Amino Acid Composition of Purified

Amino Acid Composition of Purified Proteins G. R. Tristram... 

Pradel, L.-A. Kassab, R. Conlay, C. van Thoai, N. Properties and amino acid composition of purified ATP guanidinoacetate phosphotransferase. Biochim. Biophys. Acta, 154, 305-314 (1968)... 

Determination of amino acid composition of purified polypeptide chain. 

Table 4 examines the amino acid composition of purified SCP2 [21] and compares it with the amino acid compositions of 3 other preparations of interest [47-49], The most abundant amino acid in purified SCP2 is lysine, which accounts for 14 mole%. Since the isoelectric point for SCPj is approximately 8.6, it follows that a substantial portion of the glutamic and aspartic acid residues are amidated. Also of interest is the finding that SCPj contains no arginine or tyrosine, and only small amounts (perhaps 1 residue each) of histidine and tryptophan. 

The amino acid composition of purified SCP2 is also compared in Table 4 with the amino acid composition of FABP [48]. Marked differences exist in the abundance of threonine, alanine, valine and leucine. The correlation coefficient between SCPj and FABP is 0.732. Clearly, SCP and FABP are separate and distinct proteins. Furthermore, there is no immunological cross-reactivity between SCP2 and FABP, in an extremely sensitive enzyme-linked immunosorbent assay procedure. 

In Table 4 the amino acid composition of purified SCP2 is also compared with the amino acid composition of a protein described as a nonspecific phospholipid exchange protein (CM2) [49]. The amino acid compositions of SCP2 and CM2 nearly identical. The correlation coefficient is 0.992 [21]. 

The Amino Acid Composition of Purified RBP as Reported for Various Species"... 

Seven diets were constructed from purified natural ingredients obtained from either C3 (beet sugar, rice starch, cottonseed oil, wood cellulose, Australian Cohuna brand casein, soy protein or wheat gluten for protein) or C4 foodwebs (cane sugar, corn starch, com oil, processed corn bran for fiber, Kenya casein for protein) supplemented with appropriate amounts of vitamins and minerals (Ambrose and Norr 1993 Table 3a). The amino acid compositions of wheat gluten and soy protein differ significantly from that of casein (Ambrose and Norr 1993). 

SDS-PAGE revealed only one protein band in the purified AE fractions with a MW of 42,000 D (Fig.2). Isoelectric focusing of AE showed that pi > 9. The amino acid composition of the purified AE is shown in table 1. 

With methods for the quantitative analysis of amino acids to hand, the way was now open for the determination of amino acid sequences. Purified bovine insulin was relatively freely available. On the basis of ultracentrifugal analysis (Gutfreund and Ogston), a molecular weight of 12,000 was assumed—as it later emerged, a factor of 2 too high. One of the advantages from the choice of insulin as the protein to sequence was that tryptophan is absent. A 100% recovery of the amino acids could therefore be obtained easily by simple hydrolysis with HC1. In 1948 Tristram reported the complete amino acid composition of the protein. 

Although CuBS2 elutes at approximately the same position in the salt gradient as CdBSl, this peak is not exclusively PC or glutathione. The amino acid composition of essentially pure CuBS2 (Table 2) reveals a complex mixture of amino acids although high in Glx, Cys and Gly (50%), the amino acid composition is not characteristic of other impure or purified PCs which have 75-90% Glx, Cys and Gly (Table 1 Salt et al., 1989 Tomsett etal., 1989). Furthermore, it is estimated to have a molecular mass of 2700 Da and is remarkably similar to the class I MT from A. bisporus (Table 2). 

One of the cellobiose oxidoreductases present in S. pulverulentum has been characterised and named cellobiose oxidase (Ander and Eriksson, 1978). The enzyme contains both haem and flavin co factors and binds irreversibly to concanavalin A-Sepharose, suggesting that it is a glycoprotein. Cellobiose oxidase from S. pulverulentum has now been purified to homogeneity by Morpeth (1985). The carbohydrate and amino acid compositions of the enzyme have been determined. The enzyme contains FAD and cytochrome b prosthetic groups and is a monomer with an Mr of 74400 determined by sedimentation equilibrium. 

The brain enzyme has been purified over 1000-fold and shown to be homogeneous by ultracentrifugation and electrophoresis criteria (36) the activity ratio for acetyl-P over carbamyl-P remains unchanged with purification. This enzyme is one of the smallest on record the molecular weight from physical data is 13,200 and from amino acid analysis is 12,600 the amino acid composition of the enzyme is given in Table I. The terminal amino acid is aspartic acid (25). Cystine has not been detected. 

As pointed out earlier in this article, T differs from other G proteins in that it is a peripheral membrane protein. After activation by Rho it seems to undergo subunit dissociation in which both its a subunit and its /3y complex dissociate from the Rho-containing membranes. Purification of brain G-proteins has shown that free a subunits of G0 and Gj are also water soluble, remaining in solution in the absence of detergents [74], The hydrophobicity of the whole ajSy G and Gj complexes was shown to be due to their j8y complexes 189]. Indeed, purified a subunits associate with phospholipid vesicles only if j8y complexes have been incorporated during vesicle formation [189]. Since the amino acid composition of T-/3 is equal to that of other G-j8s, but their ys differ, it follows that the principal role of y subunits should be to anchor non-T G proteins to the plasma membranes. This conclusion assumed, of course, that j8 subunits are not post-translationally modified in a tissue specific manner such that that they become water soluble in retinal photoreceptor cells and... 

Etzler and Kabat purified the agglutinating principle from Dolichos seed extracts by adsorption to insoluble polyleucyl hog A + H substance.108,517 Specific elution of the adsorbent with 2-acetamido-2-deoxy-D-galactose gave a protein that appeared homogeneous by immunodiffusion, immunoelectrophoresis, disc-gel electrophoresis under acid and alkaline conditions, and sedimentation analysis. The amino acid composition of the protein of molecular weight 141,000 reflected a high content of aspartic acid and serine, little methionine, and no cysteine. Carbohydrate analysis showed a sugar content of 2.4% of hexose, 1.6% of hexosamine, and 1.5% of 2-acetamido-2-deoxy-hexose. 

The amino acid composition of the crystalline L-arabinanase preparation isolated from Aspergillus niger showed10 relatively small proportions of proline and methionine, and rather larger proportions of serine and threonine this highly purified preparation was also found to contain some carbohydrate material (its composition was not reported) and it is, therefore, probably a glycoprotein. 

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