Endocytosis of EGF-receptor complexes at various cell cycle stages

Parameters of EGF-receptor complex endocytosis have been studied in the early and late G1 phase and in mitosis. As a model, mouse mammary epithelial cells HC11 were used, whose growth depends on EGF presence in the medium. The Scatchard analysis has demonstrated that the surface receptors are represented by two receptor populations: 4800 high affinity (KD = 10(-11) M) receptors, and 73,000 low affinity (KD = 4.10(-9) M) receptors. Incubation of cells with the growth factor (5 ng/ml) resulted in a decrease in 125I-EGF binding, with its level being low until entering the S-phase. Under these conditions, receptors disposed on the plasma membrane presented a homogeneous population (KD = 8.10(-11) M, 14,000 receptors per cell). No reliable difference was revealed between the EGF-receptor complexes, internalized in early and late G1 phases, in respect to the internalization rate, level of recycling, degradation, and dynamics of compartmentalization. However, endocytosis of EGF-receptor complexes was found to be completely blocked in mitosis at the stage of internalization.     

Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. IV. Photoinduced electron transfer

The photomodification of single-stranded DNA sensitized to visible light (450-580 nm) by a binary system of oligonucleotide conjugates complementary to adjacent DNA sequences was studied. One oligonucleotide carries a residue of the photoreagent p-azidotetrafluorobenzaldehyde hydrazone at its 3'-terminal phosphate, and the other has a residue of the sensitizer, perylene or 1,2-benzanthracene, at the 5'-terminal phosphate. The rate of photomodification sensitized by the perylene derivative is 300,000-fold higher than the rate of photomodification in the absence of the sensitizer. Since the excitation energy of perylene is lower than the energy necessary for the initiation of azide photodecomposition, it is likely that the sensitization in the complementary complex occurs by electron transfer from the azido group of the photoreagent to the excited sensitizer. The sensitization by the 1,2-benzanthracene oligonucleotide derivative occurs by means of singlet-singlet energy transfer, which enables this sensitizer to act as a unconsumable catalyst each molecule of which is able to initiate the photomodification of more than 20 DNA molecules. By both mechanisms, the photomodification occurs with high specificity on the G11 residue of the target DNA. The degree of sensitized photomodification reaches 72%.     

Point amino acid substitutions in the Ca2+-binding centers of recoverin. I. Mechanism of successive filling of Ca2+-binding centers

The molecule of photoreceptor Ca(2+)-binding protein recoverin contains four potential Ca(2+)-binding sites of the EF-hand type, but only two of them (the second and the third) can actually bind calcium ions. We studied the interaction of Ca2+ with recoverin and its mutant forms containing point amino acid substitutions at the working Ca(2+)-binding sites by measuring the intrinsic protein fluorescence and found that the substitution of Gln for Glu residues chelating Ca2+ in one (the second or the third) or simultaneously in both (the second and the third) Ca(2+)-binding sites changes the affinity of the protein to Ca2+ ions in different ways. The Gln for Glu121 substitution in the third site and the simultaneous Gln substitutions in the second (for Glu85) and in the third (for Glu121) sites result in the complete loss of the capability of recoverin for a strong binding of Ca(2+)-ions. On the other hand, the Gln for Glu85 substitution only in the second site moderately affects its affinity to the cation. Hence, we assumed that recoverin successively binds Ca(2+)-ions: the second site is filled with the cation only after the third site has been filled. The binding constants for the third and the second Ca(2+)-binding sites of recoverin determined by spectrofluorimetric titration are 3.7 x 10(6) and 3.1 x 10(5) M-1, respectively.