Biomedical subjects

P F Nielsen

Publications and source records attributed to P F Nielsen.

At least 19 recordsLinked to original sources

PubMed2003

The systems biology markup language (SBML): a medium for representation and exchange of biochemical network models.

MOTIVATION: Molecular biotechnology now makes it possible to build elaborate systems models, but the systems biology community needs information standards if models are to be shared, evaluated and developed cooperatively. RESULTS: We summarize the Systems Biology Markup Language (SBML) Level 1, a free, open, XML-based format for representing biochemical reaction networks. SBML is a software-independent language for describing models common to research in many areas of computational biology, including cell signaling pathways, metabolic pathways, gene regulation, and others. AVAILABILITY: The specification of SBML Level 1 is freely available from http://www.sbml.org/

Database Management Systems↗

PubMed2003

Isolation of peptides of the brevinin-1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii.

The emergence of strains of the human pathogen Candida albicans with resistance to commonly used antibiotics has necessitated a search for new types of antifungal agents. Six peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions from the foothill yellow-legged frog Rana boylii. Brevinin-1BYa (FLPILASLAA10KFGPKLF CLV20TKKC) was particularly potent against C. albicans [minimal inhibitory concentration (MIC) = 3 microm] and also active against Escherichia coli (MIC = 17 microm) and Staphylococcus aureus (MIC = 2 microm), but its therapeutic potential for systemic use is limited by its strong hemolytic activity (HC50 = 4 microm). The single amino acid substitution (Phe12 --> Leu) in brevinin-1BYb resulted in a fourfold lower potency against C. albicans and the additional amino acid substitutions (Lys11 --> Thr, Phe17 --> Leu and Val20 --> Ile) in brevinin-1BYc resulted in a ninefold decrease in activity. Two members of the ranatuerin-2 family and one member of the temporin family were also isolated from the secretions but showed relatively low potency against the three microorganisms tested.

Amino Acid Sequence↗

PubMed2001

Location of crosslinks in chemically stabilized horseradish peroxidase: implications for design of crosslinks.

The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 A of the EGNHS crosslinker.

Chromatography, High Pressure Liquid↗

PubMed2001

Human pS2/trefoil factor 1: production and characterization in Pichia pastoris.

The recombinant protein human trefoil factor 1 (hTFF1), formerly called hpS2, has been produced for the first time in a yeast-based expression in Pichia pastoris. hTFF1 was secreted in large amounts in the extracellular medium of P. pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The fermentation broth containing hTFF1 was concentrated by tangential flow filtration prior to purification by anion- and cation-exchange chromatography, followed by preparative high-performance liquid chromatography. The resulting hTFF1 was found to be intact by Western blot analysis. Further analysis revealed mainly the presence of the monomeric form of the hTFF1 peptide. Finally, in vitro, the recombinant hTFF1 was shown to decrease proliferation of the HCT116 cancer cells.

Amino Acid Sequence↗

PubMed2000

Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26.

A subset of prolyl oligopeptidases, including dipeptidyl-peptidase IV (DPP IV or CD26, EC ), specifically cleave off N-terminal dipeptides from substrates having proline or alanine in amino acid position 2. This enzyme activity has been implicated in the regulation of the biological activity of multiple hormones and chemokines, including the insulinotropic peptides glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Targeted inactivation of the CD26 gene yielded healthy mice that have normal blood glucose levels in the fasted state, but reduced glycemic excursion after a glucose challenge. Levels of glucose-stimulated circulating insulin and the intact insulinotropic form of GLP-1 are increased in CD26(-/-) mice. A pharmacological inhibitor of DPP IV enzymatic activity improved glucose tolerance in wild-type, but not in CD26(-/-), mice. This inhibitor also improved glucose tolerance in GLP-1 receptor(-/-) mice, indicating that CD26 contributes to blood glucose regulation by controlling the activity of GLP-1 as well as additional substrates. These data reveal a critical role for CD26 in physiological glucose homeostasis, and establish it as a potential target for therapy in type II diabetes.

Animals↗

PubMed2000

Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration.

A series of very potent derivatives of the 30-amino acid peptide hormone glucagon-like peptide-1 (GLP-1) is described. The compounds were all derivatized with fatty acids in order to protract their action by facilitating binding to serum albumin. GLP-1 had a potency (EC(50)) of 55 pM for the cloned human GLP-1 receptor. Many of the compounds had similar or even higher potencies, despite quite large substituents. All compounds derivatized with fatty acids equal to or longer than 12 carbon atoms were very protracted compared to GLP-1 and thus seem suitable for once daily administration to type 2 diabetic patients. A structure-activity relationship was obtained. GLP-1 could be derivatized with linear fatty acids up to the length of 16 carbon atoms, sometimes longer, almost anywhere in the C-terminal part without considerable loss of potency. Derivatization with two fatty acid substituents led to a considerable loss of potency. A structure-activity relationship on derivatization of specific amino acids generally was obtained. It was found that the longer the fatty acid, the more potency was lost. Simultaneous modification of the N-terminus (in order to obtain better metabolic stability) interfered with fatty acid derivatization and led to loss of potency.

Acylation↗

PubMed1999

Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat.

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered hypothalamic peptide regulated by leptin and with a potent appetite-suppressing activity. In the rat, the CART gene encodes a peptide of 116 amino acid residues (or a splice variant 13 residues longer). The predicted signal sequence is 27 amino acid residues, resulting in a prohormone of 89 residues. The CART prohormone contains several potential posttranslational processing sites in the form of mono- and dibasic sequences. In the present study we have purified CART peptides from extracts of adrenal gland, hypothalamus, nucleus accumbens, and pituitary gland (anterior and neurointermediate lobe) of the rat and determined the peptide structures by using microsequencing and mass spectrometry. In none of the tissues examined the long splice variant was found. From the adrenal gland, the CART(1-89) and CART(10-89) peptides were isolated, in contrast to the hypothalamus and nucleus accumbens, from which the shorter form peptides CART(42-89) and CART(49-89) were purified. From the anterior lobe of the pituitary gland, CART(42-89) was isolated, in contrast to the neurointermediate lobe, which contains only CART(49-89). This tissue-specific processing indicates that CART peptides may have different biological functions in the periphery and in the central nervous system.

Animals↗

PubMed1999

Molecular evolution of peptide tyrosine--tyrosine: primary structure of PYY from the lampreys Geotria australis and Lampetra fluviatilis, bichir, python and desert tortoise.

Peptide tyrosine-tyrosine (PYY) has been isolated from the intestines of two species of reptile, the desert tortoise Gopherus agassizii (Testudines) and the Burmese python Python molurus (Squamata), from the primitive Actinopterygian fish, the bichir Polypterus senegalis (Polypteriformes) and from two agnathans, the Southern-hemisphere lamprey Geotria australis (Geotriidae) and the holarctic lamprey Lampetra fluviatilis (Petromyzontidae). The primary structure of bichir PYY is identical to the proposed ancestral sequence of gnathostome PYY (YPPKPENPGE10/DAPPEELAKY20/YSALR HYINL30/ITRQRY). Tortoise and python PYY differ by six and seven residues, respectively, from the ancestral sequence consistent with the traditional view that the Testudines represent an earlier divergence from the primitive reptilian stock than the Squamates. The current views of agnathan phylogeny favor the hypothesis that the Southern-hemisphere lampreys and the holarctic lampreys arose from a common ancestral stock but their divergence is of a relatively ancient (pre-Tertiary) origin. The Geotria PYY-related peptide shows only two amino acid substitutions (Pro10-->Gln and Leu22-->Ser) compared with PYY from the holarctic lamprey Petromyzon marinus. This result was unexpected as Petromyzon PYY differs from Lampetra PYY deduced from the nucleotide sequence of a cDNA (Söderberg et al. J. Neurosci. Res. 1994;37:633-640) by 10 residues. However, a re-examination of an extract of Lampetra intestine revealed the presence of a PYY that differed in primary structure from Petromyzon PYY by only one amino acid residue (Pro10-->Ser). This result suggests that the structure of PYY has been strongly conserved during the evolution of Agnatha and that at least two genes encoding PYY-related peptides are expressed in Lampetra tissues.

Amino Acid Sequence↗

PubMed1999

Purification and characterization of galanin and scyliorhinin I from the hybrid sturgeon, Scaphirhynchus platorynchus x Scaphirhynchus albus (Acipenseriformes).

The sturgeons (order Acipenseriformes) are extant representatives of a group of ancient Actinopterygian (ray-finned) fish. Galanin and scyliorhinin I (a tachykinin with limited structural similarity to mammalian substance P) have been isolated from an extract of the gastrointestinal tract of a sturgeon (an F1 hybrid between the shovelnose sturgeon, Scaphirhynchus platorynchus, and the pallid sturgeon, Scaphirhynchus albus). The primary structure of sturgeon galanin (Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu10-Leu-Gly-Pro-His-Ala-Val -As p-Gly-His-Arg20-Ser-Leu-Ser-Asp-Lys-His-Gly-Leu-Pro.NH2) contains only two amino acid substitutions (Ser23 --> Asn and Pro29 --> Ala) compared with galanin from the bowfin, Amia calva (Amiiformes), but five amino acid substitutions compared with galanin from the trout (Teleostei). Similarly, the sturgeon tachykinin (Ser-Lys-Tyr-His-Gln-Phe-Tyr-Gly-Leu-Met.NH2) contains only one amino acid substitution (Tyr3 --> Ser) compared with scyliorhinin I previously isolated from bowfin stomach but five amino acid substitutions compared with trout substance P. The data support the hypothesis that the Acipenseriformes and the basal Neopterygians (gars and bowfin) share a close phylogenetic relationship.

Amino Acid Sequence↗

PubMed1999

Multiple forms of glucagon and somatostatin isolated from the intestine of the southern-hemisphere lamprey Geotria australis.

Current views on Agnathan phylogeny favor the hypothesis that the genera of holarctic lampreys belong to a single family (Petromyzontidae) and form an interrelated progression in which Petromyzon is near to Ichthomyzon at the base of the phylogenetic tree and Lampetra is the most derived. A stock similar to that of contemporary Ichthomyzon is considered to have given rise to the southern hemisphere lamprey Geotria australis, the sole member of the Geotriidae. In the present study, two molecular forms of glucagon were isolated from an extract of G. australis intestine that differed in structure by six amino acid residues. One form shows two amino acid substitutions (Leu14 --> Met and Ala29 --> Ser) compared with the single molecular form of glucagon isolated from the sea lamprey Petromyzon marinus and the second form shows three substitutions (Asp15 --> Glu, Ser16 --> Ala, Ile24 --> Thr) compared with the single glucagon isolated from the river lamprey Lampetra fluviatilis. As Petromyzon and Lampetra glucagons differ by six amino acid residues, the data suggest that a duplication of the glucagon gene occurred prior to or early in lamprey evolution. Although both genes are strongly expressed in G. australis, the expression of one gene predominates in P. marinus while that of the other gene predominates in L. fluviatilis. Previous work has shown that, in the islet organ of G. australis, preprosomatostatin is processed almost exclusively to somatostatin-33. However, the present study demonstrates that somatostatin-14 is the major molecular form in G. australis intestine with somatostatin-33 present only as a minor component. This result demonstrates a tissue-dependent pathway of posttranslational processing of preprosomatostatin in the Geotria enteropancreatic system.

Amino Acid Sequence↗

PubMed1999

Tachykinins (substance P and neuropeptide gamma) from the brains of the pallid sturgeon, Scaphirhynchus albus and the paddlefish, Polyodon spathula (Acipenseriformes).

A peptide with substance P-like immunoreactivity was isolated from extracts of the brains of the pallid sturgeon, Scaphirhynchus albus and the North American paddlefish, Polyodon spathula. The primary structure of the peptide (Lys-Pro-Lys-Pro-His-Gln-Phe-Phe-Gly-Leu-Met.NH(2)) is the same in both species and contains 2 amino acid substitutions (Arg(1) --> Lys and Gln(5) --> His) compared with human substance P and 1 substitution (Arg(3) --> Lys) compared with substance P from the trout (Teleostei). Scyliorhinin I, a tachykinin previously isolated from an extract of sturgeon intestine, was not detected in either brain extract. A peptide with neurokinin A-like immunoreactivity (Ser-Ser-Ala-Asn-Arg-Gln-Ile-Thr-Gly-Lys(10)Arg-Gln-Lys-Ile-Asn-Ser-P he-Val-Gly-Leu(20)Met.NH(2)) was isolated from sturgeon brain and contains 10 amino acid substitutions compared with human neuropeptide gamma (a specific product of the posttranslational processing of gamma-preprotachykinin A) but only 4 substitutions compared with trout neuropeptide gamma. It was not possible to obtain the paddlefish neurokinin A-related peptide in pure form. The structural similarity between the sturgeon and the trout tachykinins supports the hypothesis that the Acipenseriformes (sturgeons and paddlefish) represent the sister group of the Neopterygii (gars, bowfin, and teleosts).

Amino Acid Sequence↗

PubMed1999

Neuroendocrine peptides (insulin, pancreatic polypeptide, neuropeptide Y, galanin, somatostatin, substance P, and neuropeptide gamma) from the desert tortoise, Gopherus agassizii.

The traditional view that Testudines (tortoises and turtles) should be regarded as the surviving clade of the anapsid reptiles rather than classified with the diapsid reptiles (snakes, lizards, and crocodiles) has recently been challenged. Neuropeptide Y, neuropeptide gamma, and somatostatin-14 were isolated from an extract of the brain, substance P and galanin from an extract of the intestine, and insulin and pancreatic polypeptide from an extract of the pancreas of the desert tortoise, Gopherus agassizii. Despite that crocodilians did not appear until the late Triassic, the amino acid sequences of the tortoise peptides resemble those of the American alligator quite closely. The primary structures of neuropeptide Y, somatostatin-14, and neuropeptide gamma are the same in tortoise and alligator. The primary structures of substance P, insulin, galanin, and pancreatic polypeptide in the two species differ by 1, 3, 5, and 8 amino acid residues, respectively. Although fewer neurohormonal peptides from squamates (lizards and snakes) have been characterized, the primary structures of neuropeptide gamma, insulin, and pancreatic polypeptide from the Burmese python and the desert tortoise differ by 3, 8, and 18 residues, respectively. The data suggest, therefore, a closer phylogenetic relationship between Testudines and Crocodilians than that derived from 'classical' analyses based on morphological criteria and the fossil record.

Amino Acid Sequence↗

PubMed1998

Purification and characterisation of a new hypothalamic satiety peptide, cocaine and amphetamine regulated transcript (CART), produced in yeast.

Cocaine and amphetamine regulated transcript (CART) is a newly discovered hypothalamic peptide with a potent appetite suppressing activity following intracerebroventricular administration. When the mature rat CART sequence encoding CART(1-102) was inserted in the yeast expression plasmid three CART peptides could be purified from the fermentation broth reflecting processing at dibasic sequences. None of these corresponded to the naturally occurring CART(55-102). In order to obtain CART(55-102) the precursor Glu-Glu-Ile-Asp-CART(55-102) has been produced and CART(55-102) was generated by digestion of the precursor with dipeptidylaminopeptidase-1. All four generated CART peptides have been characterised by N-terminal amino acid sequencing and mass spectrometry. The CART peptides contain six cysteine residues and using the yeast expressed CART(62-102) the disulphide bond configuration was found to be I-III, II-V and IV-VI. When the four CART peptides were intracerebroventricularly injected in fasted mice (0.1 to 2.0 microg) they all produced a dose dependent inhibition of food intake.

Amino Acid Sequence↗

PubMed1998

Glycosylation profile of a recombinant urokinase-type plasminogen activator receptor expressed in Chinese hamster ovary cells.

Association of urokinase-type plasminogen activator (uPA) to cells via binding to its specific cellular receptor (uPAR) augments the potential of these cells to support plasminogen activation, a process that has been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling. The uPA receptor is a glycolipid-anchored membrane protein belonging to the Ly-6/uPAR superfamily and is the only multidomain member identified so far. We have now purified the three individual domains of a recombinant soluble uPAR variant, expressed in Chinese hamster ovary cells, after limited proteolysis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ionization and electrospray ionization mass spectrometry. Of the five potential attachment sites for asparagine-linked carbohydrate in uPAR only four are utilized, as the tryptic peptide derived from domain III containing Asn233 was quantitatively recovered without carbohydrate. The remaining four attachment sites were shown to exhibit site-specific microheterogeneity of the asparagine-linked carbohydrate. The glycosylation on Asn52 (domain I) and Asn172 (domain II) is dominated by the smaller biantennary complex-type oligosaccharides, while Asn162 (domain II) and Asn200 (domain III) predominantly carry tri- and tetraantennary complex-type oligosaccharides. The carbohydrate moiety on Asn52 in uPAR domain I could be selectively removed by N-glycanase treatment under nondenaturing conditions. This susceptibility was abrogated when uPAR participitated in a bimolecular complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proximity of the ligand-binding site to this particular carbohydrate moiety. uPAR preparations devoid of carbohydrate on domain I exhibited altered binding kinetics toward uPA (a 4-6-fold increase in Kd) as assessed by real time biomolecular interaction analysis.

Amino Acid Sequence↗

PubMed1998

Amino acid sequence diversity of pancreatic polypeptide among the amphibia.

It has been suggested that the amino acid sequence of pancreatic polypeptide (PP) may provide a useful molecular marker with which to study evolutionary relationships between tetrapods but few PP sequences from amphibia are available to test this hypothesis. PPs have been purified from the pancreata of five species belonging to the different orders of amphibians. Their amino acid sequences were established as: APSEPEHPGD10 NASPDELAKY20 YSDLWQYITF30 VGRPRY for the lesser siren, Siren intermedia (Caudata); GPTEPIHPGK10 DATPEELTKY20 YSDLYDYITL30 VGRSRW for the caecilian, Typhlonectes natans (Gymnophiona); and TPSEPQHPGD10 QASPEQLAQY20 YSDLWQYITF30 VTRPRF for the cane toad, Bufo marinus (Anura). The structure of Rana sylvatica PP is the same as that of Rana catesbeiana PP whereas PP from the green frog Rana ridibunda contains one substitution (His6 --> Gln). The data provide further support for the conclusion that the amino acid sequence of PP has been poorly conserved during evolution with only 17 residues invariant among the eight species of amphibia yet studied and only 8 residues (Pro5, Pro8, Gly9, Ala12, Leu24, Tyr27, Arg33, and Arg35) invariant among all tetrapods. A maximum parsimony analysis based upon the amino acid sequence of PP and using the sequence of frog PYY as outgroup to polarize the in-group taxa generates a consensus phylogenetic tree in which the Amniota and Amphibia form two distinct clades. However, such a tree does not permit valid conclusions to be drawn regarding branching order within the Amphibia.

Amino Acid Sequence↗

PubMed1998

Purification and characterization of insulin, glucagon, and two glucagon-like peptides with insulin-releasing activity from the pancreas of the toad, Bufo marinus.

Insulin and four peptides derived from the posttranslational processing of proglucagon have been isolated in pure form from the pancreas of the cane toad, Bufo marinus. Although Bufo insulin contains 9 amino acid substitutions, compared with human insulin, all those residues that are considered to be involved in receptor-binding and in dimer and hexamer formation have been conserved. Bufo insulin was, however, more potent (4-fold) than human insulin in inhibiting the binding of [125I-Tyr-A14] insulin to the soluble full-length recombinant human insulin receptor, which is probably a consequence of the substitution (Thr --> His) at position A-8. Bufo glucagon was isolated in two molecular forms: glucagon-29 shows only one amino acid substitution (Thr29 --> Ser), compared with human glucagon; and glucagon-36 comprises glucagon-29, extended from its C-terminus by Lys-Arg-Ser-Gly-Gly-Met-Ser. The human proglucagon gene contains one copy of glucagon-like peptide (GLP)-1, a potent insulin secretogogue, and one copy of GLP-2 that is devoid of insulin-releasing activity. In contrast, two proglucagon-derived peptides with 32- and 37-amino acid residues (GLP-32 and GLP-37), displaying greater structural similarity to human GLP-1 than to GLP-2, were isolated from Bufo pancreas. Both peptides produced concentration-dependent increases in insulin release from glucose-responsive rat insulinoma-derived BRIN-BD11 cells. The threshold concentrations producing a significant (P < 0.001) effect were 10(-8) M (GLP-32) and 10(-9) M (GLP-37), and the maximum increase in the rate of insulin release produced by 10(-6) M concentrations of both peptides was approximately 5-fold.

Amino Acid Sequence↗

PubMed1997

Pancreatic lipase structure-function relationships by domain exchange.

We designed chimeric mutants by exchanging the lid domains of the classical human pancreatic lipase (HPL) and the guinea pig pancreatic lipase related protein 2 (GPLRP2). This latter enzyme possesses naturally a large deletion within the lid domain and is not activated by lipid/water interfaces. Furthermore, GPLRP2 exhibits phospholipase A1 and lipase activities in the same order of magnitude, whereas HPL has no significant phospholipase activity and displays a clear interfacial activation. An HPL mutant [HPL(-lid)] with GPLRP2 mini-lid domain does not display interfacial activation. Its specific activity toward triglycerides is, however, dramatically reduced. A GPLRP2 mutant [GPLRP2(+lid)] with HPL full-length lid domain is not interfacially activated, and its lid domain probably exists under a permanent open conformation. Therefore, the phenomenon of interfacial activation in HPL is not only due to the presence of a full-length lid domain but also to other structural elements which probably allow the existence of stabilized closed and open conformations of the lid. GPLRP2(+lid) phospholipase activity is significantly reduced as compared to GPLRP2, whereas its lipase activity remains at the same level. Therefore, the lid domain plays a major role in substrate selectivity and can be considered as part of the active site. However, the presence of a full-length lid domain is not sufficient to explain the absence of phospholipase activity in HPL since HPL(-lid) does not display any phospholipase activity. We also produced a chimeric GPLRP2 mutant in which the C-terminal domain was substituted by the HPL C-terminal domain. The colipase effects, i.e., anchoring and stabilization of the lipase at the interface, are clearly observed with the chimera, whereas GPLRP2 is insensitive to colipase. The kinetic characterization of this chimera reveals for the first time that the interfacial stability of pancreatic lipases depends on the structure of the C-terminal domain.

Amino Acid Sequence↗

PubMed1997

Primary structure of insulin from the african lungfish, Protopterus annectens.

Among the extant Sarcopterygii, the interrelationship between the Dipnoi (lungfishes), Actinistia (coelacanths), and Tetrapoda (tetrapods) is controversial. Insulin has been purified from an extract of the pancreas of the African lungfish Protopterus annectens and its primary structure established as A-chain, Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Lys-Pro10-Cys-Ser-Leu- Tyr -Glu-Leu-Glu-Asn-Tyr-Cys20-Asn-Val-Pro; and B-chain, Ala-Val-Leu-Asn-Gln-His-Leu-Cys-Gly-Ser10-His-Leu-Val- Glu- Ala-Leu-Tyr-Leu-Val-Cys20-Ala-Asp-Asn-Gly-Phe- Phe-Tyr-Lys-Pro-Ser30-Gly. Lungfish insulin contains unusual structural features, such as the dipeptide extension to the C-terminus of the A-chain and the substitution Arg --> Asn at position B-23 in the putative receptor binding region of insulin, which may be expected to influence appreciably its biological potency relative to mammalian insulins. Lungfish insulin also contains amino acid substitutions such as Gly --> Ala at position B-21, Glu --> Asp at position B-22, and a Lys --> Ser residue at position B-30, previously found in insulins from amphibia. This observation is consistent with paleontological data suggesting that lungfish and amphibia share a close phylogenetic relationship.

Amino Acid Sequence↗