Amino acids, isolation determination

The PE2 isoform has been purified and fully sequenced (Markovic and Joumval 1986). Using this sequence data Ray et al (1988) succeeded in isolating a clone fi om a tomato fruit cDNA library. The predicted amino acid sequence fi om this clone had high homology to the actual amino acid sequence determined for PE2 but was not identical. Subsequent screening of the tomato... 

In summary, the de novo isolation of the cDNAs encoding enzymes of alkaloid biosynthesis is still achieved by using a variety of classical techniques, such as protein purification followed by partial amino acid sequence determination, and by newer techniques such as proteomics coupled to functional heterologous expression. The current status of cloned cDNAs specifically related to isoquinoline alkaloid biosynthesis is schematically presented in Figure 10.8. New additions to this list will certainly be made in the future as a result of a combination of approaches both new and old. 

High-voltage electrophoresis and subsequent paper chromatography of the fractions obtained made possible the isolation from the analyzed mixture of twenty-two components giving colored spots with ninhydrin and isatin. Among these, fourteen were identified as peptides and their amino acid composition established (Table 5). In the case of eight peptides, also N- and C-terminal amino acids were determined (Table 6). 

The successful expression of recombinant plant peroxidases such as HRP C has been a major focus of research in a number of laboratories. Three synthetic HRP C genes based on the amino acid sequence determined by Welinder (36, 47) were synthesized independently in order to initiate this work (63-65). A number of different expression systems have been evaluated (64, 66-73), a summary of which is presented in Table I. Refolding of recombinant HRP C isolated from inclusion... 

Human apoA-I is a major constituent of HDL, with an Mr of approximately 28,300, calculated from the known primary structure (Bl, B43). ApoA-I is initially synthesized as a 267-amino-acid precursor protein, pre-pro-apoA-I (G25, G26), containing an 18-amino-acid prepeptide and a 6-amino-acid propeptide [determined by nucleic acid sequence analysis of cloned apoA-I (L6), and by isolating the primary translation product of human intestinal apoA-I mRNA (G25)]. 

Incubation of a protein fraction from blood plasma with trypsin gives rise to peptides with conspicuous biological effects. Pain, dilation of peripheral blood vessels, increased coronary flow and enhanced capillary permeability were observed on administration of these protein fragments [8]. In the early sixties the nonapeptide bradykinin and its precursors, kallidin and methionyl-kalUdin were isolated in pure form and their amino acid sequences determined soon after. 

We investigated the biosynthesis of component amino acids of glutathione (GSH) in vitamin-Bi2 or folic-acid deficient rats (1). Glycine-2-C or L-cystine-S was administered intraperitoneally to normal or the deficient rats. Liver GSH was determined, then isolated, and the radioactivity of GSH and its component amino acids was determined. The extent of incorporation of either glycine-2-C or cysteine-S into the respective moieties of GSH was not appreciably affected by either deficiency, indicating that neither vitamin is involved in the sjrstems which catalyze the synthesis of GSH from glycine, cysteine, and glutamic acid, a conclusion which is in accord with those of Bloch (2) and Snoke and Rothman... 

This work was extended by the important investigations of Weigert et al. (56,57) who investigated the amino acid sequences of the variable portions of twelve different X chains. All the tryptic peptides were aligned and their amino acid compositions determined. A comparison with the sequence reported by Appella permitted the ordering of amino acids. The polypeptide chains sequenced included X chains isolated from IgA myeloma proteins, as well as urinary Bence Jones proteins from mice with serum myeloma proteins of the IgA, IgM, and IgG2a classes. Two of the proteins were obtained from the transplantable tumors that were used by Appella and Perham. 

Three clones of cDNAs encoding glycerol-P acyltransferase were isolated from Xgtll cDNA libraries made from poly(A) RNA of squash cotyledons by immunological selection and cross-hybridization. Fig. 1 shows the sequence of the isolated cDNAs which had 1,781 bp as a whole. One of the resultant clones contained an open-reading frame of 1,188 bp. The amino-acid sequence deduced from the nucleotide sequence matched the partial amino-acid sequence determined for the enzyme. The results suggest that a precursor protein of 396 amino-acid residues is synthesized first, and then processed to a mature enzyme of 368 amino-acid residues. 

We have isolated a cDNA clone encoding either AT2 or AT3 and deduced the total amino-acid sequence (Ishizaki et al. 1988). The partial amino-acid sequences determined for AT2 and AT3 were found in the deduced sequence. The amino-acid composition calculated for the deduced sequence was also in good agreement with those determined for AT2 and AT3 (Table 2). 

Hydroxylysine is an amino acid isolated from collagen. Determine the number of possible stereoisomers. 

Studies of the structure of papain were initiated by Thompson (165), who determined the amino or N-terminal amino acid sequence and the free amino groups. Crystalline papain and mercuripapain were allowed to react with l-fluoro-2,4-dinitrobenzene (FDNB) by the procedures of Sanger (136). The dinitrophenyl (DNP) proteins were subsequently hydrolyzed in 6 iV HCl and the DNP-amino acids isolated by partition chromatography of the ether extracts of the hydrolyzates. Quantitative estimates of the DNP-amino acids were made spectrophotometrically. The results are given in Table V. 

---