Structure of the antitumor protein neocarzinostatin. Purification, amino acid composition, disulfide reduction, and isolation and composition of tryptic peptides

The antitumor protein neocarzinostatin has been purified by repeated CM-cellulose chromatography and Sephadex G-50 gel filtration. The protein possesses 109 amino acid residues in a single chain, crosslinked by two disulfide bonds. Reduction and S-alkylation could not be accomplished in aqueous solution and required the use of dithiothreitol in liquid ammonia, followed by treatment with alkyl chloride. Tryptic digestion of (tetra-S-carboxymethyl)neocarzinostatin afforded five tryptic fragments which were fractionated by preparative paper chromatography and high-voltage paper electrophoresis, purified by Sephadex gel filtration and characterized by amino acid and amino end-group analysis. The total number of amino acid residues of these fragments account for the 109 residues of neocarzinostatin.     

The isolation and amino acid/sugar composition of human fibroblastoid interferon.

Human fibroblastoid interferon produced from an established human cell line was purified by controlled-pore glass and concanavalin A-Sepharose column chromatography followed by preparative two-dimensional gel electrophoresis. The purification procedure provided a 10% recovery of pure interferon with good reproducibility. The purified protein was homogeneous with respect to its molecular weight of 20,000 and net electrical charge at pH 2.5. Interferon of high specific activity of 5 x 10(8) units/mg of protein was directly demonstrated in the polyacrylamide gel before staining with Coomassie brilliant blue. Parallel purification of a sham-induced interferon preparation did not yield an equivalent product indicating the purified interferon is not derived from uninduced cells or from the fetal calf serum of the tissue culture growth medium. Pure interferon was radioiodinated by Bolton-Hunter reagent. Amino acid analysis of the pure preparation shows interferon to be a leucine-rich glycoprotein containing a high percentage of glutamic/glutamine residues and that disulfide bridges(s) are important for its biological activity.     

The isolation, and amino acid and carbohydrate composition, of polymeric collagens prepared from various human tissues

1. Insoluble polymeric collagens from various human tissues were prepared by the EDTA method. Almost all of the collagen from simple soft tissues such as dermis, tendon, submucosa, sclera and cornea could be extracted, whereas the more complex tissues such as intercostal cartilage and intervertebral disc yielded only small amounts of collagen. Amino acid and carbohydrate analysis indicated that most of the preparations were highly purified on the basis of their tyrosine, hexosamine, mannose, xylose and fucose contents. 2. Wide variation in the total hexose content was observed, the lowest being 8.5 residues/3000 amino acid residues for collagen from dermis and the highest being 42.1 residues/3000 in corneal collagen. The molar ratios of sugars also varied, submucosal collagen having a galactose/glucose ratio of 1.0 and corneal collagen having a ratio of 2.3. 3. The presence of glucosylgalactosylhydroxylysine was confirmed in submucosal collagen by compositional and chromatographic analysis of this component after its isolation from alkaline hydrolysates of the collagen. Evidence was also obtained for the presence of galactosylhydroxylysine. 4. Determination of the hydroxylysyl glycosides was carried out and it was observed that the amounts of these components varied widely from tissue to tissue. Corneal collagen contained 19.1 hydroxylysine-linked carbohydrate units/3000 amino acid residues, whereas tendon collagen contained only 4.1 units/3000. Variation in the ratio disaccharide unit/monosaccharide unit was also observed, the ratio being 1.2 in intercostal cartilage collagen and 4.1 in submucosal collagen. The proportion of the total hydroxylysine that was substituted by carbohydrate also varied from tissue to tissue.     

De novo generation of short antimicrobial peptides with simple amino acid composition

As potential therapeutic agents, antimicrobial peptides with shorter length and simpler amino acid composition can be better candidates for clinical and commercial development. Here, we attempted de novo design of short (5- to 11-residue) antimicrobial peptides with three kinds of amino acids. Amphipathic helical properties were conferred by using leucines and lysines and two tryptophan residues were positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. According to this specified rule, 12 model peptides were generated and their helical propensity was confirmed by circular dichroism spectroscopy. Antimicrobial and hemolytic activities were compared with those of the known 12-residue peptide agent, omiganan, which is currently under therapeutic and commercial development. Antimicrobial activities against Gram-negative and Gram-positive bacteria, including a multi-drug resistant strain, were observed for certain 7- to 11-residue models. Among them, the most potent activity was found for a 9-residue peptide (L₅K₂W₂), although it also had severe hemolytic activity. Alternatively, an 11-residue peptide (L₄K₅W₂) with little hemolytic activity was potentially the most useful agent, as it showed higher antibacterial activity than omiganan. These results not only suggest useful candidates for novel antibiotic development, but also provide an efficient strategy to design such peptides.     

Evaluation of iron-binding capacity,amino acid composition,functional properties of Acetes japonicus proteolysate and identification of iron-binding peptides

Recovery of functional iron-binding protein hydrolysate from Acetes japonicus employing enzymatic hydrolysis and iron-chelating peptide identification were conducted in this study. The result showed that under the optimal hydrolysis condition including Flavourzyme, pH 5, 50 °C, E:S ratio of 27.4 U/g protein and hydrolysis time of 4.8 h, the obtained proteolysate displayed the maximal iron-binding capacity (IBC) of 177.7 μgFe²⁺/g protein and comprehended 38,77 % of essential amino acids. Functional features of the Acetes proteolysate encompassing solubility, heat stability, foaming and emulsifying property, oil and water holding capacity were also noteworthy. Peptide fractionation was performed using ultrafiltration and the 1−3 kDa fraction expressed the highest IBC of 120.43 ± 0.15 μgFe²⁺/g protein, 13.7 times lower than that of disodium ethylenediaminetetraacetate (Na₂EDTA). From this fraction, two iron-binding peptides of DSVNFPVHGL (1083.53 Da) and FKVGQENTPILK (1372.77 Da) were identified utilizing nano-UHPLC-MS/MS as well as their de novo spatial structures and interaction with ferrous ion were simulated by PEP-FOLD 3. As a whole, the proteolysate/peptides could be filled as an iron chelator which could shield human body from iron deficient-related disorders or as a functional proteolysate preparation to upgrade food properties.     

Carp myogens of white and red muscles. General composition and isolation of low-molecular-weight components of abnormal amino acid composition

1. The general composition of the carp myogens of white and red muscles was examined by electrophoresis and ultracentrifugation. 2. Eight and nine peaks were found in the electrophoretic analysis at pH7·3 and I0·075 of white and red muscle respectively. Lowering of the pH to 5 or 6 did not increase the number of peaks. The electrophoretic pattern of white-muscle myogen was remarkably different from that of red-muscle myogen, though ultracentrifugal analyses of the both types of myogen gave similar diagrams, in which about one-third of the total myogen sedimented slowly. 3. The pH–mobility curves of the myogen of white muscle indicated that the net charges of the components 2, 3 and 5 vary only slightly within the pH range 7·3–5·4, suggesting that their histidine content is very low. 4. The slow-sedimenting fraction of white-muscle myogen was isolated in fairly good yield by ammonium sulphate fractionation, by taking advantage of their high salting-out range, and the fraction was shown to be composed mainly of components 2, 3 and 5. 5. The same method of fractionation was applied to red-muscle myogen and the absence of the three components was confirmed. These results bring to light a new difference between the two types of fish muscle.