Hybrid molecules containing the A-domain of insulin-like growth factor-I and the B-chain of insulin have increased mitogenic activity relative to insulin

Two synthetic insulin-like compounds consisting of the B-chain of insulin linked via disulfide bonds to A chains corresponding to the A-domain or the A- and D-domains of insulin-like growth factor I (IGF-I) have been evaluated for mitogenic activity and for binding to IGF receptors and IGF carrier proteins. Both compounds are 3- to 5-fold more potent mitogens than insulin, and have a comparably increased affinity for the type I IGF receptor that mediates these mitogenic effects in chick embryo fibroblasts. Neither compound interacts with IGF carrier proteins. These results indicate that the A-domain of IGF-I is importantly involved in its growth-promoting properties.     

Renal brush border glutamine transport: comparison between in situ and isolate membrane vesicle uptake

Glutamine uptake by renal cortical brush-border vesicles was compared to transport expressed by the functioning isolated kidney. Comparisons were made with regard to sodium dependency and the adaptive increase induced by chronic metabolic acidosis in the rat. The results show an absolute dependency upon a sodium gradient; sodium-independent glutamine uptake has no counterpart in situ. In addition, acidosis-induced adaptive increase in vesicle glutamine uptake has no counterpart in situ. Rather, the apparent adaptation reflects extravesicular gamma-glutamyltransferase-mediated conversion to glutamate and subsequent accumulation; acidosis-induced adaptation of this enzyme largely explains the apparent adaptation in glutamine uptake. Consequently the role of membrane transport in glutamine flux regulation can be assessed providing metabolic conversion is controlled.     

Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.

Enhanced resolution of rapid and complex anisotropy decays was obtained by measurement and analysis of data from progressively quenched samples. Collisional quenching by acrylamide was used to vary the mean decay time of indole or of the tryptophan fluorescence from melittin. Anisotropy decays were obtained from the frequency-response of the polarized emission at frequencies from 4 to 2,000 MHz. Quenching increases the fraction of the total emission, which occurs on the subnanosecond timescale, and thereby provides increased information on picosecond rotational motions or local motions in proteins. For monoexponential subnanosecond anisotropy decays, enhanced resolution is obtained by measurement of the most highly quenched samples. For complex anisotropy decays, such as those due to both local motions and overall protein rotational diffusion, superior resolution is obtained by simultaneous analysis of data from quenched and unquenched samples. We demonstrate that measurement of quenched samples greatly reduces the uncertainty of the 50-ps correlation time of indole in water at 20 degrees C, and allows resolution of the anisotropic rotation of indole with correlation times of 140 and 720 ps. The method was applied to melittin in the monomeric and tetrameric forms. With increased quenching, the anisotropy data showed decreasing contributions from overall protein rotation and increased contribution from picosecond tryptophan motions. The tryptophan residues in both the monomeric and the tetrameric forms of melittin displayed substantial local motions with correlation times near 0.16 and 0.06 ns, respectively. The amplitude of the local motion is twofold less in the tetramer. These highly resolved anisotropy decays should be valuable for comparison with molecular dynamics simulations of melittin.     

Binding of Eu3+ to cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase-laser excited Eu3+ spectroscopic studies.

The binding of Eu3+ with Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ([Ca2+ + Mg2+]-ATPase) of cardiac sarcoplasmic reticulum (SR) has been investigated using direct laser excited Eu3+ luminescence. Eu3+ is found to inhibit both Ca2+-dependent ATPase activity and Ca2+-uptake in a parallel manner. This is attributed to the binding of Eu3+ to the high affinity Ca2+-binding sites. The Ki for Ca2+-dependent ATPase is approximately 50 nM. The 7F0----5D0 excitation spectrum of Eu3+ in cardiac SR shows a peak at 579.3 nm, as compared to 578.8 nm in potassium-morpholino propane sulfonic acid (K-MOPS) pH 6.8. Upon binding with cardiac SR, Eu3+ shows an increase in fluorescence intensity as well as in lifetime values. The fluorescence decay of bound Eu3+ exhibits a double-exponential curve. The apparent number of water molecules in the first coordination sphere of Eu3+ in SR is 2.8 for the short component and 1.0 for the long component. In the presence of ATP, a further increase in fluorescence lifetimes is observed, and the number of water molecules in the first coordination sphere of Eu3+ is reduced further to 1.3 and 0.5. The double exponential nature of the decay curve and the different number of water molecules coordinated to Eu3+ for both decay components suggest that Eu3+ binds to two sites and that these are heterogeneous. The reduction in the number of H2O ligands in the presence of ATP shows a change in the molecular environment of the Eu3+-binding sites upon phosphoenzyme formation, with a movement of Eu3+ to an occluded site on the enzyme.     

Structures of shorthorn sculpin antifreeze polypeptides

The amino acid sequences of the two major antifreeze polypeptides (AFP) from the shorthorn sculpin have been determined using an automatic protein sequencer and enzymic digestion. These two polypeptides, SS-3 and SS-8, consist of 33 and 45 amino acid residues respectively. The N-terminal methionyl residue is blocked in both the polypeptides. When aligned for maximum structural similarity these two AFP are 80% homologous, and there appears a deletion of 12 amino acid residues at the N-terminal portion of SS-3. Like the winter flounder AFP, both the sculpin AFP also contain the 11-amino-acid repeat sequences. The secondary structure of the sculpin AFP is mainly alpha-helical as deduced from circular dichroic spectral data. The helical content of SS-8 is high (73%), while that of SS-3 is moderate (about 45%). The latter exhibits a relatively weak antifreeze activity. Removal of the blocked N-terminal residue in SS-8 did not alter the helical content significantly but did reduce the antifreeze activity. Helical contents of proteolytically generated fragments of AFP are much lower, and they are devoid of activity. The alpha-helix in the SS-8 component is seen to be amphiphilic in character. The relevance of this feature to the mechanism of the antifreeze action is briefly discussed.     

Biotransformation of 3-methoxy-8,14-seco-1,3,5(10),9(11) estratetraen-14,17-dione (secodione) to its 17beta-hydroxy derivative (secol) using immobilized yeast cells (Pichia farinosa Y-118)

The yeast cells of Pichia farinosa Y-118 were immobilized in polyacrylamide gel and used for 17 beta-oxidoreduction of secondione to secol. The loss of hydroxysteroid oxidoreductase activity of cells was found to be insignificant during immobilization. The preparation exhibited greater temperature stability as compared to free cells. The ratio of reaction volume to the volume of immobilized biocatalyst in the range 1.4-1.9 was found to be satisfactory for the reaction conditions studied. This ratio played a significant role in the stability of the catalyst particle, since beyond a critical value the disintegration of gel granules was rapid resulting in sharp decline of activity. The immobilized cell preparation could be used 50 times over a period of 100 days without loss of activity. However, the activity declined in further reuses, leaving the preparation 50 and 35% active after its 60th and 70th uses, respectively.     

Tension and compression in the cytoskeleton of PC 12 neurites

We report in this article that the retraction of PC 12 neurites, unlike that of other cultured neurons, is due to tension within the neurite. Retraction is rapid and independent of metabolic energy. Transection of one arm of a branched neurite immediately causes the remaining arm to take up a new equilibrium position between attachment points. Similarly, detachment of one growth cone of a cell causes the cell body to move to a new equilibrium position between the remaining neurites. These observations provide direct evidence for the suspension of the cell soma among a network of tensioned neurites. We used retraction as an assay for neurite tension to examine the role of actin filaments and microtubules in neurite support and elongation. Our data suggest that microtubules (MTs) within PC 12 neurites are under compression, supporting tension within the actin network. Treatment of cells with drugs that disrupt actin networks, cytochalasin D or erythro-9-[3-(2-hydroxynonyl)]adenosine eliminates retraction regardless of the absence of MTs, lack of adhesion to the substratum, or integrity of the neurite. Conversely, stimulation of actin polymerization by injection of phalloidin causes retraction of neurites. Treatments that depolymerize MTs, nocodazole or cold, cause retraction of neurites, which suggests that microtubules support this tension, i.e., are under compression. Stabilization of MTs with taxol stabilizes neurites to retraction and under appropriate circumstances can drive neurite extension. Taxol-stimulated neurite extension is augmented by combined treatment with anti-actin drugs. This is consistent with the actin network's normally exerting a force opposite that of MT assembly. Cytochalasin and erythro-9-[3-(2-hydroxynonyl)] adenosine were found to increase slightly the dose of nocodazole required for MT depolymerization. This is consistent with the postulated balance of forces and also suggests that alteration of the compression borne by the microtubules could serve as a local regulator for MT polymerization during neurite outgrowth.     

Structural features involved in the biological activity of insulin and the insulin-like growth factors: A27 insulin/BIGF-I

A synthetic insulin-like compound consisting of the A-chain of insulin extended at its carboxyl terminus with the hexapeptide "D-domain" of insulin-like Growth Factor II, linked via disulfide bonds to a B-chain corresponding to the "B-domain" of insulin-like Growth Factor I, has been examined for insulin-like metabolic activity and for mitogenic activity. The synthetic material (A27 insulin/BIGF-I) is less potent than insulin in metabolic assays, and less potent than both insulin and IGF-I in mitogenic assays. It is proposed that neither the "D-domain" nor the "B-domain" of the IGFs is a major contributor to mitogenic activity. Their presence in the same molecule does not result in significant growth-promoting activity.     

Comparison of three mammalian cell-lines with respect to their sensitivities to hyperthermia, gamma-rays and U.V.-radiation.

Three types of cultured mammalian cells, from a rat ureter carcinoma (RUC-2), from a mouse mammary carcinoma (M8013), and from a Chinese hamster lung cell-line (CH-V79), have been compared with respect to various treatments. Using cloning assay, the sensitivity of these cell-lines was measured for ionizing radiation, U.V.-light and for temperatures of 41 degrees, 42 degrees, 43 degrees, 44 degrees and 45 degrees C. RUC-2 cells are the least sensitive to these treatments, except for 45 degrees C. M8013 is sensitive to gamma-rays and U.V.-irradiation. CH-V79 is sensitive to gamma-radiation and hyperthermia. Since sensitivity to one type of treatment does not correlate with similar susceptibility to the others, it is concluded that different mechanisms may be involved in inactivation by these treatments.