Isolation and partial amino acid sequence of three subunit species of porcine spleen ferritin: evidence of multiple H subunits.

A partial amino acid sequence for three different subunits of the iron storage protein, ferritin, has been determined. Ferritin (Mr approximately 480,000) was isolated from porcine spleen and dissociated into its component subunits (Mr approximately 20,000). The subunits, in turn, were separated into three fractions by reversed-phase HPLC. The fractions appeared to be of equal size by sedimentation velocity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and size-exclusion chromatography in 6 M guanidinium chloride. All three fractions were shown to be monomeric and to have no covalently attached carbohydrate (J. F. Collawn et al. (1984) Arch. Biochem. Biophys. 233, 260-266). Determination of the amino acid sequence of the C-terminal 70-80 residues from each of the fractions demonstrated three different sequences. Comparison with human liver H and L subunit sequences indicates that two of the porcine ferritin subunits are H-type subunits and one is an L-type subunit. Application of the Chou-Fasman algorithm on the three partial sequences suggests that these respective regions from each of the three subunits would probably adopt the same conformation.

Purification and characterization of a unique high molecular weight form of insulin-like growth factor II.

A form of insulin-like growth factor II (IGF-II) with a mol wt of 15,000 has been purified to homogeneity from human Cohn fraction IV1-4. This protein has an amino-terminal sequence through the first 28 residues that is identical to 7.5K IGF-II. The amino acid composition of 15K IGF-II, however, indicates that its carboxyl-terminal region may be different from that predicted from the analysis of IGF-II cDNA clones. The affinities of 15K IGF-II for receptors on rat placental membranes and for an IGF-binding protein that was isolated from the medium of cultured buffalo rat liver cells were similar to those of the 7.5K form of the growth factor. A best-fit analysis of data from the binding of the two mol wt forms of IGF-II to receptors on rat placental membranes by the LIGAND program was consistent with a model in which 7.5K and 15K IGF-II bound to one site with Kd values of 0.27 +/- 0.03 and 0.38 +/- 0.04, respectively. There was an indication that 15K IGF-II also bound to a second low affinity site on the membrane. In mitogenesis assays performed on human fibroblasts isolated from the skin of two fetuses of an early gestational age, 15K IGF-II stimulated the incorporation of [3H]thymidine into DNA at a half-maximal concentration, i.e. ED50, of 5.7 and 5.0 nM. In these experiments, the ED50 values for 7.5K IGF-II were 8.7 and 15 nM.

Isolation, purification, and the sequence of relaxin from spiny dogfish (Squalus acanthias).

A relaxin-like molecule has been isolated from the ovaries of the spiny dogfish (Squalus acanthias) which consists, like porcine relaxin, of two chains linked by the insulin-type disulfide bonds. The total number of amino acids is 54 of which 24 are in the A chain and 30 in the B chain. The molecular masses, calculated from the amino acid compositions, are 2510 Da for the A chain and 3370 Da for the B chain, making a total of 5880 Da. The N-terminus of the B chain is protected by a 5-oxoproline (pyrrolidone carboxylic acid) residue which is also found in the same position in the relaxins of sand tiger shark, pig, and man, whereas the relaxin of the rat has its 5-oxoproline residue at the N-terminal of the A chain. By all available criteria, S. acanthias relaxin is a typical member of the relaxin family although the sequence homology to mammalian relaxins is limited to about 45% of its amino acid residues. In contrast, the dogfish relaxin shows about 80% homology with sand tiger shark relaxin (the first such interspecies similarity to be observed) and has about twice the biological activity (mouse pubic symphysis test) when compared to sand tiger relaxin.

Coypu insulin. Primary structure, conformation and biological properties of a hystricomorph rodent insulin.

Insulin from a hystricomorph rodent, coypu (Myocaster coypus), was isolated and purified to near homogeneity. Like the other insulins that have been characterized in this Suborder of Rodentia, coypu insulin also exhibits a very low (3%) biological potency, relative to pig insulin, on lipogenesis in isolated rat fat-cells. The receptor-binding affinity is significantly higher (5-8%) in rat fat-cells, in rat liver plasma membranes and in pig liver cells, indicating that the efficacy of coypu insulin on receptors is about 2-fold lower than that of pig insulin. The primary structures of the oxidized A- and B-chains were determined, and our sequence analysis confirms a previous report [Smith (1972) Diabetes 21, Suppl. 2, 457-460] that the C-terminus of the A-chain is extended by a single residue (i.e. aspartate-A22), in contrast with most other insulin sequences, which terminate at residue A21. In spite of a large number of amino acid substitutions (relative to mammalian insulins), computer-graphics model-building studies suggest a similar spatial arrangement for coypu insulin to that for pig insulin. The substitution of the zinc-co-ordinating site (B10-His----Gln) along with various substitutions on the intermolecular surfaces involved in the formation of higher aggregates are consistent with the observation that this insulin is predominantly 'monomeric' in nature. The c.d. spectrum of coypu insulin is relatively similar to those of casiragua insulin and of bovine insulin at low concentration.

Dogfish insulin. Primary structure, conformation and biological properties of an elasmobranchial insulin.

Insulin from an elasmobranch, the spiny dogfish (Squalus acanthias) has been purified to near homogeneity by means of acid-ethanol extraction and salt precipitation. The amino acid sequences of the performic-acid-oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A21 asparagine. The B10 histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co-ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A5Gln leads to His, B21Glu leads to Pro, B22Arg leads to Lys, B25Phe leads to Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half-maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin. The use of interactive computer graphics model-building predicts that the dogfish insulin can attain a three-dimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model-building studies.

Isolation and characterization of relaxin from the sand tiger shark (Odontaspis taurus).

A peptide with relaxin activity in guinea pigs but not in mice has been extracted from the ovaries of pregnant sand tiger sharks (Odontaspis taurus). The structural similarity of this peptide to porcine relaxin includes molecular size approximately 6000 daltons), number of chains, and possibly, disulfide cross-links. The relaxin-type peptide isolated from shark ovaries contains the amino acid residues tyrosine, proline, and histidine, which are absent in the porcine hormone. The amino acid composition of shark relaxin, therefore, resembles that of porcine insulin to a greater extent than does the amino acid composition of porcine relaxin. This finding supports the idea that shark relaxin may be a primitive relaxin that has undergone fewer mutations than porcine relaxin since the putative duplication of the insulin gene. The data presented here suggest that the putative duplication of the insulin gene, which might have given rise to relaxin, has occurred much earlier than the separation of sharks from the general branch of animals that eventually gave rise to mammals.