Large Tryptic Fragments Research Articles

Analysis of the Primary Structure of the Strongly Hydrophobic Brain Myelin Proteolipid Apoprotein (Lipophilin). Isolation and Amino Acid Sequence Determination of Proteolytic Fragments

Proteolipid aproprotein (lipophilin) and DM-20 protein from bovine brain white matter proved to be identical in polyacrylamide gel electrophoresis and automated Edman degradation of the N-terminal end over 20 cycles. Lipophilin can be hydrolysed by trypsin, thermolysis, chymotrypsin and subtilisin. We describe here a new, effective and rapid high-performance liquid chromatographic separation method for hydrophilic polypeptides according to molecular mass on an analytical and preparative scale. Three large and several small peptides have been isolated from the tryptic and thermolysinolytic hydrolysate and purified by combined molecular sieve and high-performance chromatographic separation and purification for automated Edman degradation. 40 amino acid residues of the large tryptic fragment and sequences of 43 and 22 amino acids of two thermolysinolytic fragments have been determined. These three polypeptides are partial structures of the l4 kDa large tryptophan fragment 1 or the cyanogen bromide fragment I (18-19 kDa). Thermolysin also releases a polypeptide from incompletely reductively carboxymethylated lipophilin which is cleaved into the large thermolysin fragment mentioned, 22 residues of which were analysed, and a 14 amino acids long sequence of tryptophan fragment IV, described in the previous paper. Reductively carboxymethylated liprophilin, the lysine side chains of which were blocked with maleic anhydride, can be cleaved at arginine specific sites. Bio-Gel P-150 and high-performance chromatographic purification yielded a polypeptide, which upon performic acid oxidation was split into a 15 kDa and a 7.8 kDa polypeptide. The 15 kDa polypeptide resembles the N-terminal end as proven by 31 cycles in Edman degradation. The 7.8 kDa polypeptide corresponds to the 72 amino acid C-terminal sequence, which equals cyanogen bromide fragments II, III and IV and embraces tryptophan fragment IV.

Digestion of insulin receptors with proteolytic and glycosidic enzymes-effects on purified and membrane-associated receptor subunits

Insulin receptors, photoaffinity-labeled in rat liver and human placenta membranes, and receptor purified from rat liver membranes, were treated with proteolytic and glycosidic enzymes and analyzed by SDS-polyacrylamide gel electrophoresis. The protease fragments from the purified receptor were similar to those from the photoaffinity-labeled receptors, if these were solubilized prior to digestion. However, the intact photoaffinity-labeled membranes were less sensitive to proteases, and different fragments were produced. Thus, solubilization exposes sites very sensitive to proteolysis. The labeled tryptic fragments of placenta membranes remained membrane-bound. Neuraminidase altered the mobility of the intact receptor and of several of the large tryptic fragments, indicating that these contain sialic acid and are probably exposed on the outer surface of the membrane.

Primary structure of porcine tropoelastin.

Structural studies on porcine aortic tropoelastin as obtained from copper-deficient swine have been performed on the whole protein as well as fragments produced by trypsin, subtilisin and thrombin digestion. Tryptic fragments fall into two catagories, those large, over 20 residues, and those small, 4 residues and less. Analyses of the small peptides has been accomplished by hand sequencing methods and by mass spectrometry. Analyses of the large fragments has been accomplished on the automated spinning cup amino acid sequencer. There is a striking difference in the composition of the two groups of peptides. The small ones contain a large amount of alanine, and appear to be present in sufficient amounts to indicate repeating sequences of these within the protein. The large tryptic fragments can be grouped together as those beginning with tyrosine, those beginning with alanine, and those beginning with other residues such as glycine, valine, phenylalanine and possibly leucine. The most striking feature of the large tryptic fragments is that they contain repeating structures of a tetrapeptlde, Pro-Gly-Gly-Val, of a pentapeptlde, Pro-Gly-Val-Gly-Val and of a hexapeptide, ProGly-Val-Gly-Val-Ala. They also appear to be deficient in alanine at their NH2-termini, but enriched in this amino acid at their C-terminals. Subtilisin digestion of these large peptides gives arginine and lysine-containing fragments similar in composition to the small tryptic peptides. Only a few of the large tryptic peptides have been completely sequenced, but we do have sufficient information to indicate that at least 70% of these large fragments are over 50 residues in size. Some are over 100 residues long.KeywordsLysine ResidueTryptic PeptideSmall PeptideArginine ResidueAlanine ResidueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Crystals of partially trypsin-digested elongation factor Tu

Limited tryptic digestion of elongation factor Tu from Escherichia coli and Bacillus stearothermophilus at room temperature produces a small number of scissions without concomitant loss of GDP binding activity. The small number of large tryptic fragments produced are not separated by gel filtration under non-denaturing conditions and they coelute with the GDP binding activity. Crystals of the trypsin-treated elongation factor Tu from E. coli obtained from polyethylene glycol solutions are apparently identical to the pseudotetragonal crystals previously reported (Sneden et al., 1973).

Does a bacterial elongation factor share a common evolutionary ancestor with actin?

Protein synthesis elongation factor Tu from E. coli shares several physical, chemical, and functional properties with actin-like proteins. Limited tryptic degradation indicates that the two polypeptides have a similar molecular architecture. These observations suggest that they could have evolved from a common ancestor, although more information will be necessary to prove or disprove this hypothesis. A partial sequence, comprising 22 aminoacid residues from the aminoterminal end of the large tryptic fragment of elongation factor Tu is presented.

Amino-acid sequence of lac repressor from Escherichia coli. Isolation, sequence analysis and sequence assembly of tryptic peptides and cyanogen-bromide fragments.

The lac repressor from Escherichia coli, composed of four identical subunits with a molecular weight of 37160, was carboxymethylated and fragmented by tryptic digestion and cyanogen bromide treatment. Using ion-exchange chromatography, gel filtration and preparative thin-layer electrophoresis and chromatography 29 of the 30 tryptic peptides were isolated in pure form. Direct Edman degradation and the dansyl-Edman technique were used to determine the sequence of the small tryptic peptides. Special emphasis was put on the sequence determination of the six large tryptic fragments which together account for 177 residues, corresponding to 51% of the repressor subunit with its 347 residues. The large tryptic fragments were analyzed after fragmentation with chymotrypsin, thermolysin and dipeptidyl aminopeptidase I. Thus the sequence of all 30 tryptic peptides could be deduced. The complete sequences of all cyanogen bromide fragments were deduced from peptides obtained by tryptic, chymotryptic and thermolytic digestion of the individual fragments and by automated stepwise Edman degradation of lac repressor and of the large cyanogen bromide fragments. The order of the cyanogen bromide fragments was given by overlapping tryptic peptides. The resulting amino acid composition of the monomer is Asp15, Asn11, Thr18, Ser30, Glu14, Gln27, Pro13, Gly22, Ala44, Cys3, Val33, Met9, Ile17, Leu40, Tyr8, Phe4, Trp2, Lys11, His7, Arg19. The sequence of lac repressor shows no similarities with that of other proteins known to bind to DNA or RNA. The N-terminal 55 residues contain two homologous regions. This part of the sequence which is involved in lac operator binding might have been formed by gene duplication.

Serine-containing active center of alkaline proteinase of Aspergillus flavus

1. 1. From the soluble portion of the tryptic digest of oxidized diisopropyl [ 32P]-phosphoryl-derivative of alkaline proteinase of Aspergillus flavus a radioactive fragment was isolated and its amino acid composition determined to be Asp 7, Met 1, Thr 5, Ser 8, Glu 4, Pro 3, Gly 7, Ala 9, Val 6, Ile 5, Leu 6, Tyr 2 and Phe 2. 2. 2. Four radioactive peptides were isolated from a partial acid hydrolysate of the slightly soluble portion of the tryptic digest. These peptides provides evidence of the amino acid sequence Gly-Thr-Ser-Met-Ala around the reactive serine residue of alkaline proteinase. This amino acid sequence represents a part of the large tryptic fragment. 3. 3. Similarities in the amino acid sequences of serine-containing active centers of microbial proteinases are discussed.

Simulation of rain environment : ( U.S.A.) M. H. Simpson, Proc. Inst. Environ. Sci., 1961, 65–74

Limited tryptic digestion of elongation factor Tu from Escherichia coli and Bacillus stearothermophilus at room temperature produces a small number of scissions without concomitant loss of GDP binding activity. The small number of large tryptic fragments produced are not separated by gel filtration under non-denaturing conditions and they coelute with the GDP binding activity. Crystals of the trypsin-treated elongation factor Tu from E. coli obtained from polyethylene glycol solutions are apparently identical to the pseudotetragonal crystals previously reported (Sneden et al., 1973).

A high vacuum orbital simulator : ( U.S.A.) W. G. Camack and C. A. Ellis, Proc. Inst. Environ. Sci., 1961, 349–357

Limited tryptic digestion of elongation factor Tu from Escherichia coli and Bacillus stearothermophilus at room temperature produces a small number of scissions without concomitant loss of GDP binding activity. The small number of large tryptic fragments produced are not separated by gel filtration under non-denaturing conditions and they coelute with the GDP binding activity. Crystals of the trypsin-treated elongation factor Tu from E. coli obtained from polyethylene glycol solutions are apparently identical to the pseudotetragonal crystals previously reported (Sneden et al., 1973).