scholarly journals Structure and receptor-binding activity of insulin from a holostean fish, the bowfin (Amia calva)

1991 ◽  
Vol 276 (1) ◽  
pp. 261-264 ◽  
Author(s):  
J M Conlon ◽  
J H Youson ◽  
J Whittaker
Keyword(s):  
Biological Activity ◽  
Human Insulin ◽  
Binding Activity ◽  
Vertebrate Species ◽  
3T3 Cells ◽  
Receptor Binding Activity ◽  
Human Insulin Receptor ◽  
A Chain ◽  
Amia Calva ◽  
Nih 3T3

The holostean fishes are the extant representatives of the primitive ray-finned fishes from which the present-day teleosts may have evolved. The primary structure of insulin from a holostean fish, the bowfin (Amia calva), was established as: A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Leu-Lys-Pro-Cys-Thr-Ile-Tyr-Glu-Met-Glu- Lys-Tyr-Cys-Asn B-chain: Ala-Ala-Ser-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Phe-Leu- Val-Cys-Gly-Glu-Ser-Gly-Phe-Phe-Tyr-Asn-Pro-Asn-Lys-Ser This amino acid sequence contains several substitutions (methionine at A16, phenylalanine at B16 and serine at B22) at sites that have been strongly conserved in other vertebrate species and that may be expected to influence biological activity. Consistent with this prediction, bowfin insulin was approx. 14-fold less potent than pig insulin in inhibiting the binding of [125I-Tyr-A14](human insulin) to transfected mouse NIH 3T3 cells expressing the human insulin receptor.

scholarly journals The (ProB27, ThrB28) human insulin analogue is more potent and more rapidly absorbed from subcutis than human insulin

2002 ◽  
pp. 227-233 ◽  
Author(s):  
R Clausen ◽  
TG Jorgensen ◽  
KH Jorgensen ◽  
AH Johnsen ◽  
JJ Led ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Insulin Receptor ◽  
Human Insulin ◽  
Binding Activity ◽  
Glucose Lowering ◽  
Recombinant Receptors ◽  
Receptor Binding Activity ◽  
Human Insulin Receptor ◽  
Igf Receptors

OBJECTIVE: To test the physiological properties of human insulin in which the amino acids Thr (B27) and Pro (B28) are interchanged (PT insulin). This was hypothesised to prevent dimerisation and accelerate the absorption from s.c. tissue without altering the affinity for the insulin receptor. DESIGN: PT insulin was expressed in Pichia pastoris and processed in vitro. The purified compound was used for physiological investigations. METHODS: Receptor binding activity to insulin and IGF receptors was evaluated in a competition assay using iodinated PT insulin and recombinant receptors while growth induction properties were evaluated by thymidine incorporation. Absorption kinetics from pig subcutis was investigated by measuring the disappearance of iodinated PT insulin. The potency was evaluated by measuring the blood glucose lowering activity in mice. RESULTS: The absorption of PT insulin was accelerated compared with human insulin, although still slower than Asp (B28) insulin. Human and PT insulin had similar affinities for the human insulin receptor (K(d)=3.6 x 10(-12) vs 5.2 x 10(-12) mol/l) while the affinity for the IGF receptor was four times higher for PT insulin than for human insulin (K(d)=3.4 x 10(-8) vs 1.3 x 10(-7) mol/l). This resulted in a slightly higher DNA synthesis when assayed in intermediary insulin concentrations. The blood glucose lowering effect in mice exceeded the effect of human insulin (integral 0-60 min: 61.4+/-7 vs 30+/-4, n=6, P=0.046). CONCLUSIONS: PT insulin is absorbed faster and is more potent than human insulin. Although PT insulin stimulates growth more than human insulin, this will not prevent its use in the clinic, but the main interest will probably focus on investigations to clarify the paradox of full biological activity in connection with the recently described lack of structure in the B-chain.

scholarly journals High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells.

1987 ◽  
Vol 84 (15) ◽  
pp. 5237-5241 ◽  
Author(s):  
J. Whittaker ◽  
A. K. Okamoto ◽  
R. Thys ◽  
G. I. Bell ◽  
D. F. Steiner ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Human Insulin ◽  
3T3 Cells ◽  
High Level Expression ◽  
Human Insulin Receptor ◽  
Receptor Cdna ◽  
High Level ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals N Terminus of Sos1 Ras Exchange Factor: Critical Roles for the Dbl and Pleckstrin Homology Domains

1998 ◽  
Vol 18 (2) ◽  
pp. 771-778 ◽  
Author(s):  
Xiaolan Qian ◽  
William C. Vass ◽  
Alex G. Papageorge ◽  
Pieter H. Anborgh ◽  
Douglas R. Lowy
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Map Kinase ◽  
Stable Complex ◽  
Egf Receptors ◽  
Pleckstrin Homology ◽  
3T3 Cells ◽  
N Terminus ◽  
Ph Domains ◽  
Nih 3T3

ABSTRACT We have studied the functional importance of the N terminus of mouse Sos1 (mSos1), a ubiquitously expressed Ras-specific guanine nucleotide exchange factor whose C-terminal sequences bind Grb-2. Consistent with previous reports, addition of a myristoylation signal to mSos1 (MyrSos1) rendered it transforming for NIH 3T3 cells and deletion of the mSos C terminus (MyrSos1-ΔC) did not interfere with this activity. However, an N-terminally deleted myristoylated mSos1 protein (MyrSos1-ΔN) was transformation defective, although the protein was stable and localized to the membrane. Site-directed mutagenesis was used to examine the role of the Dbl and pleckstrin homology (PH) domains located in the N terminus. When mutations in the PH domain were introduced into two conserved amino acids either singly or together in MyrSos1 or MyrSos1-ΔC, the transforming activity was severely impaired. An analogous reduction in biological activity was seen when a cluster of point mutations was engineered into the Dbl domain. The mitogen-activation protein (MAP) kinase activities induced by the various Dbl and PH mutants of MyrSos1 correlated with their biological activities. When NIH 3T3 cells were transfected with a myristoylated Sos N terminus, their growth response to epidermal growth factor (EGF), platelet-derived growth factor, lysophosphatidic acid or serum was greatly impaired. The dominant inhibitory biological activity of the N terminus correlated with its ability to impair EGF-dependent activation of GTP-Ras and of MAP kinase, as well with the ability of endogenous Sos to form a stable complex with activated EGF receptors. The N terminus with mutations in the Dbl and PH domains was much less inhibitory in these biological and biochemical assays. In contrast to wild-type Sos1, nonmyristoylated versions of Sos1-ΔN and Sos1-ΔC did not form a stable complex with activated EGF receptors. We conclude that the Dbl and PH domains are critical for Sos function and that stable association of Sos with activated EGF receptors requires both the Sos N and C termini.

Expression of a human insulin-like growth factor II cDNA in NIH-3T3 cells

1990 ◽  
Vol 169 (3) ◽  
pp. 832-839 ◽  
Author(s):  
Daniel M. Buergisser ◽  
Birgit V. Roth ◽  
Christine Luethi ◽  
Hans Peter Gerber ◽  
Annemarie Honegger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Human Insulin ◽  
Insulin Like Growth Factor ◽  
3T3 Cells ◽  
Factor Ii ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities.

1991 ◽  
Vol 11 (2) ◽  
pp. 604-610 ◽  
Author(s):  
N Yew ◽  
M Oskarsson ◽  
I Daar ◽  
D G Blair ◽  
G F Vande Woude
Keyword(s):  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Kinase Activity ◽  
Vertebrate Species ◽  
3T3 Cells ◽  
Transforming Activity ◽  
Cytostatic Factor ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v-mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes.

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