Pig Insulin Research Articles

Ir gene control of carrier recognition. III. Cooperative recognition of two or more carrier determinants on insulins of different species.

AbstractWhen crosses between different nonresponder strains to pig and bovine insulin were tested, some cases of complementation could only be achieved if one of the strains had C3H bavkgrounmd,e.g.(C3H.SW × C3H/He)F1 and (C57BL/6 × C3H/He)F1 show complementation, whereas (C57BL/10 × B10.BR)F1 do not.It is suggested that three genes are necessary for the complementation, two genes in the H‐2 region and an additional gene in the C3H bacjground. It was therefore supposed that this type of complementation might be due to a cooperative recognition byone of the parental strains. This possibitility was tested by coimmunization experiments performed with the parental strains C3H/He and C57BL/6.Insulin to which these strains respond (sheep or bovine insulin, respectively) was injected together with an excess of an insulin nonimmunogenic in the parental strins but immunogenic to the hybrids (pig insulin).It proved that under these conditions the response of C3H/He mice to sheep insulin was reduced by more than 90%, whereas C57BL/6 mice gave a normal response to bovine insulin. However, both strains were now able to respond to the pig insulin as shown bythe antibody production to fluoresceinthiocarbamyl groups coupled to pig insulin. This shows that the missing carrier determinant can be provided by a different molecule.The results are discussed in terms of a multideterminant recognition of the antigen at the carrier level. The recognition of each carrier determinant can be provided by a different molecule.

PHYLOGENY OF INSULIN:Some Evolutionary Aspects of Insulin Production with Particular Regard to the Biosynthesis of Insulin in Myxine glutinosa

Preceding phylogenetic studies on the occurrence of insulin have shown--e.g. by bioassays and immunocytochemical procedures--that insulin producing B-cells are present in all vertebrates and even in several invertebrates, both protostomian and deuterostomian. The most original B-cells are obviously endocrine cells of open type, situated in the mucosa of the alimentary tract. Moreover, the results of these studies show that insulin is not only a polypeptide hormone of considerable age but also that the insulin molecule seems to have been kept surprisingly stable during evolution. Best known of all non-mammalian insulins is that from the hagfish, Myxine glutinosa. It is probably the most original insulin of all in the vertebrate series. Both the amino-acid sequence and the three-dimensional structure of the dimer of hagfish insulin differ only little from those of pig insulin. The biosynthesis occurs via proinsulin and is also in most respects similar to mammalian insulin biosynthesis. There are, however, some differences. Although it readily crystallizes as tetragonal bipyramids, hagfish insulin does not form hexamers. In a test system, with isolated rat fat cells, its binding affinity is 23% and its potency 5% of that of pig insulin, a discrepancy indicating a "partial antagonism" on the receptors. Although the conversion of proinsulin to insulin seems to occur in the secretion granules, they contain no crystalline cores. Since a strictly tryptic-like enzyme was found to destroy hagfish insulin rapidly, the enzyme converting proinsulin to insulin must--in addition to a carboxy-peptidase-B-like activity--have a different specificity in Myxine.

Semisynthetic analogues of insulin. The use of N-substituted derivatives of methionine as acid-stable protecting groups.

1. We describe the use of benzyloxycarbonylmethionine and ethoxycarbonylmethionine for the selective protection of the amino groups of glycine-A1 and lysine-B29 of pig insulin. We have used the Edman method to remove residues from the N-terminal and of the B-chain of the N(A1)N(B29)-di-protected derivatives. The benzyloxycarbonyl group shows slight but noticeable lability in the acid-cleavage step, but the ethoxycarbonyl group remained intact even after five cycles of degradation. 2. We have prepared the following truncated forms of insulin via the di(ethoxycarbonylmethionyl) derivative: des-Phe(B1)-insulin;des-(Phe(B1)-Val(B2))-insulin; des-(Phe(B1)-Val(B2)-Asn(B3))-insulin;des- (Phe(B1)-Val(B2)-Asn(B3)-Gln(B4))-insulin; des-(Phe(B1)-Val(B2)-Asn(B3) -Gln(B4)-His(B5))-insulin. 3. Insulin was re-synthesized from the di-protected des-Phe(B1)-insulin by reaction with an active ester of t-butoxycarbonyl-l-phenylalanine. The product after deprotection crystallized, and the immunoreactivity of the crystalline material was identical with that of the native protein. 4. We have prepared the following analogues of insulin in a similar manner: [l-Ala(B1)]insulin; [l-Val(B1)]insulin; [l-Tyr(B1)]insulin; [m-F-l-Phe(B1)]insulin; [o-F-l-Phe(B1)]-insulin; [o-F-l-Phe(B2)]des-Phe(B1)-insulin. All had between 34 and 62% of the activity of insulin in the fat-cell test. 5. We have also investigated the use of the benzyol, toluene-p-sulphonyl, p-nitrobenzyloxycarbonyl and 2,4-dinitrophenyl groups for the N-protection of the methionine active esters. Each should have had some particular advantage over the benzyloxycarbonyl and ethoxycarbonyl groups, but all proved in practice to have disadvantages that more than outweighed anything in their favour.

Ir Gene Control of Carrier Recognition II. Unexpected Low Immunogenicity of Guinea Pig Insulin

The immune response of mice to guinea pig insulin (as well as to insulins from other species) is controlled by immune response genes. The immunogenicity of guinea pig insulin is generally much lower than that of bovine insulin, though there are 17 sequence differences between mouse insulin and guinea pig insulin as compared to 5 sequence differences between mouse insulin and bovine insulin, respectively. The low immunogenicity of guinea pig insulin is indicated by a low frequency of responder strains and the requirement for high antigen doses for immunisation. Guinea pig and bovine insulin do not cross-react at the antibody level, but there seems to be some cross-recognition at the carrier level, as indicated by the capability of guinea pig insulin primed spleen cells to provide help for the pig insulin carrier in cooperative cell transfer.

Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells.

Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.

Structure and function of insulin: preparation and biological activity of guinea pig des-B-Asp30,des-A-Asn21-insulin

Bovine des-B-Ala30,des-A-Asn21-insulin and guinea pig des-B-Asp30,des-A-Asn21-insulin were prepared from bovine and guinea pig insulin by digestion with carboxypeptidase A (EC 3.4.12.2). As reported by other investigators, the biological activity of bovine des-Ala30,des-Asn21-insulin was less than 10% that of bovine insulin. Contrary to theoretical consideration, removal of A-Asn21 and B-Asp30 from the carbosyl termini of guinea pig insulin resulted in a loss of more than 90% of the biological activity. Receptor binding studies of these insulin derivatives demonstrated a good correlation between the loss of biological activity and the decrease in binding affinity. It is suggested that the carboxyl terminal A-Asn21 of insulin may interact directly with the insulin receptor.

Near-ultraviolet tyrosyl circular dichroism of pig insulin monomers, dimers, and hexamers. Dipole-dipole coupling calculations in the monopole approximation.

The tyrosyl circular dichroism (CD) has been calculated using the conformation of pig insulin observed in rhombohedral crystals containing 2 zinc atoms per hexamer. These calculations predict that the tyrosyl CD at 275 nm will be enhanced disproportionally as monomers interact to form dimers and as dimers interact to form hexamers. This enhanced tyrosyl CD (delta epsilon per 5800 molecular weight) results from new coupling interactions generated in the regions of contact between monomers and between dimers. These calculations illustrate that a large CD enhancement may accompany aggregation even in the absence of a conformation change in eith monomer. The tyrosyl CD intensities calculated for monomers, dimers, and hexamers of 2-zinc pig insulin are compatible with the experimentally observed CD spectra which are enhanced about fourfold in the hexamer compared with the monomer. Zinc ions and other metals do not contribute directly to the tyrosyl CD but only influence the optical properties by promoting the hexameric state. The relation of the integrity of the molecule to dimer formation and the biological activity of the molecules are discussed. The largest calculated contributions to tryosyl CD arise from interactions with far-ultraviolet transitions of neighboring aromatic groups. In the hexamer, about half of the tyrosyl CD intensity is calculated to arise from Tyr-A14.

Structure of insulin in 4-zinc insulin.

DURING his experiments on slow acting clinical preparations of insulin, Schlichtkrull found that a second form of rhombohedral insulin crystals appeared when the sodium chloride ion concentration in the solution was 6% or more1. The new crystals contained four zinc ions per hexamer of insulin compared with two in the original crystals. Both forms crystallised in the space group R3 and had similar lattice constants: a=82.5 A, c=34.0 A for 2-zinc insulin, and a=80.7 A, c=37.6 A for 4-zinc insulin crystals2. Both, from an early application of the rotation function3, also seemed to be very similar in structure, and to contain in each asymmetric unit, two insulin molecules related to one another by non-crystallographic twofold axes of symmetry. We have now found, from electron density calculations on 4-zinc pig insulin, that the apparent similarity of the crystal structures conceals a surprisingly large change in the conformation of one only of the two insulin molecules in the unit.

Deviations from compositional randomness in eukaryotic and prokaryotic proteins: The hypothesis of selective-stochastic stability and a principle of charge conservation

Eight proteins of diverse lengths, functions, and origin, are examined for compositional non-randomness amino acid by amino acid. The proteins investigated are human fibrinopeptide A, guinea pig Insulin, rattlesnake cytochrome c, MS2 phage coat protein, rabbit triosephosphate isomerase, bovine pancreatic deoxyribonuclease A, bovine glutamate dehydrogenase, and Bacillus thermoproteolyticus thermolysin. As a result of this study the experimentally testable hypothesis is put forth that for a large class of proteins the ratio of that fraction of the molecule which exhibits compositional non-randomness to that fraction which does not is on the average, stable about a mean value (estimated as 0.32 plus or minus 0.17) and (nearly) independent of protein length. Stochastic and selective evolutionary forces are viewed as interacting rather than independent phenomena. With respect to amino acid composition, this coupling ameliorates the current controversy over Darwinian vs. non-Darwinian evolution, selectionist vs. neutralist, in favor of neither: Within the context of the quantitative data, the evolution of real proteins is seen as a compromise between the two viewpoints, both important. The compositional fluctuations of the electrically charged amino acids glutamic and aspartic acid, lysine and arginine, are examined in depth for over eighty protein families, both prokaryotic and eukaryotic. For both taxa, each of the acidic amino acids is present in amounts roughly twice that predicted from the genetic code. The presence of an excess of glutamic acid is independent of the presence of an excess of aspartic acid and vice versa.

The reaction of penicillin with proteins.

The mode of reaction of benzylpenicillin with two proteins was studied, with particular reference to the allergenicity of penicillin. These reactions, with pig insulin, and with hen's-egg-white lysozyme, were carried out in neutral solution at 37 degrees C. High concentrations of penicillin are needed to label the proteins, owing to concurrent hydrolysis of penicillin. Evidence has been obtained that the penicillin-reactive sites on the insulin molecule are the alpha-amino group at the N-terminus of the A chain and the epsilon-amino group of the lysine residue; whereas a site of reaction with lysozyme appears to be the epsilon-amino group of lysine-116.

Guinea pig proinsulin. Primary structure of the C-peptide isolated from pancreas

The amino acid sequence of the proinsulin C-peptide isolated from guinea pig pancreas was determined and experimental data are presented. Digestion of the C-peptide with chymotrypsin provided two dodecapeptides, a tetrapeptide, and glutamine, which account for the intact chain. Reaction of the C-peptide with cyanogen bromide resulted in cleavage at the single methionine and provided two additional fragments. Digestion of the large peptides with papain provided a variety of small peptides and the complete sequence was assigned by identification of the fragments. Although guinea pig insulin differs markedly from mammalian insulins, guinea pig C-peptide has many features of primary structure in common with the C-peptides of other mammals. The conservation of specific residues in C-peptides indicates that these residues form essential elements in the three-dimensional structure of proinsulin.

The relation of conformation and association of insulin to receptor binding; x-ray and circular-dichroism studies on bovine and hystricomorph insulins.

Crystal and solution structure studies on insulins of different sequences and of widely different receptor binding affinities are reported. Bovine insulin, studied as a control, has a circular dichroism spectrum which is dependent both on protein concentration and zinc concentration. The spectrum appears to be related to the level of association of the insulin molecules. This implies that when using circular dichroism to compare solution structures of insulin derivatives or species variants one must make the comparison at equivalent levels of association and not merely at the same concentration. Changes in circular dichroism are related to the known crystal structure of zinc insulin hexamers. The chinchilla insulin spectrum shows a reduced zinc dependence in low-salt conditions which correlates with the inability to form crystals in similar conditions. This is attributed to an amino acid substitution at position B4. Crystals are obtained in high-salt conditions and X-ray diffraction patterns show these to be isomorphous with bovine 4Zn insulin crystals. Guinea pig insulin failed to crystallise under conditions which are normally conducive to the formation of crystals of zinc insulin hexamers and the circular dichroism showed no zinc dependence. This is consistent with a monomeric structure. The significance of the association behaviour of chinchilla and guinea pig insulins may be in the storage of the hormone in vivo. Whereas the monomeric form of chinchilla insulin has a structure closely related to bovine insulin, the circular dichroism indicates a gross structural difference for guinea pig insulin. This may be similar to that in des-A21, des-B30-insulin, as both lack the Arg-B22--Asn-A21 carboxylate ion pair. The similarity of structure of chinchilla and bovine insulins is reflected in their receptor binding whereas the low receptor binding of guinea pig insulin probably results from the changes in its conformation rather than an alteration in residues of a receptor binding region.

Binding of insuling and other hormones to non-receptor materials: Saturability, specificity and apparent “negative cooperativity”

Summary Studies of interactions of 125 I-insulin with non-tissue materials (talc, silica, protein-agarose derivatives, glass test tubes) are presented as examples of non- receptor (“nonspecific”) interactions which share, at least superficially, the criteria (saturability, specificity, high affinity and reversibility) commonly attributed to “specific” hormone-receptor interactions. Such “specific” nonreceptor interactions, which can potentially complicate interpretations of receptor binding studies, also exhibit characteristics analogous to those observed in liver membranes where the data have been interpreted (3) to indicate “negative cooperativity” between receptors. Similar data are presented here for insulin and epidermal growth factor binding to placenta membranes, and the effects of porcine insulin are compared to those of guinea pig insulin, a derivative which dimerizes very poorly. The “negative cooperativity” observed in the nonreceptor systems is most likely attributable to insulin-insulin interactions. Wherever ligand (e.g., hormone) self-association or isomerization occurs, the binding data (e.g. Scatchard plot) may yield nonlinear relationships which can falsely be interpreted as indicating cooperative interactions between receptors, or the presence of “additional groups” of binding sites.

Low resolution crystal structure of hagfish insulin

Insulin from the Atlantic hagfish, Myxine glutinosa, crystallizes in space group P4 12 12 with a monomer in the asymmetric unit. The application of the Rossmann & Blow (1962) rotation function, utilizing the known 2-zinc pig insulin crystal structure, has established the existence of an insulin dimer containing a crystallographic 2-fold axis. The position of the hagfish insulin molecule in the unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 Å resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.

Guinea Pig Insulin: II. BIOLOGICAL ACTIVITY

Abstract The biological activity of guinea pig insulin was determined to be between 1 and 3 i.u. per mg when measured in vitro using isolated rat tissues and in vivo by mouse convulsion. However, when assayed by blood glucose lowering in the guinea pig, guinea pig insulin has a potency of 8.5 to 9.0 i.u. per mg. The low activity in vitro was not due to a more rapid degradation of guinea pig insulin by the rat tissues. The dose-response relationship in the isolated epididymal fat cell assay showed that guinea pig insulin had an intrinsic biological activity comparable to that of bovine insulin, but a reduced affinity for the tissue receptor sites.

Guinea Pig Insulin: I. PURIFICATION AND PHYSICAL PROPERTIES

Abstract A method is described for the preparation of homogeneous insulin from the pancreatic tissue of the guinea pig. The amino acid composition of this insulin and of the separated S-sulfonated chains demonstrated that the primary structure of the molecule was intact. Guinea pig insulin sedimented as a monomer in analytical ultracentrifugation, even in the presence of equimolar zinc ion. Ultraviolet absorbance difference spectrophotometry showed that certain hydrogen bonds which may be involved in stabilizing the monomer, and possibly the hexameric aggregate, in crystalline bovine or porcine insulin may not exist in guinea pig insulin. The reason for the failure of guinea pig insulin to dimerize may be related to the replacement of the B-22 arginine by aspartic acid.

New method of calculating evolutionary rates of proteins applied to insulin and C-peptides.

The optimal number of differences between sequences for phylogenetic studies is derived, and the developed theory is used to show that a sequenced peptide assumed to be duck proinsulin C‐peptide had the expected differences from the mammalian C‐peptides. The theory is used to separate the guinea pig insulin from other insulins, indicating that the pressure of selection on guinea pig insulin has been significantly lower than that on other insulins. Finally, calculation of the half‐life of a protein as a measure of the pressure of selection rather than the evolutionary rate constant is proposed.

Binding of parathyroid hormone to bovine kidney-cortex plasma membranes.

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with (125)I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. (75)Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca(2+) inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.

Hyperglycemia, hypoglycemic attacks, and production of anti-insulin antibodies without previous known immunization. Immunological and functional studies in a patient.

A forty-two-year-old man with hyperglycemia and pronounced reactive hypoglycemia after meals and oral glucose is reported. He produced anti-insulin antibodies without previous known immunization. Labeled insulin, when mixed with the patient's serum, passed with the serum proteins on gel filtration, and was found among the 7S globulins on preparative ultracentrifugation, in the anodic γ-region on agar electrophoresis, and among the anodic γG-globulins on immunoelectrophoresis. The clearance of labeled insulin in vivo was very slow, and the glucose response to intravenous insulin was absent. The free binding capacity in serum was several thousand μU./ml. Acid splitting of the insulin—anti-insulin complexes revealed 7,000 μU./ml. or more of endogenous insulin, and antibodies which bound human and pig insulin better than bovine insulin. After oral glucose the patient's serum insulin increased by 2,300 μU./ml. Some new principles are introduced in the quantitative studies on serum insulin and antibodies. Theoretical restrictions on these and other quantitations are discussed. The hyperglycemia may be explained by the antibodies binding secreted insulin, and the hypoglycemia by an insulin overshoot from maximally stimulated β-cells when saturation of the binding capacity was approached. Carbohydrate restriction prevented the hypoglycemic attacks. The cause of the antibody production and the properties of the antibodies are discussed.

Amino acid sequences of insulins.

The amino acid sequences of insulins determined to date are summarized. Guinea pig, coypu and rat insulins are discussed in particular.