radioactivity (using [14C]benzylpenicillin) or from the recovery of the transpeptidase activity. These observations supported the conclusion that the high-affinity binding sites in the isolated membranes were the transpeptidase molecules. All the complexes formed between the membranes and the various penicillins and cephalosporins examined were reversible at 37°C and in 0.017 M K2HPO4 at least with regard to the transpeptidase. Depending upon the antibiotics, the rate constants for the dissociation of these complexes varied from 3.3 × 10−3 to 0.73 × 10−4 s−1. The radioactivity released through the dissociation of [14C]benzylpenicillin membrane complex occurred mainly in the form of a compound which behaved as [W]-benzylpenicilloic acid both by paper electrophoresis and thin-layer chromatography. It was impossible to choose between several possible mechanisms for the release of the antibiotic molecule.