Evolution of the insulin molecule: insights into structure-activity and phylogenetic relationships.

The conformation of insulin in the crystalline state has been known for more than 30 years but there remains uncertainty regarding the biologically active conformation and the structural features that constitute the receptor-binding domain. The primary structure of insulin has been determined for at least 100 vertebrate species. In addition to the invariant cysteines, only ten amino acids (GlyA1, IleA2, ValA3, TyrA19, LeuB6, GlyB8, LeuB11, ValB12, GlyB23 and PheB24) have been fully conserved during vertebrate evolution. This observation supports the hypothesis derived from alanine-scanning mutagenesis studies that five of these invariant residues (IleA2, ValA3, TyrA19, GlyB23, and Phe24) interact directly with the receptor and five additional conserved residues (LeuB6, GlyB8, LeuB11, GluB13 and PheB25) are important in maintaining the receptor-binding conformation. With the exception of the hagfish, only conservative substitutions are found at B13 (Glu --> Asp) and B25(Phe --> Tyr). In contrast, amino acid residues that were also considered to be important in receptor binding based upon the crystal structure of insulin (GluA4, GlnA5, AsnA21, TyrB16, TyrB26) have been much less well conserved and are probably not components of the receptor-binding domain. The hypothesis that LeuA13 and LeuB17 form part of a second receptor-binding site in the insulin molecule finds some support in terms of their conservation during vertebrate evolution, although the site is probably absent in some hystricomorph insulins. In general, the amino acid sequences of insulins are not useful in cladistic analyses especially when evolutionary distant taxa are compared but, among related species in a particular order or family, the presence of unusual structural features in the insulin molecule may permit a meaningful phylogenetic inference. For example, analysis of insulin sequences supports monophyletic status for Dipnoi, Elasmobranchii, Holocephali and Petromyzontiformes.     

Mutations at the dimer, hexamer, and receptor-binding surfaces of insulin independently affect insulin-insulin and insulin-receptor interactions.

Mutagenesis of the dimer- and hexamer-forming surfaces of insulin yields analogues with reduced tendencies to aggregate and dramatically altered pharmacokinetic properties. We recently showed that one such analogue, HisB10----Asp, ProB28----Lys, LysB29----Pro human insulin (DKP-insulin), has enhanced affinity for the insulin receptor and is useful for studying the structure of the insulin monomer under physiologic solvent conditions [Weiss, M. A., Hua, Q. X., Lynch, C. S., Frank, B. H., & Shoelson, S. E. (1991) Biochemistry 30, 7373-7389]. DKP-insulin retains native secondary and tertiary structure in solution and may therefore provide an appropriate baseline for further studies of related analogues containing additional substitutions within the receptor-binding surface of insulin. To test this, we prepared a family of DKP analogues having potency-altering substitutions at the B24 and B25 positions using a streamlined approach to enzymatic semisynthesis which negates the need for amino-group protection. For comparison, similar analogues of native human insulin were prepared by standard semisynthetic methods. The DKP analogues show a reduced tendency to self-associate, as indicated by 1H-NMR resonance line widths. In addition, CD spectra indicate that (with one exception) the native insulin fold is retained in each analogue; the exception, PheB24----Gly, induces similar perturbations in both native insulin and DKP-insulin backgrounds. Notably, analogous substitutions exhibit parallel trends in receptor-binding potency over a wide range of affinities: D-PheB24 greater than unsubstituted greater than GlyB24 greater than SerB24 greater than AlaB25 greater than LeuB25 greater than SerB25, whether the substitution was in a native human or DKP-insulin background. Such "template independence" reflects an absence of functional interactions between the B24 and B25 sites and additional substitutions in DKP-insulin and demonstrates that mutations in discrete surfaces of insulin have independent effects on protein structure and function. In particular, the respective receptor-recognition (PheB24, PheB25), hexamer-forming (HisB10), and dimer-forming (ProB28, LysB29) surfaces of insulin may be regarded as independent targets for protein design. DKP-insulin provides an appropriate biophysical model for defining structure-function relationships in a monomeric template.     

Mechanisms of leucine- and theophylline-stimulated insulin biosynthesis in isolated rat pancreatic islets.

To extend previous observations on the mechanisms of translational regulation of insulin biosynthesis [Welsh, Scherberg, Gilmore & Steiner (1986) Biochem. J. 235, 459-467], we have now compared the intracellular distributions of insulin mRNA after stimulation of insulin biosynthesis by glucose, leucine or theophylline. In comparison with low glucose (3.3 mM) only, the presence of 10 mM-leucine + 3.3 mM-glucose resulted in the transfer of insulin mRNA from the pool of the uninitiated mRNA to the free polysome/monosome fraction and an increase in the amount of insulin mRNA associated with the microsomal fraction. Islets exposed to 5 mM-theophylline + 3.3 mM-glucose also showed a decreased content of uninitiated insulin mRNA in the cytosol, but these islets showed no increase in insulin mRNA in the microsomal fraction. These results suggest that leucine, a nutrient stimulant of insulin biosynthesis, acts essentially by the same mechanisms as those of glucose, whereas theophylline acts only to stimulate initiation rates.     

Two distinct pathways of formation of 4-hydroxynonenal. Mechanisms of nonenzymatic transformation of the 9- and 13-hydroperoxides of linoleic acid to 4-hydroxyalkenals

The mechanism of formation of 4-hydroxy-2E-nonenal (4-HNE) has been a matter of debate since it was discovered as a major cytotoxic product of lipid peroxidation in 1980. Recent evidence points to 4-hydroperoxy-2E-nonenal (4-HPNE) as the immediate precursor of 4-HNE (Lee, S. H., and Blair, I. A. (2000) Chem. Res. Toxicol. 13, 698-702; Noordermeer, M. A., Feussner, I., Kolbe, A., Veldink, G. A., and Vliegenthart, J. F. G. (2000) Biochem. Biophys. Res. Commun. 277, 112-116), and a pathway via 9-hydroperoxylinoleic acid and 3Z-nonenal is recognized in plant extracts. Using the 9- and 13-hydroperoxides of linoleic acid as starting material, we find that two distinct mechanisms lead to the formation of 4-H(P)NE and the corresponding 4-hydro(pero)xyalkenal that retains the original carboxyl group (9-hydroperoxy-12-oxo-10E-dodecenoic acid). Chiral analysis revealed that 4-HPNE formed from 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) retains >90% S configuration, whereas it is nearly racemic from 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9S-HPODE). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid is >90% S when derived from 9S-HPODE and almost racemic from 13S-HPODE. Through analysis of intermediates and products, we provide evidence that (i) allylic hydrogen abstraction at C-8 of 13S-HPODE leads to a 10,13-dihydroperoxide that undergoes cleavage between C-9 and C-10 to give 4S-HPNE, whereas direct Hock cleavage of the 13S-HPODE gives 12-oxo-9Z-dodecenoic acid, which oxygenates to racemic 9-hydroperoxy-12-oxo-10E-dodecenoic acid; by contrast, (ii) 9S-HPODE cleaves directly to 3Z-nonenal as a precursor of racemic 4-HPNE, whereas allylic hydrogen abstraction at C-14 and oxygenation to a 9,12-dihydroperoxide leads to chiral 9S-hydroperoxy-12-oxo-10E-dodecenoic acid. Our results distinguish two major pathways to the formation of 4-HNE that should apply also to other fatty acid hydroperoxides. Slight ( approximately 10%) differences in the observed chiralities from those predicted in the above mechanisms suggest the existence of additional routes to the 4-hydroxyalkenals.     

Spectroscopic evidence for an intermediate in the T6 to R6 allosteric transition of the Co(II)-substituted insulin hexamer.

The phenol-induced conformational transition in the insulin hexamer is known to involve a large change in structure wherein residues 1-8 of the insulin B-chain are transformed from an extended coil (T-state) to a helix (R-state). This change in protein conformation both exposes a cryptic protein pocket on each subunit to which phenol binds and forces the HisB10 zinc sites to undergo a change in coordination geometry from octahedral to tetrahedral [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 593-596]. Substitution of Co(II) for Zn(II) at the HisB10 sites introduces a sensitive chromophoric probe of the structural and chemical events that occur during this allosteric transition [Roy, M., Brader, M. L., Lee, R. W.-K., Kaarsholm, N. C., Hansen, J. F., & Dunn, M. F. (1989) J. Biol. Chem. 264, 19081-19085]. In this study, using rapid-scannig stopped-flow (RSSF) UV-visible spectroscopic studies, we demonstrate that a transient chemical intermediate is formed during the phenol-induced conversion of Co(II)-substituted hexamer from the T-state to the R-state. Decomposition of the RSSF spectra gave a spectrum for the intermediate with d-d transitions consistent with the assignment of the intermediate as either a distorted tetrahedral or a 5-coordinate Co(II) species. Possible structures for the intermediate and the implications of these findings to the allosteric mechanism are considered.     

Properties and distribution of the protein inhibitor (Mr 17,000) of protein kinase C.

Ca2+-dependent hydrophobic-interaction chromatography is a powerful tool for the identification and isolation of a variety of Ca2+-binding proteins which expose a hydrophobic site(s) in the presence of Ca2+ [Gopalakrishna & Anderson (1982) Biochem. Biophys. Res. Commun. 104, 830-836; Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127; McDonald & Walsh (1985) Biochem. J. 232, 559-567]. Using this approach, we isolated two potent and specific protein inhibitors of protein kinase C, of 17 kDa [McDonald & Walsh (1985) Biochem. J. 232, 559-567] and 12 kDa [McDonald & Walsh (1986) Biochem. Soc. Trans. 14, 585-586]. Although these inhibitors were purified by Ca2+-dependent hydrophobic-interaction chromatography and exhibit properties similar to those of calmodulin and related Ca2+-binding proteins, we were unable to demonstrate high-affinity Ca2+ binding to these inhibitors, using equilibrium dialysis. Protein kinase C exhibited half-maximal activity at 0.6 microM-Ca2+ in the presence of phospholipid and diacylglycerol, and complete inhibition by both inhibitors was observed over the range of Ca2+ concentrations examined (10 nM-10 microM). These observations suggest that the inhibitory action of these proteins does not require Ca2+. The inclusion of proteinase inhibitors during isolation of the kinase C inhibitors, as well as two-dimensional peptide mapping and amino acid analysis of the isolated proteins, suggested that the 12 kDa inhibitor is a proteolytic fragment of the 17 kDa protein which is generated during purification. Antibodies raised in rabbits against the bovine brain 17 kDa inhibitor were shown to be specific by Western immunoblotting and the competitive enzyme-linked immunosorbent assay method and were used to study the tissue and species distribution of this protein. The inhibitor was found to be present in several bovine, murine, avian and human tissues, consistent with a role in the regulation of a variety of physiological functions involving the widely distributed protein kinase C.     

An improved procedure, involving mass spectrometry, for N-terminal amino acid sequence determination of proteins which are N alpha-blocked.

A modification to a previously described procedure [Gray & del Valle (1970) Biochemistry 9, 2134-2137; Rose, Simona & Offord (1983) Biochem. J. 215, 261-272] for mass-spectral identification of the N-terminal regions of proteins is shown to be useful in cases where the N-terminus is blocked. Three proteins were studied: vesicular-stomatitis-virus N protein, Sendai-virus NP protein, and a rabbit immunoglobulin lambda-light chain. These proteins, found to be blocked at the N-terminus with either the acetyl group or a pyroglutamic acid residue, had all failed to yield to attempted Edman degradation, in one case even after attempted enzymic removal of the pyroglutamic acid residue. The N-terminal regions of all three proteins were sequenced by using the new procedure.     

Labelling of chlorophylls and precursors by [2-14C]glycine and 2-[1-14C]oxoglutarate in Rhodopseudomonas spheroides and Zea mays. Resolution of the C5 and Shemin pathways of 5-aminolaevulinate biosynthesis by thin-layer radiochromatography

The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-14C]glycine or 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spheroides and Zea mays (maize), respectively, as examples of each pathway. Both [2-14C]glycine and, to a lesser extent 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a formed during adaptation of respiring R. spheroides cells to photosynthetic (anaerobic, illuminated) conditions. This and earlier evidence suggested augmentation of the Shemin pathway by a minor C5 pathway contribution. The present studies revealed only Shemin pathway activity: with laevulinate present, [2-14C]glycine formed 5-[5-14C]aminolaevulinate as proved by H14CHO production during periodate cleavage. These methods were sufficiently sensitive also to detect the incorporation of 14CO2, from degradation of either substrate, into 5-aminolaevulinate via the Shemin pathway thus labelling the succinate fragment produced with periodate: this explains bacteriochlorophyll a labelling by 2-[1-14C]oxoglutarate and proves double labelling of 5-aminolaevulinate by [2-14C]glycine. The same techniques were applied to etiolated maize leaves exposed to aerobic illuminated conditions with laevulinate and either 2-[1-14C]oxoglutarate or [2-14C]glycine as substrates. Only the C5 pathway was detected: 2-[1-14C]oxoglutarate was converted to 5-[5-14C]aminolaevulinate, which yielded H14CHO on periodate cleavage. This is not inconsistent with our earlier 13C-NMR studies [Porra, R.J., Klein, O. and Wright, P. E. (1983) Eur. J. Biochem. 130, 509-516] showing that the C5 pathway formed all the 5-aminolaevulinate for chlorophyll biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of thrombomodulin on the cleavage of fibrinogen and fibrinogen fragments by thrombin

Thrombomodulin acts as a linear competitive inhibitor of thrombin with respect to the substrate fibrinogen. In the present study the effect of thrombomodulin on the activity of thrombin with fragments of the A alpha and B beta chain of fibrinogen has been examined. The cleavage of fibrinopeptide A from the N-terminal disulphide knot, fragment 1-44 and fragment 1-51 of the A alpha chain was inhibited by thrombomodulin. The average value for the inhibition constant obtained with these substrates was 0.83 +/- 0.09 nM, which was in good agreement with the values obtained previously for the inhibition of thrombin by thrombomodulin with native fibrinogen as the substrate [Hofsteenge, J., Taguchi, H. & Stone, S. R. (1986) Biochem. J. 237, 243-251]. In contrast, the cleavage of fibrinopeptide A from fragment 1-23 and fragment 1-29 of the A alpha chain was not affected by thrombomodulin. Although the cleavage of the B beta chain in the intact fibrinogen molecule was inhibited by thrombomodulin [Hofsteenge, J., Taguchi, H. & Stone, S. R. (1986) Biochem. J. 237, 243-251], the release of fibrinopeptide B from the N-terminal disulphide knot and the N-terminal 118-residue fragment of the B beta chain was not inhibited by thrombomodulin. In addition, we determined the second-order rate constants of cleavage of these substrates using human thrombin. Fragments of the A alpha chain whose cleavage was inhibited by thrombomodulin were found to have values for kcat/Km that were within one order of magnitude of that for the native fibrinogen, whereas those for A alpha chain fragments whose cleavage was not inhibited by thrombomodulin were found to be more than two orders of magnitudes lower. From these results we conclude that only a relatively small portion of the A alpha chain of the fibrinogen molecule is responsible for the specific binding to thrombin that is affected by thrombomodulin. Moreover, residues 30-44 of the A alpha chain play an important role in this thrombin-fibrinogen interaction.     

Enzyme kinetic studies from progress curves.

A method is described for fitting the velocities obtained from progress curves to a steady-state rate equation. It is based on the method of Markus & Plesser [(1981) in Kinetic Data Analysis: Design and Analysis of Enzyme and Kinetic Data (Edrenyi, ed.), pp. 317-339, Plenum Press, New York]. The obstacle of needing good initial estimates of kinetic parameters is removed by using the parameters provided graphically by a minor modification of the method of Yun & Suelter [(1977) Biochim, Biophys. Acta 480, 1-13]. This progress-curved-based method allows the same discrimination among rival models as do the initial-velocity-based methods, with a great saving of experimental time. The BASIC and FORTRAN 77 programs are deposited as Supplementary Publication SUP 50132 (17 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5-6.     

Exogenous adenosine 3': 5'-monophosphate can release yeast from catabolite repression.

A new simple fast and reproducible purification procedure for the proteinase from rat liver mitochondria has been worked out. The specificity of cleavage of peptide bonds in glucagon, oxidized A and B chains of insulin and yeast proteinase B inhibitor by the proteinase of the inner mitochondrial membrane has been studied. The proteinase hydrolyzed three peptide bonds in glucagon, Tyr (13) - Leu (14), Trp (25) - Leu (26) and Phe (22) - Val (23) (minor cleavage site); none in the insulin A chain; one in the B chain of insulin, Tyr (16) - Leu (17); and three in the yeast proteinase B inhibitor, Phe (4) - Ile (5), Phe (20) - Leu (21) and Tyr (41) - Thr (42) (minor cleavage site).Thus, the mitochondrial proteinase cleaves peptide bonds at the carboxyl site of an aromatic amino acid and the amino site of a leucine, isoleucine, threonine or valine. The comparison with chymotrypsin A shows that the mitochondrial proteinase cleaves peptide bonds in a more restricted manner.     

Molecular Evolution of Insulin in Non-Mammalian Vertebrates

The traditional view, based primarily on X-ray crystallographicdata, is that the amino acid residues at positions B12, B16,B23-B26, A1-A5, A19 and A21 in the insulin molecule comprisethe receptor-binding domain. More recently, however, it hasbeen proposed that the conformation adopted by insulin in thecrystal structure is an inactive one. The results of alanine-scanningmutagenesis studies suggest that GlyB23, PheB24, IleA2, ValA3,and TyrA19 interact directly with the receptor with LeuB6, GlyB8,LeuB11, GluB13 and PheB25, although not part of the bindingepitope, being important in maintaining the receptor-bindingconformation. A comparison of the primary structures of insulinsfrom a wide range of non-mammalian vertebrates, from hagfishto birds, provides support for this revised view by demonstratingthat strong evolutionary pressure has acted to conserve thoseamino acids postulated to be important in the biologically activeconformation. In addition to the cysteine residues, the aminoacids at B6, B8, B11, B23, B24, A2, A3, and A19 are invariantin all species yet studied with only conservative substitutions(Glu Asp) at B13 and (Phe Tyr) at B25. In contrast, severalinsulins containing substitutions at positions B16, A5 and A21,sites of importance in maintaining the crystal structure conformation,have been identified. Although the amino acid sequences of insulinare not generally useful as molecular markers for inferringphylogenetic relationships between species, the presence ofcommon structural features in insulins from closely relatedspecies may permit a valid inference. For example, the presenceof an N-terminal pentapeptide extension to the B-chains of insulinsisolated from both holarctic and southern hemisphere lampreyssupports the monophyletic status of the Petromyzontiformes.     

Detailed analysis of base preferences at the cleavage site of a trans-acting HDV ribozyme: a mutation that changes cleavage site specificity.

In our previous attempt at in vitro selection of a trans - acting human hepatitis delta virus (HDV) ribozyme, we found that one of the variants, G10-68-725G, cleaved a 13 nt substrate, HDVS1, at two sites [Nishikawa,F., Kawakami,J., Chiba,A., Shirai,M., Kumar,P.K.R. and Nishikawa,S. (1996) Eur. J. Biochem., 237, 712-718]. One site was the normal cleavage site and the other site was shifted 1 nt toward the 3'-end. To clarify the interactions between nucleotides around the cleavage site of the trans -acting HDV ribozyme, we analyzed the efficiency of the reaction for every possible base pair between the substrate and the ribozyme at positions -1 (-1N:726N) and +1 (+1N:725N) relative to the cleavage site using the genomic HDV ribozyme, TdS4(Xho), and derivatives of the most active variant, G10-68. These mutagenesis analyses revealed that the +1 base of the substrate affects the structure of the catalytic core in the complex with G10-68-725G, substrate and divalent metal ions, and it shifts the cleavage site. In a comparison with other variants of the trans -acting HDV ribozyme, we found that this cleavage site shift occurred only with G10-68-725G.     

Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N-terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].     

Identification of insulin intermediates and sites of cleavage of native insulin by insulin protease from human fibroblasts

We have studied the time sequence degradation of native insulin by insulin protease from human fibroblast using multiple steps involving purification of the products by high performance liquid chromatography, determination of peak composition by amino acid sequence analysis, and confirmation of structure by mass spectrometry and thus elucidated the sites of cleavage of insulin by human insulin protease. We observed that as early as 0.5 min of incubation, three major new peptide peaks, intact insulin, and four smaller peptide peaks can be detected. The major peptides are portions of the insulin molecule, with the amino ends of the A and B chains or the carboxyl ends of the A and B chains still connected by disulfide bonds. Peptide peak I is A1-13-B1-9. Peptide peak II is A1-14-B1-9. Peptide peak III is A14-21-B14-30. The smaller peptide peaks are A14-21-B17-30, A15-21-B14-30, A15-21-B10-30, and A14-21-B10-30. The major peptide bond cleavage sites therefore consist of A13-14, A14-15, B9-10, B13-14, and B10-17. With longer incubation times, peptide peak II appears to lose the A14 tyrosine to form peptide peak I. This peptide I, which is the amino end of the A and B chains, is not further degraded even after 1.5 h of incubation. With longer incubation times, the peptides containing the carboxyl ends of the A and B chains are further degraded to form products from cleavage at the A18-19, B14-15, B25-26, and a small amount of A19-20, B10-11, and B24-25 cleavage and the emergence of 2-5-amino acid peptide chains, tyrosine, alanine, histidine, and leucine-tyrosine. We conclude, based on the three-dimensional structure of insulin, that human insulin protease recognizes the alpha-helical regions around leucine-tyrosine bonds and that final degradation steps to small peptides do not require lysosomal involvement.     

Removal of covalently bound inositol from Torpedo acetylcholinesterase and mammalian alkaline phosphatases by deamination with nitrous acid. Evidence for a common membrane-anchoring structure.

Our earlier evidence suggested that both acetylcholinesterase and alkaline phosphatase are anchored to the cell surface via covalently-attached phosphatidylinositol [Low, Futerman, Ferguson & Silman (1986) Trends Biochem. Sci. 11, 212-215]. We now present chemical data, based upon a nitrous acid deamination reaction, showing that in both proteins the phosphatidylinositol moiety is attached through a glycosidic linkage to a sugar residue bearing a free amino group.     

High-pressure-liquid-chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin.

1. A method of obtaining serial arterial and mammary-venous blood samples was used to identify possible factors involved in the regulation of glucose uptake in the gland of the lactating rat. 2. Administration of insulin alone increased the arteriovenous glucose difference across the mammary gland of starved rats, but the time course of the recovery could not account for the restoration of arteriovenous glucose difference observed during refeeding [Page & Kuhn (1986). Biochem. J. 239, 269-274]. 3. A crude extract of the gastrointestinal tract (stomach-ileum) from lactating rats enhanced the change in mammary glucose uptake observed with insulin, but only when large amounts (100 munits/rat) of insulin were used. To achieve a similar recovery of arteriovenous glucose difference using near-physiological amounts (5 munits/rat) of insulin it was necessary to sever the mammary nerves. 4. A peptide fraction (of less than 10 kDa) isolated from the gut extract enhanced the effect of insulin in a similar manner to the crude extract. 5. It is suggested that in addition to insulin at least another component, probably a gut peptide, is required for the restoration of mammary glucose uptake during refeeding. An inhibitory component may also contribute to the regulation of mammary glucose extraction in the lactating rat.     

Heteronuclear 2D NMR studies of an engineered insulin monomer: assignment and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design

Insulin provides an important model for the application of genetic engineering to rational protein design and has been well characterized in the crystal state. However, self-association of insulin in solution has precluded complementary 2D NMR study under physiological conditions. We demonstrate here that such limitations may be circumvented by the use of a monomeric analogue that contains three amino acid substitutions on the protein surface (HisB10----Asp, ProB28----Lys, and LysB29----Pro); this analogue (designated DKP-insulin) retains native receptor-binding potency. Comparative 1H NMR studies of native human insulin and a series of three related analogues--(i) the singly substituted analogue [HisB10----Asp], (ii) the doubly substituted analogue [ProB28----Lys; LysB29----Pro], and (iii) DKP-insulin--demonstrate progressive reduction in concentration-dependent line-broadening in accord with the results of analytical ultracentrifugation. Extensive nonlocal interactions are observed in the NOESY spectrum of DKP-insulin, indicating that this analogue adopts a compact and stably folded structure as a monomer in overall accord with crystal models. Site-specific 2H and 13C isotopic labels are introduced by semisynthesis as probes for the structure and dynamics of the receptor-binding surface. These studies confirm and extend under physiological conditions the results of a previous 2D NMR analysis of native insulin in 20% acetic acid [Hua, Q. X., & Weiss, M. A. (1991) Biochemistry 30, 5505-5515]. Implications for the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogues.     

Effects of dexamethasone treatment on insulin-stimulated rates of glycolysis and glycogen synthesis in isolated incubated skeletal muscles of the rat.

1. Rats were treated with dexamethasone for 4 days before measurement of the rates of lactate formation [which is an index of hexose transport; see Challiss, Lozeman, Leighton & Newsholme (1986) Biochem. J. 233, 377-381] and glycogen synthesis in response to various concentrations of insulin in isolated incubated soleus and extensor digitorum longus muscle preparations. 2. The concentration of insulin required to stimulate these processes half-maximally in soleus and extensor digitorum longus muscles isolated from control rats was about 100 muunits/ml. 3. Dexamethasone increases the concentration of insulin required to stimulate glycolysis half-maximally in soleus and extensor digitorum longus preparations to 250 and 300 muunits/ml respectively. The respective insulin concentrations necessary to stimulate glycogen synthesis half-maximally were about 430 and 370 muunits/ml for soleus and extensor digitorum longus muscle preparations isolated from steroid-treated rats. 5. Dexamethasone treatment did not change the amount of insulin bound to soleus muscle.     

On the disulphide bonds of rhodopsins.

Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones).