Purification and amino acid composition of monomeric and polymeric collagens

The preparations and amino acid compositions of highly purified tropocollagen and insoluble polymerized collagen are described. These collagens appear to be very suitable for comparative studies in an investigation of the cross-linkages that are introduced into tropocollagen during the formation of polymerized collagen.     

The purification and amino acid composition of human uterus collagens, rheumatoid-arthritis-nodule collagen and ox tendon collagen

Collagens and gelatins were isolated from human post-menopausal uterus, puerperal (post-partum) uterus, rheumatoid-arthritis-nodule and ox tendon. Different means of purifying collagen were studied and a method was devised that enables highly purified collagen to be obtained, even from the uterus. This method involves the use of a number of aqueous and organic extractants as well as digestion with elastase to eliminate elastin. The purity of the collagen preparations was assessed and they were used to study the amino acid composition of collagen. The amino acid compositions of all the collagens studied were similar to those of human bone and tendon collagen, but certain small differences were noted and are discussed. The soluble collagen extracted from some of the tissues was also studied.     

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.     

Separation, isolation and amino acid composition of uremic peptides

Gel filtration and thin layer chromatography were conducted on sera from uremic patients and normal subjects for the isolation of nitrogenous substances unique to uremia. Many ninhydrin-positive substances were found in greater amounts in uremic patients compared to normal subjects. Some of these ninhydrin-positive substances were also detected by staining with chlorine-tolidine. Amino acid analysis of these substances showed considerable qualitative and quantitative differences, perhaps reflecting interference with enzymatic activity by the uremic environment.     

The isolation of ovomucoid variants differing in carbohydrate composition

Three major and two minor species of ovomucoid were separated by chromatography on sulphoethyl-Sephadex. The predominant sialic acid-free species was further resolved into three fractions by DEAE-cellulose chromatography. Although all species of ovomucoid had closely similar trypsin-inhibiting activity, immunochemical properties and amino acid composition, they differ in carbohydrate composition. Wide variation was observed in the content of galactose, N-acetylglucosamine and sialic acid. Charge heterogeneity was related, in part, to variation in sialic acid content. The implications of variable carbohydrate composition for the structure and function of ovomucoid are discussed.     

Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition

The seven major desmosomal polypeptides from isolated bovine muzzle desmosomes ranging from Mr 75 000 to 250 000 were separated by gel electrophoresis, isolated and characterized with respect to their amino acid composition and sugar content. The two largest polypeptides (bands 1 and 2), i.e. desmoplakins I and II, are similar in their amino acid composition, confirming our previous immunological and biochemical data, and display a relatively high glycine content. In contrast, the other two cytoplasmic components also believed to be associated with the desmosomal plaque, i.e. polypeptides of bands 5 (Mr 83 000) and 6 (Mr 75 000), differ significantly in their amino acid composition from the desmoplakins and from each other. All four candidate polypeptides for plaque association, i.e. bands 1, 2, 5, and 6, show no significant glycosylation. The glycoproteins 4a and 4b (Mr 115 000 and 130 000) are similar in their amino acid composition, peptide analysis and immunological reactivity. Both are relatively rich in mannose and galactose but also contain sialic acid. Our determinations also indicate that the two polypeptides differ significantly in their N-acetylglucosamine and mannose content. Most, if not all, of the sugar residues are associated with a water-soluble fragment of Mr 15 500 obtained after limited digestion with V8 protease. The glycopolypeptides obtained in band 3 (Mr 164 000-175 000) are distinct from the glycopolypeptides 4a and 4b in amino acid composition, sugar content, isoelectric pH values, certain antigenic determinants and in their pattern of cleavage products obtained by treatment with proteases or cyanogen bromide. The results identify polypeptides of bands 3, 4a and 4b as glycosylated with characteristic sugar compositions. It is suggested that the major glycoproteins (bands 3, 4a, 4b) of the desmosome are integral membrane components arranged in a special way conferring resistance to detergent treatment. The possible roles of these glycoproteins in cell recognition and in adhesive functions of the desmosome are discussed.     

Chemical composition, mineral content and amino acid and lipid profiles in bones from various fish species

Insulin-like growth factor-I (IGF-I) is an important regulator of growth and development in vertebrates. Both the endocrine and paracrine actions of IGF-I are mediated through ligand-binding to a membrane-bound IGF-I receptor (IGF-IR). The characterization of this receptor and subsequent expression studies thus help elucidate the endocrine regulation of developmental processes. As other flatfish species, the Atlantic halibut (Hippoglossus hippoglossus) undergoes a dramatic larval metamorphosis. This process is largely under endocrine control, and data indicate that IGF-I could be a key regulator. IGF-I content increases up to late pre-metamorphosis and decreases during metamorphosis. The IGF-IR has, however, not been studied during flatfish metamorphosis. To examine IGF-IR gene expression, two IGF-IR mRNA were cloned and sequenced. These partial sequences share high identity (>or=95%) and similarity (>or=97%) with other fish IGF-IR and lower identity (>or=77%) and similarity (>or=83.5%) with Japanese flounder insulin receptors. The expression of mRNA for both IGF-IR was analyzed by quantitative real-time RT-PCR during six larval developmental stages from pre- to post-metamorphosis. IGF-IR1 and IGF-IR2 mRNA are differentially expressed during metamorphosis, but if this indicates an isoform-specific regulation of developmental processes by circulating and/or locally-secreted IGF-I is unclear. Both IGF-IR genes are down-regulated in halibut larvae experiencing arrested metamorphosis, suggesting the IGF-I system is critical for metamorphic success in halibut.