Contribution of the B16 and B26 tyrosine residues to the biological activity of insulin

We report the synthesis and biological evaluation of five insulin analogues in which one or both of the B-chain tyrosine residues have been substituted. [B16 Phe]insulin and [B16 Trp]insulin display a very modest reduction in potency (c. 65%) relative to porcine insulin; [B26 Phe]insulin is less active (30–50%), and the doubly substituted [B16 Phe, B26 Phe]insulin displays still lower potency (c. 35%). The further substitution of Asp for B10 His in [B16 Phe, B26 Phe]insulin raises its activity to approximately twofold greater than natural insulin, an increase of approximately fivefold over the parent compound. We conclude that the bulk and/or aromaticity of the amino acid residue at position B16, but not its hydrogen-bonding capacity, contributes to the biological activity of the hormone. We further conclude that hydrogen-bonding capacity or special side-chain packing characteristics are required at the B26 position for insulin to display high biological activity.     

The A14 position of insulin tolerates considerable structural alterations with modest effects on the biological behavior of the hormone

As part of our aim to investigate the contribution of the tyrosine residue found in the 14 position of the A-chain to the biological activity of insulin, we have synthesized six insulin analogues in which the A14 Tyr has been substituted by a variety of amino acid residues. We have selected three hydrophilic and charged residues—glutamic acid, histidine, and lysine—as well as three hydrophobic residues—cycloleucine, cyclohexylalanine, and naphthyl-(1)-alanine—to replace the A14 Tyr. All six analogues exhibit full agonist activity, reaching the same maximum stimulation of lipogenesis as is achieved with procine insulin. The potency for five of the six analogues, [A14 Glu]-, [A14 His]-, [A14 Lys]-, [A14 cycloleucine]-, and [A14 naphthyl-(1)-alanine]-insulins in receptor binding assays ranges from 40–71% and in stimulation of lipogenesis ranges from 35-120% relative to porcine insulin. In contrast, the potency of the sixth analogue, [A14 cyclohexylalanine]insulin, in both types of assays is less than 1% of the natural hormone. The retention time on reversed-phase high-performance liquid chromatography for the first five analogues is similar to that of bovine insulin, whereas for the sixth analogue, [A14 cyclohexylalanine]insulin, it is approximately 11 min longer than that of the natural hormone. This suggests a profound change in conformation of the latter analogue. Apparently, the A14 position of insulin can tolerate a wide latitude of structural alterations without substantial decrease in potency. This suggests that the A14 position does not participate directly in insulin receptor interaction. Only when a substitution which has the potential to disrupt the conformation of the molecule is made at this position, is the affinity for the receptor, and hence the biological potency, greatly reduced.     

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys¹⁰-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys¹⁰-B] insulin was found to possess a potency of ～17% compared to insulin. We have shown previously that substitution of the B¹⁰ polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays ～50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B¹⁰, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.     

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys¹⁰-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys¹⁰-B] insulin was found to possess a potency of 17% compared to insulin. We have shown previously that substitution of the B¹⁰ polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays 50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B¹⁰, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.For the previous paper in this series see Schwartzet al. (1981).     

Determination of the Amino Acid Sequence of a New Phospholipase A<Subscript>2</Subscript> (MIDCA1) Isolated from <Emphasis Type="Italic">Micrurus dumerilii carinicauda</Emphasis> Venom

A new Phospholipase A₂ (PLA₂) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA₂ showed a low enzymatic activity when compared with other PLA₂s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA₂s, such as Naja naja, showed significant differences in the β-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA₂, supporting the view that other regions of the protein are involved in the biological effects.     

Inhibition of Platelet Aggregation of a Mutant Proinsulin Chimera Engineered by Introduction of a Native Lys-Gly-Asp-containing Sequence

An eight amino acid sequence, CAKGDWNC, from disintegrin barbourin, was introduced into an inactive human proinsulin molecule between the B28 and A2 sites to construct a chimeric, anti-thrombosis recombinant protein. The constructed Lys-Gly-Asp (KGD)-proinsulin gene was expressed in Escherichia coli and then purified. The KGD-proinsulin chimera protein inhibits human platelet aggregation, induced by ADP, with an IC₅₀ value (molar concentration causing 50% inhibition of platelet aggregation) of 830 nM and demonstrates also specific affinity to glycoprotein IIb/IIIa receptor. Its insulin receptor binding activity remaines as low as 0.04% with native insulin as a control.     

Complete amino acid sequence of the FK506 and rapamycin binding protein, FKBP, isolated from calf thymus

FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (K _d=0.4 nM) and rapamycin (K _d=0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFVLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other post-translational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaertet al., 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent -strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes.     

Expression of a Recombinant Molt-Inhibiting Hormone of the Kuruma Prawn Penaeus japonicus in Escherichia coli

The crustacean molt-inhibiting hormone (MIH) suppresses ecdysteroid synthesis by the Y-organ. The MIH of the kuruma prawn Penaeus japonicus has recently been isolated and its cDNA cloned. In this study, we expressed the MIH in Escherichia coli to obtain a large quantity of this hormone with biological activity. The MIH cDNA was processed and ligated into an expression plasmid. E. coli was transformed with this plasmid, and then the recombinant MIH (r-MIH) was expressed. The r-MIH was put through the refolding reaction and was purified by reverse-phase HPLC. N-terminal amino acid sequence and time-of-flight mass spectral analyses supported the idea that the r-MIH had the entire sequence. By in vitro bioassay using the Y-organ of the crayfish, the r-MIH was found to be comparable to natural MIH in inhibiting ecdysteroid synthesis.     

Preparation and physical-chemical characterization of poly-N-vinylpyrrolidone-insulin

In an attempt to modify the structure of insulin, the hormone was coupled by covalent linkage to poly-N-vinylpyrrolidone (PVP). PVP-insulin is soluble in aqueous solution and was purified by column chromatography. Special care was taken to check for a possible leakage of insulin from the synthetic polymer. The amount of bound insulin was determined by quantitative amino acid analysis after acid hydrolysis. After protection of the N-terminal glycine residue of the A-chain by citraconylation, a biologically active PVP-insulin was obtained. The molecular weight determined by analytical ultracentrifugation and column chromatography was 50 000-60 000. The biological activity of the coupled hormone was between 0.5 and 7% when compared with native insulin in the system in vitro. The immunological activity was about 20-50%.     

Synthetic peptide VKGFY and its cyclic analog stimulate macrophage bactericidal activity through non-opioid beta-endorphin receptors

We synthesized linear and cyclic pentapeptides corresponding to the sequence 369-373 of human immunoglobulin G heavy chain—VKGFY (referred to as pentarphin and cyclopentarphin, respectively). The effect of pentarphin and cyclopentarphin on phagocytosis of Salmonella typhimurium virulent 415 strain bacteria by mouse peritoneal macrophages in vitro was studied. Control experiments showed that macrophages actively captured these bacteria, but did not digest them: the captured microbes were viable and continued to proliferate inside the phagocytes; within 12 h all macrophage monolayer was destroyed (incomplete phagocytosis). If 1 nM pentarphin or cyclopentarphin was added to the cultivation medium, macrophage bactericidal activity was significantly increased and they digested all captured microorganisms within 6 h (complete phagocytosis). To study the receptor binding properties of pentarphin and cyclopentarphin we prepared ¹²⁵I-labeled pentarphin (179 Ci/mmol specific activity). The binding of ¹²⁵I-labeled pentarphin to mouse peritoneal macrophages was highaffinity (K _d = 3.6 ± 0.3 nM) and saturable. Studies on binding specificity revealed that this binding was insensitive to naloxone and [Met⁵]enkephalin, but completely inhibited by unlabeled cyclopentarphin (K _i = 2.6 ± 0.3 nM), immunorphin (K _i = 3.2 ± 0.3 nM), and -endorphin (K _i = 2.8 ± 0.2 nM). Thus, the effects of pentarphin and cyclopentarphin on macrophages are mediated by naloxone-insensitive receptors common for pentarphin, cyclopentarphin, immunorphin, and -endorphin.     

The relationship between the connecting peptide of recombined single chain insulin and its biological function

To investigate the relationship between the biological activity of recombined single chain insulin and the length of the connecting peptide, we designed and prepared three single chain insulin molecules, namely, PIP, [A]₅PIP and [A]₁₀PIP, by site-directed mutagenesis, in which B30 and A1 were linked through dipeptide A-K, heptapeptide A-A-A-A-A-A-K, and dodecapeptide A-A-A-A-A-A-A-A-A-A-A-K, respectively. Their receptor binding capacities were 0.14%, 14.3% and 11.1% of that of insulin respectively and theirin vivo biological activities were in consistence with their receptor binding capacity; whereas their growth promoting activities were 17%, 116.3% and 38% of that of insulin. These results suggested the following conclusions. (i) The recombined single chain insulin could also possess the same metabolic and mitogenic function as insulin. (ii) The receptor binding capacity of recombined single chain insulin to insulin receptor was closely related to the length and amino acid composition of the connecting peptide and could change from 0 to 100% of insulin depending on the different connecting peptides. This result further illustrated the necessity of B chain C-terminus swaying away from A chain N-terminus when insulin binds to its receptor. (iii) The mitogenic activity of recombined single chain insulin also depended on the length and the amino acid composition of the connecting peptide and was higher than its metabolic activity.     

The biological activity of catfish pancreatic somatostatin

Catfish pancreatic somatostatin, which contains eight additional amino acids on the amino terminus of a tetradecapeptide with considerable homology to tetradecapeptide somatostatin (SRIF), is a naturally occurring homology of the hypothalamic peptide. The purpose of these studies was to determibe the biological activity of this somatostatin homolog. Inhibition of ¹²⁵I-labelled tyr¹-SRIF binding to bovine pituitart plasma membranes by catfish pancreatic somatostatin was approximately 33% that of SRIF. Pancreatic somatostatin has full biological activity measured by inhibition of growth hormone release from isolated rat pituitary cells, but 0.01–0.1% the potency of SRIF. Pancreatic somatostatin at 100 ng/ml produced a 50–60% inhibition of insulin and glucagon secretion from perfused rat pancreas, while SRIF produced comparable inhibition at 10 ng/ml. This report demonstrates that a larger molecular form and natural homolog of SRIF, isolated from fish pancreas, has the same (but reduced) biological activities in rat assay systems as somatostatin originally isolated from sheep hypothalamus.     

Induction of tyrosine aminotransferase and amino acid transport in rat hepatoma cells by insulin and the insulin-like growth factor,multiplication-stimulating activity: Mediation by insulin and multiplication-stimulating activity receptors

Insulin stimulates a 2-fold increase in the amount of tyrosine aminotransferase and a 5–10-fold increase in the rate of amino acid transport in dexamethasone-treated rat hepatoma cells. In order to determine whether these effects are mediated by insulin receptors or receptors for insulin-like growth factors, we have examined the binding of ¹²⁵I-labeled insulin and ¹²⁵I-labeled multiplication-stimulating activity, a prototype insulin-like growth factor, and compared the biological effects of these polypeptides. Insulin and multiplication-stimulating activity cause an identical increase in transaminase activity and transport velocity; half-maximal biological effects were observed at 35 ng/ml (5.5 nM) insulin and 140 ng/ml multiplication-stimulating activity. The hepatoma cells display typical insulin receptors of appropriate specificity; half-maximal displacement of tracer insulin binding occured at 33 ng/ml unlabeled insulin, but only at 2500 ng/ml unlabeled multiplication-stimulating activity. Specific multiplication-stimulating activity receptors also were demonstrated with which insulin did not interact even at 10 μg/ml. Half-maximal displacement of tracer multiplication-stimulating activity occured at 200 ng/ml unlabeled multiplication-stimulating activity. We conclude that insulin cannot act via the multiplication-stimulating activity receptor and presumably acts via typical insulin receptors. The effects of multiplication-stimulating activity on enzyme induction and amino acid transport are probably mediated primarily via the multiplication-stimulating activity receptor.     

21-arginine-A] insulin: a biologically active analog.

An analog of sheep insulin which differs from the parent molecule in that the C-terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg21] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S-sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S-sulfonated B chain of bovine (sheep) insulin yielded [Arg21-A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg21-A] sheep insulin shows potencies of 10.5--12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23--25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A21 asparagine participates in salt bridge- and hydrogen bond-forming interactions which are critical in the biological activity of the hormone. Although the [Arg21-A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the above mentioned interactions come into play, which involve the side chain of the A21 amino acid residue and affect the biological activity of the hormone.     

Characterization and molecular cloning of a novel endoglucanase from Trichoderma sp. C-4

A fungal strain, C-4, was isolated from etiolated leaves. Based on taxonomic studies, the fungus C-4 can be classified as a strain of Trichoderma species. When strain C-4 was cultured in Mandels medium at 28°C for 6 days, the enzyme activities detected in the broth corresponded to 8.2 U/ml (28.1 U/mg) carboxymethylcellulase activity. An endoglucanase (EG; F-I-II) was purified from the culture filtrate of the strain through a four-step procedure—chromatography on Sephacryl S-200, DEAE-Sephadex A-50, Con A-Sepharose, and Chromatofocusing on Mono-P (HPLC). The molecular weight of this EG, which was called C4endoII, was determined to be about 51 kDa. The optimum temperature and pH of C4endoII were 50°C and 5.0, respectively. Incubation at 50°C for 24 h did not destroy the cellulose degradation activity. Amino acid sequence analysis revealed the N-terminal sequence of an internal peptide of C4endoII to be Phe-Ala-Gly-Ile-Asn-Ile-Ala-Gly-Phe-Asp-Phe, which is homologous to EGII from Trichoderma reesei. A C4endoII cDNA (C4endoII) was cloned from a cDNA library constructed using the mRNA of the strain cultivated in a cellulase-induction medium. The deduced protein sequence of C4endoII was 417 amino acids long and had a putative signal sequence of 21 amino acids with a predicted cleavage site after Ala-21. A single potential N-glycosylation site was present in the amino acid sequence.     

Binding of all-trans-retinoic acid to MLTC-1 proteins

The covalent incorporation of [³H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells. The maximum retinoylation activity of MLTC-1 cell proteins was 710 ± 29 mean ± SD) fmoles/8 × 10⁴ cells at 37 °C. About 90% of [³H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65–75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC₅₀ values of 0.9 μM and 0.65 μM, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 μM); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 μM lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [³H]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [³H]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells. (Mol Cell Biochem 276: 55–60, 2005)This paper is dedicated to the memory of Prof. E. Quagliariello.     

Growth hormone stimulates hepatic lipid mobilization in rainbow trout,Oncorhynchus mykiss

Rainbow trout were used to characterize the direct influence of growth hormone on hepatic lipid mobilization in vitro. Liver was removed from fish fasted 72 h, sliced into 1-mm³ pieces and incubated in Hanks' medium containing ovine or salmon growth hormone (0.28 ng·ml^-1–28 g·ml^-1). Lipid mobilization, resulting from triacylglycerol hydrolysis, was evaluated by fatty acid and glycerol release into culture medium. Control rates of fatty acid release and glycerol release were 0.95±0.16 (mean ± SE) and 0.88±0.28 mol·l^-1·mg fresh weight, respectively. Both ovine growth hormone (28 ng·ml^-1) and salmon growth hormone (28 ng·ml^-1) stimulated fatty acid release into culture medium, increasing rates by 112% and 97%, respectively, during the course of a 24-h incubation. Glycerol release was similarly augmented by growth hormone treatment. Growth hormone-stimulated metabolite release became evident within 12 h and persisted for up to 72 h. The presence of amino acids in the culture medium potentiated the lipolytic action of growth hormone. Ovine growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 108% increase in glycerol, release over rates observed in the absence of amino acids. Salmon growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 44% increase in glycerol, release over rates observed in the absence of amino acids. Ovine growth hormone (28 ng·ml^-1) also stimulated gluconeogenesis, as indicated by a 75% increase in phosphoenolpyruvate carboxykinase activity in liver pieces incubated in the presence of amino acids. These results indicate that growth hormone directly stimulates lipid breakdown in the liver of trout and that amino acids potentiate growth hormone-stimulated lipolysis.Abbreviations AA amino acid(s) - dGDP deoxy-guanosine diphosphate - ED ₅₀ 50% effective dose - FA fatty acid(s) - fw fesh weight - GH growth hormone - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MS-222 tricaine methanesulfonate - MEM minimum essential medium - oGH ovine growth hormone - PEPCK phosphoenolpyruvate carboxykinase - PK_c protein kinase C - rpm revolutions per minute - sGH salmon growth hormone - TG triacylglycerol - w/v weight per volume This paper was presented in abstract form at the Annual Meeting of the American Society of Zoologists, Dec. 26–30, 1991, Atlanta, GA     

Chlorella chloroplast DNA sequence containing a gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a part of a possible gene for the β′ subunit of RNA polymerase

The sequence of a 2782 bp fragment of the chloroplast genome of Chlorella ellipsoidea has been determined. The region includes the entire gene (rbcL) for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase and a sequence (rpoC-like) similar to part of the gene for the subunit of E. coli RNA polymerase which is oriented in same direction as rbcL. The arrangement is rpoC-like — 446 bp — rbcL. The rbcL gene codes for a polypeptide of 475 amino acids whose sequence shows 88% homology with those of tobacco and spinach, 94% homology with that of Chlamydomonas, and 85% homology with that of Anacystis. The putative rbcL promoter sequence has homology with E. coli promoter sequences and its putative terminator sequence is capable of forming a stem-and-loop structure.     

Purification and biochemical characterization of a fibrinolytic enzyme from Bacillus subtilis BK-17

A fibrinolytic enzyme from Bacillus subtilis BK-17 has been purified to homogeneity by gel-filtration and ion-exchange chromatography. Compared to the crude enzyme extract, the specific activity of the enzyme increased 929-fold with a recovery of 29%. The subunit molecular mass of the purified enzyme was estimated to be 31 kDa by SDS–PAGE. The N-terminal amino acid sequence of the purified fibrinolytic enzyme was: A-Q-S-V-P-Y-G-V-S-Q-I-K-A-P-A-A-H-N. The sequence was highly homologous to the fibrinolytic enzymes nattokinase, subtilisin J and subtilisin E from Bacillus spp. However, there was a substitution of three amino acid residues in the N-terminal sequence. The amidolytic activity of the purified enzyme for several substrates was assessed. In comparison with nattokinase and CK (fibrinolytic enzyme from a Bacillus spp.), which showed strong fibrinolytic activity, the amidolytic activity of the enzyme for the synthetic substrate, kallikrein (H-D-Val-Leu-Arg-pNA, S-2266) increased 2.4- and 11.8-fold, respectively.