Succinyl Residues Research Articles

An experimental study of the conformational energies of poly(neopentyl glycol succinate)

Values of the dipole moment ratio 〈µ2〉/nm2 of poly(neopentyl glycol succinate), obtained from dielectric measurements on dilute solutions of the polymer in benzene, varied from 0.548–0.586 in the temperature range 30–60 °C. From thermoelastic experiments, performed on polymer networks prepared by cross-linking hydroxy-terminated poly(neopentyl glycol succinate) chains, a value of 0.52 × 10–3 K–1 was obtained for the temperature coefficient of the unperturbed dimensions. Critical analysis of the thermoelastic and dielectric results, using the rotational isomeric state model, suggests that whereas the gauche states about the C–C bonds of the neopentyl glycol residue have an energy of ca. 0.6 kcal mol–1 lower than that of the alternative trans states, the former conformational states are disfavoured with respect to the corresponding trans states in the case of the central C–C bond of the succinyl residue. This study also suggests that the skeletal bonds of the type OC–C bonds are almost freely rotating.

Enzyme immobilization via monoclonal antibodies I. Preparation of a highly active immobilized carboxypeptidase A

A novel method for the preparation of highly active immobilized enzymes is described. It is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The applicability of the method forwarded has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations as follows: A mouse monoclonal antibody (mAb 100)to CPA that binds to the enzyme with a high-affinity constant without affecting its catalytic activity was prepared, purified, and characterized. Covalent binding of this monoclonal antibody to Eupergit C (EC) or noncovalent binding to Sepharose-protein A (SPA)yielded the conjugated carriers EC-mAb and SPA.mAb, respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb.CPA and SPA.mAb.CPA displaying full catalytic activity and improved stability. At pH 7.5 and a temperature range of 4-37 degrees C an apparent binding constant of approximately 10(8)M(-1) characterizing the interaction of CPA with EC-mAb and SPA.mAb, was obtained. To compare the properties of EC-mAb.CPA and SPA.mAb.CPA with those of immobilized CPA preparations obtained by some representative techniques of covalent binding of the enzyme with a corresponding carrier, the following immobilized CPA preparations were obtained and their properties investigated: EC-CPA (I), a preparation obtained by direct binding of EC with CPA; EC-NH-GA-CPA (II), a derivative obtained by covalent binding of CPA to aminated EC via glutaraldehyde; EC-NH-Su-CPA (III), a CPA derivative obtained by binding the enzyme to aminated EC via a succinyl residue; and EC-HMD-GA-CPA (IV), obtained by binding the enzyme via glutaraldehyde to a hexamethylene diamine derivative of EC. Full enzymic activity for all of the bound enzyme, such as that recorded for the immobilized CPA preparations EC-mAb.CPA and SPA.mAb.CPA, was not detected in any of the insoluble covalently bound enzyme preparations.

Comparison of the chemical composition of lipomannan fromMicrococcus agilismembranes with that ofMicrococcus luteusstrains

A neutral lipomannan has been isolated from the membranes of Micrococcus agilis. In contrast to the lipomannans from 2 strains of Micrococcus luteus, which contained succinic acid ranging from 5.1%–8.0%, the M. agilis lipomannan had no detectable succinyl residues and exhibited neutral behaviour on Concanavalin A-agarose rocket electrophoresis. As with the M. luteus lipomannans, mannose was the only sugar detectable (as alditol acetate) by GLC analysis in the purified M. agilis lipomannan. Fatty acids accounted for 2% of the M. agilis lipomannan and were predominantly C15 branched-chain acids, with higher amounts of C16iso and C17anteiso than that found in the M. luteus polymers. Neither conditions of growth of the organism nor the method of membrane preparation appeared to be responsible for the absence of succinyl residues. This appears to be the first report of a neutral membrane amphiphile.

Inactivation of the 2-ketoglutarate and pyruvate dehydrogenase complexes of beef heart by branched chain keto acids.

Incubation of 2-ketoglutarate dehydrogenase complex with 2-ketoisovalerate, 2-keto-4-methylvalerate, or 2-keto-3-methylvalerate leads to the appearance of a lag phase and of a progressive loss of activity in subsequent measurements of the initial rate of oxidation of 2-ketoglutarate. In the case of 2-ketoisovalerate these effects are shown to be due to the formation of an isobutyryllipoate derivative of the enzyme, as a result of the very slow oxidation of 2-ketoisovalerate by the enzyme complex (Vmax congruent to 0.15% of that for 2-ketoglutarate). Incubation of the enzyme complex with 2-keto[14C]isovalerate or 2-keto[14C]glutarate results in comparable incorporation of radioactivity, amounting to 3.5 to 5.3 nmol of isobutyryl or succinyl residues per mg of protein in the complex. Isobutyryl residues are also incorporated in the enzyme during the simultaneous oxidation of both of these substrates. During the early phase of incubation of the complex with 2-ketoisovalerate the incorporation of isobutyryl residues is much faster than the loss of enzyme activity. This observation seems to support the suggestion that each 2-ketoglutarate decarboxylase subunit of the complex may catalyze the succinylation of more than one lipoate succinyltransferase subunit. Results are also presented showing the inactivation of pyruvate dehydrogenase complex on preincubation with 2-ketoisovalerate and of 2-ketoglutarate dehydrogenase complex with methylenecyclopropylpyruvate, the keto acid corresponding to the toxic amino acid hypoglycin. The relevance of covalent modifications of the two keto acid dehydrogenase complexes to the pathological manifestations of maple syrup urine disease are discussed.

Intra and inter generic homology in polysaccharide structure ofRhizobium andAlcaligenes

A study was conducted on the structure of extracellular, water-soluble polysaccharides from 5 different strains ofRhizobium viz. R. trifolii J60 andR. meliloti strains J7017, 202, 204 and 207. All these polysaccharides were found to contain glucose and galactose in the approximate molar ratio of 7:1. Methylation analysis revealed these polysaccharides to contain (1 → 3), (1 → 6), (1 → 4), (1 → 4, 1 → 6)-linked D-glucose residues, (1 → 3)-linked D-galactose and nonreducing terminal D-glucose attached to pyruvate. These polysaccharides were also found to be acylated by both acetyl and succinyl residue. This structure was found to be similar to that of succinoglycan, a succinic acid-containing water-soluble, extra-cellular polysaccharide elaborated byAlcaligenes faecalis var.myxogenes 10C3. This similarity in structure of polysaccharides from two different species ofRhizobium and also the polysaccharide produced byAlcaligenes has been discussed.

Effects of Phosphate Limitation of Growth on the Cell-wall and Lipid Composition of Saccharomyces cerevisiae

The phosphorus content of phosphate-limited Saccharomyces cerevisiae was only 71% of that of non-limited yeast. Walls prepared from phosphate-limited cells contained slightly less phosphorus than control walls. No evidence was obtained for the presence in these walls of uronic acid or succinyl residues. The carbohydrate content of walls of phosphate-limited yeast was less than that of non-limited walls, and this was reflected in a decreased glucan content. There was only a slight decrease in glucosamine content while the protein content increased. The major change in the lipid composition of phosphate-limited yeast was a decrease in both sterol esters and triacylglycerols. There was a decrease in total lipid content, but increased production of phosphatidylethanolamine and phosphatidylcholine. The phosphatidylserine content was decreased. These results suggest that there are fewer intracellular low-density vesicles in phosphate-limited yeast.

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80°C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem. Biophys. Res. Commun. 63, 875–880). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB 3H 4. The polysaccharide was only slightly degraded (2%) by jack bean α-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymer was also detected in the cell “periplasm” and was secreted from cells grown in defined media during the period of decelerating growth.

The biological activity and the binding affinity of modified insulins determined on isolated rat fat cells

A number of modified insulins were assayed for biological activity (increase in synthesis of lipids from glucose) and binding affinity (inhibition of receptorbinding of125I-insulin) on rat fat cells and the following was found: -1. The modified insulins tested showed the same maximal effect as native insulin. Decrease in biological activity induced by modifications was interpreted as decrease in potency. -2. Removal of 2 amino acids from the amino-terminal end of the B chain caused little or no decrease in potency. Removal of 5 amino acids from the carboxy-terminal end of the B chain caused a decrease in potency to about 17% of that of insulin. Removal of one amino acid (glycine) from the amino-terminal end of the A chain caused a decrease in potency to about 1 %. -3. Substitutions with acetyl or succinyl residues at position Al had a greater effect on the potency than substitution at position B1 or B29. -4. Cross linkage between positions A1 and B29 caused decreases in potency on fat cells to between 2 and 10%. Cross linkage between positions A1 and B1 almost abolished biological activity. −5. 9 of the modified insulins were tested for ability to inhibit binding of125I-insulin to fat cell receptor sites. The decrease in receptor binding affinity induced by chemical modifications was, in all cases, in agreement with the decrease in biological potency.-6. It is concluded that the binding affinity of insulin to the receptor, and therefore the potency, is dependent on (1.) a largely unchanged tertiary structure of the insulin molecule and (2.) free access to the region of the amino-terminal end of the A chain.

Biological activities of chemically modified endotoxins.

The cell wall glycolipid (somatic antigen) of the Re mutant of Salmonella minnesota is comparable in its endotoxic activity to the complete wild type lipopolysaccharide. The glycolipid is composed mainly of lipid A and 2‐keto‐3‐deoxyoctonate (KDO); smaller amounts of ethanolamine and 4‐aminoarabinose, and traces of amino acids are also present. The serological specificity is determined by KDO residues. In order to determine if non‐lipid A constituents contribute to endotoxicity, amino groups and KDO units in the glycolipid were chemically modified by the introduction of substituents. When free amino groups were dinitrophenylated or succinylated the serological specificity was unaffected. By contrast, the original serological specificity was completely lost when the free hydroxyl groups of the KDO region were substituted with succinyl residues or the carboxyl groups converted to the methyl esters. In biological tests, however, the modified preparations exhibited full endotoxic activity. It is concluded that the polar groups, which determine the serological specificity of the KDO region of the glycolipid, do not play a specific role in endotoxicity.

Some immunochemical properties of succinylated human transferrin and related proteins

Fully succinylated transferrin, immune globulins, and orosomucoid lost their ability to form precipitates with antisera to their native counterparts. If used to immunize rabbits, the succinylated transferrin gave rise primarily to antibodies specific for the succinyl residues, hence, several otherwise different succinylated proteins formed precipitin patterns with partial identity characteristics on double diffusion slides. On the other hand, if succinylation was not complete ( e.g., iron-saturated transferrin subjected to succinylation), antibodies to the non-succinylated area (“active site”?) were formed in addition to the succinyl residues.