Biomedical subjects
A Neuberger
Publications and source records attributed to A Neuberger.
Purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of its properties with lectins of similar specificity.
1. The lectin from the broad bean (Vicia faba) was purified by affinity chromatography by using 3-O-methylglucosamine covalently attached through the amino group to CH-Sepharose (an omega-hexanoic acid derivative of agarose). Its composition and the nature of its subunits were compared with concanavalin A and the lectins from pea and lentil. 2. Unlike the other three lectins, broad-bean lectin is a glycoprotein; a glycopeptide containing glucosamine and mannose was isolated from a proteolytic digest. 3. The mol.wt. is about 47500; the glycoprotein consists of two apprently identical subunits, held together by non-covalent forces. Fragments of the subunits, similar to those found in concanavalin A and soya-bean agglutinin, were found in active preparations. 4. Broad-bean lectin was compared with concanavalin A and the lectins from pea and lentil in an investigation of the inhibition of their action by a number of monosaccharides, methyl ethers of monosaccharides, disaccharides and glycopeptides. The most striking differences concern 3-O-substituted monosaccharides, which are strong inhibitors of the action of broad-bean, pea and lentil lectins but not of the action of concanavalin A. There is, however, no strong inhibition of the action of these lectins by 3-Olinked disaccharides.
Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides.
Rhodopseudomonas spheroides can grow in a defined medium with either light or oxygen as an energy source. Cells grown anaerobically or at very low oxygen tensions are rich in the photosynthetic pigment bacteriochlorophyll, whereas this pigment is virtually absent in cells grown under high oxygen tensions. Aminolaevulinate synthetase, the first enzyme on the pathway to bacteriochlorophyll, appears to play an important role in the control of bacteriochlorophyll synthesis. Thus, the enzyme has a high activity in extracts of pigmented cells and a low activity in extracts of non-pigmented cells. Further, oxygenation of a pigmented culture causes immediate cessation of pigment synthesis and produces a rapid fall in the activity of aminolaevulinate synthetase. This loss of activity appears to be due to the loss of an endogenous activator of the enzyme. Thus, pigmented cells contain cystine trisulphide, which at muM concentrations is an activator of aminolaevulinate synthetase, while oxygenation causes a rapid fall in the cellular content of this trisulphide. Cystathionase (EC 4.2.1.15) extracted from pigmented cells can catalyse the formation of cystine trisulphide from cystine, while rhodanese (EC 2.8.1.1) extracted from the same cells can catalyse the degradation of cystine trisulphide in the presence of sulphite to form cystine and thiosulphate. It is proposed that the cellular content of cystine trisulphide is controlled by changes in the levels of substrates for cystathionase and possibly rhodanese rather than changes in the amounts of these enzymes. Cystine trisulphide controls the activity of aminolaevulinate synthetase by converting a low-activity form of the enzyme (b-form) into a high-activity form (a-form). The fall in aminolaevulinate synthetase activity on oxygenation appears to be the result of cessation of conversion of b-form into a-form, along with a conversion of a-form into b-form. Factors affecting the equilibrium between the forms and the possible mechanisms for their interconversion are discussed.
The separation of methyl ethers of methyl 2-acetamido-2-deoxy-D-glucopyranosides on a strongly basic ion-exchange resin.
The methyl ethers of methyl 2-acetamido-2-deoxy-alpha- and -beta-D-glucopyranoside can be isolated on a preparative scale by chromatography on Dowex-1(HO-) resin. This procedure greatly simplifies the purification of methyl ethers, and has been used to isolate the methyl ethers produced by partial methylation of methyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The separations are thought to depend on an ion-exchange process in which all the free hydroxyl groups are involved. It is concluded that the following acidity sequence holds: HO-4 greater than HO-3 greater than HO-6.
The quantitation of glucosamine and galactosamine in glycoproteins after hydrolysis in p-toluenesulphonic acid.
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A simple method for the preparation of an affinity absorbent for soybean agglutinin using galactosamine and CH-Sepharose.
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Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides a role for trisulphides.
1. The aminolaevulinate synthetase activator of fresh extracts of semi-anaerobically grown Rhodopseudomonas spheroids was resolved into two fractions by ion-exchange chromatography. One fraction was identified as cystine trisulphide (CySSSCy). Analysis of the other fraction indicated the presence of about equal amounts of glutathione trisulphide (GSSSG) and the mixed trisulphide of glutathione and cystine (GSSSCy). 2. Four further fractions with activator activity were observed on ion-exchange chromatography of extracts prepared by methods similar to those described earlier [Neuberger et al. (1973)Biochem. J. 136,491-499]. These activators were generated by the extraction procedure. Two of them have been identified as trisulphanedisulphonate (S5O62-) and additional cystine trisulphide. 3. For the series of polysulphanedisulphonates (-O3S-Sn-SO3-, n greater than or equal to 1), activator activity at muM concentrations was exhibited only by compounds with n greater than 3. This, together with the results described above, indicates that for a compound R-Sn-R' (where R and R' are organic or inorganic groups) the only structural requirement for activity is n greater than or equal to 3. 4. Oxygenation of a semipanaerobic culture of R. spheroids for 1.5h before harvesting the cells produced a decrease of more than 90% in the cellular content of cystine trisulphide and glutathione trisulphides. 5. Chromatography on DEAE-Sephadex confirmed the presence of multiple forms of aminolaevulinate synthetase in extracts of R. spheroides [Tuboi et al. (1970) Arch.Biochem. Biophys. 138,147-154]. Oxygenation of a semi-anaerobic culture resulted in the disappearance of high-activity enzyme (a-form) and the accumulation of low-activity enzyme (b-form) in the cell. Spontaneous activation [Marriott et al. (1969) Biochem. J. 111,385-394] And activation by cystine trisulphide both resulted in the almost complete conversion of the b-form into the a-form.
Enzyme induction.
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A protein, immunologically similar to Tamm-Horsfall glycoprotein, produced by cultured baby hamster kidney cells.
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The purification, composition and specificity of wheat-germ agglutinin.
1. The purification of wheat-germ agglutinin from commercial wheat germ is described. By ion-exchange chromatography three active proteins (isolectins) were separated, one of which was examined in detail. 2. The amino acid composition is unusual, as 20% of residues are half-cystine and 21% are glycine. Unlike most lectins and contrary to previous reports, this protein is not a glycoprotein. 3. The efficiency of various saccharides as inhibitors of the agglutination reaction was investigated and from this the specificity of the binding site was inferred. Of monosaccharides, only derivatives of glucose with a 2-acetamido group and a free 3-hydroxyl group are effective inhibitors, and glycosides of either anomeric configuration are bound. Oligosaccharides are much more powerful inhibitors of agglutination than are monosaccharides. 4. It is proposed that the binding site consists of three or four subsites with differing specificities, in a cleft in the molecule resembling that proposed for hen's-egg-white lysozyme.
Polypyrroles formed from porphobilinogen and amines by uroporphyrinogen synthetase of Rhodopseudomonas spheroides.
1. Uroporphyrinogen I synthetase of Rhodopseudomonas spheroides was purified more than 200-fold from the soluble protein of broken bacterial cells. The enzyme had molecular weight 36000, an isoelectric point of 4.46 and migrated as a single active protein band on disc-gel electrophoresis at pH7.5 and 8.9. 2. The enzyme consumed porphobilinogen and formed uroporphyrinogen at pH8.2 without the accumulation of intermediates. In the presence of hydroxylamine, ammonia or methoxyamine the production of porphyrinogen was inhibited and the enzyme formed open-chain polypyrroles instead. 3. These polypyrroles behaved like uroporphyrinogen on Sephadex G-25; they were colourless and had unsubstituted alpha-pyrrolic positions. The inhibitory amines were incorporated into the molecules. 4. The polypyrroles formed porphyrins non-enzymically and the cyclization reaction was accompanied by the release of the inhibitory amine. Exchange of the amino function of the original porphobilinogen in the polypyrrole was complete with hydroxylamine and almost complete with methoxyamine, both ammonia and methoxyamine being present in the polypyrrolic material. 5. The behaviour, properties and composition of the radioactive hydroxylamine derivative were consistent with a tetrapyrrolic structure, probably a pyrrylmethane, that was not cyclized, rather than with di-, tri- or penta-pyrrolic structures. No monopyrrolic or dipyrrolic Ehrlich-positive material was released on cyclization. The ammonia and methoxyamine derivatives had properties similar to the hydroxylamine derivative. 6. Another modified pyrrole was detected only in experiments with hydroxylamine. It differed from both porphobilinogen and known dipyrroles and appeared to be a monopyrrole. 7. The participation of positively charged reaction centres in the enzymic mechanism, particularly in the cyclization step, is discussed.
Kinetic studies on the acid hydrolysis of the methyl ketoside of unsubstituted and O-acetylated N-acetylneuraminic acid.
The hydrolysis of the model compound 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl-alpha-d-neuraminic acid and neuraminidase (Vibrio cholerae) closely resembled that of the O-acetylated sialic acid residues of rabbit Tamm-Horsfall glycoprotein. This confirmed that O-acetylation was responsible for the unusually slow rate of acid hydrolysis of O-acetylated sialic acid residues observed in rabbit Tamm-Horsfall glycoprotein and their resistance to hydrolysis by neuraminidase. The first-order rate constant of hydrolysis of 2-methyl-N-acetyl-alpha-d-neuraminic acid by 0.05m-H(2)SO(4) was 56-fold greater than that of 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl -alpha-d-neuraminic acid. Kinetic studies have shown that in the pH range 1.00-3.30, the observed rate of hydrolysis of 2-methyl-N-acetyl-alpha-d-neuraminic acid can be attributed to acid-catalysed hydrolysis of the negatively charged CO(2) (-) form of the methyl ketoside.
The purification and properties of the lectin from potato tubers, a hydroxyproline-containing glycoprotein.
1. Potato lectin has been purified and shown to be a glycoprotein containing about 50% of carbohydrate. Most of the sugar residues (92%) are arabinose; small amounts of galactose, glucose and glucosamine are also present. 2. The most abundant amino acid is hydroxyproline (16% of the residues), 11.5% of the residues are half-cystine and phenylalanine is absent. The lectin also contains about one residue/molecule of a basic amino acid, not usually found in proteins, which has been tentatively identified as ornithine. There is indirect evidence that the components of the glycoprotein are linked through hydroxyproline and arabinose. 3. By gel filtration in 6m-guanidine-HCl on Sepharose 4B, it was found that both the native glycoprotein and its S-carboxymethylated derivative had subunit molecular weights of 46000 (+/-5000). In a non-denaturing solution, two of these units appear to be associated. 4. The lectin is specifically inhibited in its agglutination reaction by oligosaccharides that contain N-acetylglucosamine. Its specificity is similar to, but not identical with, that of wheat-germ agglutinin.
Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism.
1. The ;initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ;maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80-90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65-75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ;low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ;high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.
Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The purification and properties of an endogenous activator of the enzyme.
1. A low-molecular-weight activator of 5-aminolaevulinate synthetase was detected in extracts of Rhodopseudomonas spheroides. The compound activates the enzyme extracted from oxygenated semi-anaerobically grown organisms by a factor of 6-8. 2. The activator was extensively purified, but owing to the exceedingly small amounts that could be extracted in the active form its structure was not determined. 3. The activator contains an acetylatable amino group; it is more stable at acid than at alkaline pH values; it is stable to treatment with I(2)-KI or potassium ferricyanide, but irreversibly inactivated by Na(2)S(2)O(4) or NaBH(4). 4. The chromatographic, electrophoretic, chemical and stability properties of the activator are similar to those of pteridines; purified activator preparations contain pteridines, as shown by their fluorescence spectrum. This does not, however, constitute an identification of the activator. 5. The activator enhances the activity of crude and partially purified enzyme and does not appear to require other endogenous factors or a supply of air to produce activation. Activation of the purified enzyme, however, requires the presence of either pyridoxal phosphate or sodium succinate. In the absence of both these factors the activator produces a time- and temperature-dependent decay of activity.
The turnover rate of rabbit urinary Tamm-Horsfall glycoprotein.
1. The turnover rate of urinary Tamm-Horsfall glycoprotein in rabbits was determined by two different methods. The first involved measurement of the pool size of the glycoprotein in rabbit kidney and the daily urinary excretion rate by a radioimmunoassay from which the turnover rate was calculated. 2. The second method made use of the incorporation in vivo of Na(2) (14)CO(3) and sodium [(14)C]acetate. After a single intramuscular injection of one of these compounds, urine collections were made every 24h and the glycoprotein was isolated and its specific radioactivity was determined. 3. Incorporation of the label into urinary HCO(3) (-), urea and plasma fibrinogen was also examined. The specific radio-activities of the O-acetyl, sialic acid, aspartic acid and glutamic acid residues isolated from the Tamm-Horsfall glycoprotein were compared and their half-lives were compared with that of the intact glycoprotein. The two methods gave results in quite close agreement and indicated a half-life for the glycoprotein of approx. 9h. 4. An attempt was made to localize the glycoprotein within the kidney and within the cell. It is present throughout the kidney, but was not detected in the brush-border fraction isolated from the proximal tubules. From differential cell-centrifugation studies, the glycoprotein seemed to be predominantly present in the soluble fraction (100000g supernatant). This suggests that it is either largely a soluble cytoplasmic component or is very loosely bound to a membrane, being readily released under the gentlest homogenization procedure. 5. The half-life of Tamm-Horsfall glycoprotein in human kidney was found by the radioimmunoassay method to be approx. 16h. The similarity between the composition of Tamm-Horsfall glycoprotein and human erythropoietin is discussed.
The development of a radioimmunoassay for the measurement of urinary Tamm-Horsfall glycoprotein in the presence of sodium dodecyl sulphate.
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