1. Growth factor-dependent proliferative stimulation of hematopoietic cells is associated with the modulation of cytoplasmic and nuclear 68-Kd calmodulin-binding protein
G P Reddy, W C Reed, D H Deacon, P J Quesenberry Blood. 1992 Apr 15;79(8):1946-55.
Calcium and calmodulin (CaM) are known to play critical roles in controlling cell cycle progression in a variety of cells. We observed that the CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), inhibited 3H-thymidine incorporation into DNA of factor-dependent hematopoietic cells. To delineate the role of CaM in proliferation of hematopoietic cells, we have investigated intracellular distribution of specific CaM-binding proteins (CaM-BPs) in response to hematopoietic growth factors in FDC-P1, 32D, NFS-60, and T1165 cells. Each of these cell lines, when deprived of cytokines for 16 to 18 hours, essentially ceased proliferation, even in the presence of fetal calf serum. Concomitant to the cessation of proliferation, there was a dramatic depletion of a specific CaM-BP of about 68 Kd in both their cytoplasmic and nuclear fractions. Within 6 to 12 hours of reexposure to proliferation-specific cytokines, there was a restoration of the nuclear as well as cytoplasmic 68-Kd CaM-BP. Furthermore, such an induction and nuclear localization of the 68-Kd CaM-BP by the cytokines coincided temporally with the progression of synchronized FDC-P1 cells from G1 to S phase. By contrast, colony-stimulating factor-1 (CSF-1)-dependent bone marrow macrophages and BAC-1 cells did not exhibit 68-Kd CaM-BP in the nuclear or cytoplasmic fractions. These studies suggest that while hematopoietic growth factor granulocyte CSF-, granulocyte-macrophage CSF-, interleukin-3 (IL-3)-, or IL-6-, whose receptors are members of the hematopoietin receptor family, induced cell proliferation is associated with a common mechanism involving nuclear localization of the 68-Kd CaM-BP, the CSF-1-induced proliferation seems to involve 68-Kd CaM-BP-independent pathways.
2. Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives
A Nureddin, T Inagami Biochem J. 1975 Apr;147(1):71-81. doi: 10.1042/bj1470071.
1. Isoionic chemical modification of amino groups of trypsin (EC 18.104.22.168) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.
3. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: investigation of aliphatic residues
L Herrmann, B E Bowler, A Dong, W S Caughey Biochemistry. 1995 Mar 7;34(9):3040-7. doi: 10.1021/bi00009a035.
A series of hydrophilic to hydrophobic surface mutations were prepared at the highly solvent-exposed lysine 73 of iso-1-cytochrome c to assess the ability of such mutants to affect the energetics of the denatured state. In this report, the aliphatic hydrophobics (leucine, isoleucine, valine, alanine, glycine) were studied. The thermodynamic stability of each of these mutants was determined by guanidine hydrochloride denaturation. Both the free energy of unfolding in the absence of denaturant, delta GouH2O, and the slope, m, of a plot of the free energy of unfolding, delta Gou, versus [guanidine hydrochloride] show significant negative correlations with the 1-octanol to water transfer free energy, delta Gtr, of the amino acid side chain at position 73. A negative correlation with hydrophobicity is consistent with these mutants leading to more extensive hydrophobic clustering in the denatured state, consistent with the predictions of heteropolymer theory for compact denatured states; an effect operating on the native state energetics should produce a positive correlation of delta GouH2O with hydrophobicity. Infrared amide I spectroscopy indicated native state structural perturbations for the glycine 73 and isoleucine 73 mutants. A moderate correlation of delta GouH2O was also found with alpha-helix propensity, suggesting that both hydrophobic effects acting on the denatured state and alpha-helix propensity are affecting the delta GouH2O values for these mutants.