Succinylated proteins

The only disadvantage of the succinylation procedure (which is practical and amenable to conventional cell disruption processes) is that the final product is a succinylated protein. Succinyl groups cannot be removed from the succinylated proteins under mild conditions. This could be a problem if succinylated yeast protein was a major source of dietary proteins. Therefore we explored the feasibility of using reversible modifying reagents (citraconic anhydride and maleic anhydride) to separate proteins from NAs and subsequently remove the modifying groups under mild acidic conditions. 

Chemical modification of yeast protein has received limited attention though as described above it has potential as a method for facilitating recovery of yeast protein. Current studies are concerned with determination of the functional properties of proteins succinylated during the extraction. The composition of yeast proteins prepared by different methods is shown (Table 8). Noteworthy is the protein and nucleic acid concentration in the yeast isolate which differed from the concentrate in that cell wall material was removed by centrifugation. 

Functional properties of canola protein products can be improved by succinylation (130,131). Controlled acetylation can reduce undesirable phenoHc constituents as well (132). However, antinutrients in canola and other vegetable protein products such as glucosinolates, phytic acid, and phenoHc compounds have severely limited food appHcations of these products. 

Figure 16-2. The citric acid cycle the major catabolic pathway for acetyl-CoA in aerobic organisms. Acetyl-CoA, the product of carbohydrate, protein, and lipid catabolism, is taken into the cycle, together with HjO, and oxidized to CO2 with the release of reducing equivalents (2H). Subsequent oxidation of 2H in the respiratory chain leads to coupled phosphorylation of ADP to ATP. For one turn of the cycle, 11 are generated via oxidative phosphorylation and one arises at substrate level from the conversion of succinyl-CoA to succinate.

The following protocol is based on the method of Morris and Saelinger (1984) for the labeling of succinylated avidin with gold particles of 5.2nm diameter. Succinylated avidin was used to reduce the pi of the protein, thus eliminating nonspecific binding due to the strong positive... 

Prepare 5 ml of a lmg/ml succinylated avidin solution by dissolving the protein in 50mM sodium phosphate, pH 7.5. 

Klotz, I.M., and Keresztes-Nagy, S. (1962) Dissociation of proteins into subunits by succinylation haem-erythrin. Nature (London) 195, 900. 

The thermodynamic activation parameters for the enzyme-catalysed reaction are very different from those for the uncatalysed process (Albers et at., 1990). For the isomerization of succinyl-alanyl-leucyl-prolylphenylalanyl-p-nitroanilide catalysed by recombinant human FK binding protein AH = 5.85 kcal mol"1 and AS = -44. e.u. This compares with figures of 18.9 kcal mol 1 and — l.lbe.u. for the uncatalysed reaction of the same substrate. Probably a different step is rate determining in the enzyme-catalysed reaction. 

In addition to their well known role in protein structure, amino acids also act as precursors to a number of other important biological molecules. For example, the synthesis of haem (see also Section 5.3.1), which occurs in, among other tissues, the liver begins with glycine and succinyl-CoA. The amino acid tyrosine which maybe produced in the liver from metabolism of phenylalanine is the precursor of thyroid hormones, melanin, adrenaline (epinephrine), noradrenaline (norepinephrine) and dopamine. The biosynthesis of some of these signalling molecules is described in Section 4.4. 

The viability and function tests described above are used to evaluate the hepatocytes within the slice. Up to now, tests to measure the viability of the non-parenchymal cells have not been reported. The presence of the latter cell types is one of the conceptual advantages of slices as compared to isolated hepatocytes. As some drug targeting devices are designed to target non-parenchymal cells in the liver, the development of tests for the sinusoidal cell types deserves more attention. For example, the uptake of substrates such as succinylated human serum albumin (Suc-HSA,which is specifically endocytosed by endothelial cells [79]), or hyaluronic acid [80], can be used to assess the functionality of endocytotic pathways in the endothelial cells in the liver [81]. Other modified proteins that are specifically taken up by Kupffer cells such as mannosylated HSA, may be used to assess the functionality of the endocytotic pathway in Kupffer cells [79]. Another parameter which can be used to assess the functionality of these non-parenchymal liver cells, is the excretion of cytokines in response to pro-inflammatory stimuli. Non-parench5mal cell function in liver slices will be described in more detail in the Section 12.7. 

The smallest member of a new family of prolyl iso-merases (unrelated to the cyclophilins or the FK-506 binding proteins) that catalyzes the proline-limited folding of a variant of ribonuclease T1 with a KJK value of 30,000 M s With the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide as a substrate in parvulin-catalyzed prolyl isomerization, this parameter is 1.1 x 10 M s Parvulin also accelerates its own refolding in an autocatalytic fashion. 

Soy protein is a low-cost food protein with good nutritional value, but its uses in foods are limited because of inferior functional properties as compared to those of commonly used animal proteins such as casein and albumin (1.2). Therefore, modifications are often required to make soy protein more suitable for food use. Improved functional properties, particularly in the pH range of 3 to 7 where most food systems belong, have been achieved by non-enzymatic methods, including succinylation (3-5), deamidation (6.7), and phosphorylation (8.9). 

Lahti CJ, d Oliveira CE, Johnson PJ (1992) 3-Succinyl-coenzyme-A synthetase from Trichomonas vaginalis is a soluble hydrogenosomal protein with an amino-terminal sequence that resembles mitochondrial presequences. J Bacteriol 174 6822-6830... 

Fig. 2 SDS-polyacrylamide gel electrophoresis of T. vaginalis hydrogenosomes purified by isopycnic centrifugation on a Percoll gradient. 1 PFOR 2 malic enzyme, 64-kDa hy-drogenase, and Cpn 60 3 succinyl CoA synthetase fi subunit 4 Hmp35 5 succinyl CoA synthetase a subunit 6 Hmp31 (ATP/ADP carrier) 7 adenylate kinase 8 thiol peroxidase. Well-resolved but unmarked bands mostly belong to malic enzyme fragments or unknown proteins. Proteins were identified by mass spectroscopy. The 12% gel is stained with Coomassie Brilliant Blue R 250 (authors original)...

Succinyl-CoA synthetase (SCS), also known as succinate thiokinase (STK) or succinate CoA ligase ( 6.2.1.4-5), is so far the only known hydrogenosomal enzyme directly involved in energy conservation. The protein catalyzes the reversible, substrate-level phosphorylation of ADP or GDP to the respective triphosphate at the expense of the high-energy thioester bond of succinyl-CoA. Succinate and CoA are released in the reaction. The I vaginalis enzyme... 

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