Platypus insulin: indications from the amino acid sequence of significant differences in structure from porcine insulin.

Insulin from a monotreme, the platypus (Ornithorhynchus anatinus), was isolated and the amino acid sequence determined. It differs from pig insulin at eleven amino acid sites, mainly on the surface of the monomer. Substitutions relative to pig insulin occur in the monomer-monomer interface, the dimer-dimer interface and the receptor binding region. The residues A5 Glu, A8 Lys and A13 Met have not been reported before in any insulin. Multiple sequence comparison studies reveal a relatively close relationship with the nearest group of relatives to the platypus, the mammals. The relationship of the platypus sequence to reptilian insulin sequences (and amphibian and avian insulin sequences in this case) is sufficiently close to support the observation that platypus has retained some ancient reptilian characteristics over the course of evolution. Model building the platypus insulin sequence on the structure of porcine insulin indicates that there may be some interesting differences.

Self-association of kangaroo and chicken insulins, chicken despentapeptide insulin and deamidated insulin.

Intact and modified metal-free insulins from kangaroo, chicken and pig have been purified by HPLC and studied by equilibrium sedimentation at pH 7. The aggregation of deamidated pig insulin is lowered significantly, consistent with its modified electrostatic charge. Native kangaroo and chicken insulins self-associate to the same extent as pig and cow insulins under these conditions. Despentapeptide chicken insulin does not self-associate at pH 7.

Purification of a marsupial insulin: amino-acid sequence of insulin from the eastern grey kangaroo Macropus giganteus.

Insulin has been purified from kangaroo pancreas by acidic ethanol extraction, diethyl ether precipitation and gel filtration. The amino-acid sequence of this, the first marsupial insulin to be studied, is reported. It differs from human insulin by only four amino-acid substitutions, all in regions of the molecule previously known to be variable. However, it should be noted that one of these, asparagine for threonine at A8, has not been reported before. Computer comparisons of all 43 insulin sequences reported to date with kangaroo insulin show it to be most closely related to a group of mammalian insulins (dog, pig, cow, human) known to be of high biological potency. The measurement of blood glucose lowering in the rabbit by kangaroo insulin is consistent with this conclusion. Comparisons of amino-acid sequences of other proteins with their kangaroo counterparts show a greater difference, in line with the time of divergence of marsupials. The limited differences observed in insulin and cytochrome c suggest that their structures need to be closely conserved in order to maintain function.

Molecular weights of mitochondrial and cytoplasmic aminoacyl-tRNA synthetases of beef liver and their complexes.

In eukaryotes, multienzyme complexes containing five to nine aminoacyl-tRNA synthetase activities have frequently been reported. In this study, we report the existence, in bovine liver cytoplasm, of a multienzyme complex containing at least 16 activities which can be disrupted by homogenization to give rise to smaller complexes and noncomplexed synthetases. Determination of the size and component activity of these complexes and of the molecular weights of all 20 free synthetases suggests that the smaller complexes and free activities normally identified arise from the larger complex by well-defined stages during homogenization. We also show that similar, though not identical, complexes are found in bovine liver mitochondria and give the molecular weights of 16 mitochondrial synthetases.

"Whey" proteins of milk of the red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroo.

1. A comparison is made of gel electrophoretic patterns of the "whey" proteins of the milk of red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroos at various stages of lactation. Qualitative and quantitative changes occur with time during the mature phase of lactation of both types. Their onset is related solely to the stage of lactation. "Whey" proteins are isolated and characterised and the nature of protein changes determined for the first time. 2. The anodic electrophoretic pattern is divided into 6 main zones (designated A F in order of decreasing mobility) and 2 cathodic zones (G and H) that are only detected in the milk of M. giganteus. 3. Zones A, B and C are milk specific. Zone B is present throughout lactation in both species and is an alpha-lactalbumin. Zones A and C are present only in late lactation, zone C, usually, but not always, appearing first. Zone A is an alpha-lactalbumin in M. giganteus, but is not an alpha-lactalbumin in M. rufus. Zone C appears to be the same protein in both species and is possibly a beta-lactoglobulin. 4. Zone D is kangaroo serum albumin and zone E is possibly a beta 2-microglobulin. Zone F contains three main iron (III) binding bands whose relative intensity varies with stage of lactation. Their intensity differs from the corresponding blood serum transferrin bands. 5. Zone H of Macropus giganteus is a lysozyme. 6. Lactose is present in the milk, but is not the principal sugar. 7. The significance of the results is discussed.

Aggregation patterns in Cherax destructor hemocyanin: control of oligomer distribution by incorporation of specific subunits.

Recent polyacrylamide gel electrophoresis studies on Cherax destructor hemocyanin have demonstrated the presence of three further constituent fractions in the alkaline dissociation product in addition to the three subunits reported in earlier work. Two of these recently discovered subunits are monomeric with molecular weights around 750000, while the third subunit is of similar size to the previously identified dimeric subunit M3' with a molecular weight near 150 000. The aggregation process is influenced by the presence of calcium ions, particularly in the distribution of hybrid hexameric species. However, the relative proportions, as well as the types of subunits present initially, are of primary importance in determining the oligomer distribution pattern obtained upon reconstitution from alkaline pH to pH 7.8 of selected mixtures of subunits. An additional significant factor in the assembly process has been proposed: the operation of different relative rates of aggregation between different types of subunits. Reconstitution experiments based on these findings substantially explain the complex distribution of oligomeric forms in C. destructor hemolymph.

Hemocyanin from the Australian freshwater crayfish Cherax destructor. Oxygen binding studies of major components.

Oxygen binding curves have been obtained for unfractionated hemocyanin from Cherax destructor and it major components, the 25S and 17S forms. In all cases the binding was characterized by positive cooperativity at pH 7.8 with a P50 of approximately 4 mmHg and a Hill coefficient, nH, of approximately 3. There was no evidence of concentration dependence of the binding curves in the range 0.6-6 mg/mL, a finding which excludes a dynamic equilibrium between polymeric forms of different oxygen affinity as a source of the cooperative binding. A positive Bohr effect operates between pH 6.8 and pH 7.8 and removal of calcium ions from the 25S and 17S aggregates markedly reduces their affinities for oxygen. Cooperativity is retained in these circumstances though nH drops to about 2.5 in the case of the 25S and 2.0 in the case of the 17S form. The two major monomers M1 and M2, from which the 25S and 17S complexes are constructed, may be reconstituted into the hexamers (M1)6 and (M2)6. These show oxygen binding behavior perfectly consistent with that expected of native hexamers as studied in the 17S fraction, a mixed population of hexamers. The monomer M1 can also be studied in monomeric form and was found to show indistinguishable oxygen binding at pH 7.8 and pH 10, the curve being a rectangular hyperbola as expected. The oxygen binding curve of the single subunit hexamer (M1)6 was fitted adequately by a polynomial expression of order 6 as required for a molecule with six binding sites. Further interpretation in terms of a particular binding model was not attempted because available knowledge of the structures of arthropod hemocyanin aggregates and their oxygen binding sites does not yet justify it.

Hemocyanin from the Australian freshwater crayfish Cherax destructor. Characterization of a dimeric subunit and its involvement in the formation of the 25S component.

The molecular weight of a dimeric subunit, M3', isolated from Cherax destructor hemocyanin has been measured by sedimentation equilibrium to be 144 000. Peptide mapping and end-group analysis together with gel electrophoresis show that the dimer consists of two very similar or identical monomers, cross-linked by disulfide bridges. Dissociation of the 25S component of the hemocyanin shows that it contains the dimer and two previously identified monomers, M1 and M2. Its molecular weight is 900 000 by sedimentation equilibrium, and reconstitution studies show that the dimer is essential for its formation. Analysis of the results of polyacrylamide disc gel electrophoresis experiments with the 25S component indicates that it consists of a population of 11 compositional isomers. These all contain one dimeric subunit and ten monomeric subunits, the latter being present in all the combinations of M1 and M2.