Phosphorylation of ankyrin decreases its affinity for spectrin tetramer

The effects of phosphorylation on the interaction between spectrin and ankyrin were investigated. Spectrin and ankyrin were phosphorylated using purified human erythrocyte membrane and cytosolic (casein kinase A) kinases. These two kinases have similar properties as well as activities toward spectrin and ankyrin. Both kinases catalyzed the incorporation of about 2 mol of phosphate/mol of spectrin and about 7 mol of phosphate/mol of ankyrin. These phosphates were incorporated primarily into seryl and threonyl residues of the proteins. The phosphopeptide maps of ankyrin phosphorylated by the membrane kinase and casein kinase A were identical. Binding studies indicate that ankyrin exhibits different affinities for spectrin dimers (KD = 2.5 +/- 0.9 X 10(-6) M) and tetramers (KD = 2.7 +/- 0.8 X 10(-7) M). These dissociation constants were not appreciably affected by the phosphorylation of spectrin. On the other hand, phosphorylation of ankyrin was found to significantly reduce its affinity for either phosphorylated or unphosphorylated spectrin tetramers (KD = 1.2 +/- 0.1 X 10(-6) M) but not spectrin dimers (KD = 2.5 +/- 0.4 X 10(-6) M). The same results were obtained using either the membrane kinase or casein kinase A as the phosphorylating enzyme. The above observation suggests that ankyrin phosphorylation may provide an important mechanism for the regulation of the erythrocyte membrane cytoskeletal network.     

Temperature control of initiation of protein synthesis in Escherichia coli

When an exponentially growing culture of Escherichia coli is cooled to below 8 °C, initiation of protein synthesis appears to be blocked, while the elongation of initiated proteins continues until they are completed. This is demonstrated here by showing that nascent polypeptide chains increase in size during a 5 °C incubation and that f2 viral coat protein is completed, but not initiated. Upon rewarming, the cells initiate protein synthesis synchronously. This is demonstrated by a transient rise in the incorporation of methionine which is used to initiate protein synthesis.     

Genetic Recombination in Coprinus. IV. a Kinetic Study of the Temperature Effect on Recombination Frequency

At the restrictive conditions (35 degrees under continuous light) Coprinus lagopus is unable to initiate premeiotic S phase which takes place normally within 8-10 h of karyogamy. A shift-up to the restrictive conditions causes an arrest of the basidiocarps at this critical stage. A prolonged arrest causes a reversal to mitosis (Lu 1974b). Incubation of basidiocarps at the restrictive conditions before this critical stage causes no increase in recombination frequency (R.F.) in the loci studied. An arrest of 4 h at the critical stage still causes no R.F. increase, but 12-13 h and 18-19 h arrests cause increases of 50% and 90% over the controls, respectively. Thus R.F. can be increased even before the cells are fully committed to meiosis.-A 3-h heat treatment at the beginning of S phase (or 8 h before karyogamy) also causes some (30%) increase in R.F. while the same treatment at late S phase (or 3 h before karyogamy) causes a substantial (164%) increase in R.F. over the controls. A 3-h heat treatment before S phase causes no increase in R.F.-Pachytene is also responsive to temperature treatments (Lu 1969). The maximum R.f. increase is 100% by heat and 220% by cold treatment. The shortest time that can cause the maximum increase in recombination by high temperature is 3 h and that by cold treatment is 7 h. These durations are correlated with the length of the pachytene stage under the treatment conditions. The kinetic data show that the increase in R.F. caused by high and low temperatures follows two-hit kinetics and their rate of increase is almost identical. The higher increase in R.F. by low temperature can be attributed to the increased duration of pachytene and therefore R.F. is a function of time. The longer the homologous chromosomes are held together, the higher the recombination frequency.     

Role of recurrent hydrophobic residues in catalysis of helix formation by T cell-presented peptides in the presence of lipid vesicles

We tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an alpha helix, can lead to helical nucleation on a hydrophobic surface. The hydrophobic surface could anchor such residues, whereas the peptide sequence grows in a helical configuration that is stabilized by hydrogen bonds among carbonyl and amido NH groups along the peptidyl backbone of the helix, and by other intercycle interactions among amino acid side chains. Such bound, helical structures might protect peptides from proteases and/or facilitate transport to a MHC-containing compartment and thus be reflected in the selection of T cell-presented segments. Helical structure in a series of HPLC-purified peptides was estimated from circular dichroism measurements in: 1) 0.01 M phosphate buffer, pH 7.0, 2) that buffer with 45% trifluoroethanol (TFE), and 3) that buffer with di-O-hexadecyl phosphatidylcholine vesicles. By decreasing the dielectric constant of the buffer, TFE enhances intrapeptide interactions generally, whereas the lipid vesicles only provide a surface for hydrophobic interactions. The peptides varied in their strip-of-helix hydrophobicity indices (SOHHI; the mean Kyte-Doolittle hydrophobicities of residues in an axial strip of an alpha helix) and in proline content. Structural order for peptides with helical circular dichroism spectra was estimated as percentage helicity from circular dichroism theta 222 nm values and peptide concentration. A prototypic alpha helical peptide with three cycles plus two amino acids and an axial hydrophobic strip of four leucyl residues (SOHHI = 3.8) was disordered in phosphate buffer, 58% helical in that buffer with 48% TFE, and 36% helical in that buffer with vesicles. Percentage helicity in the presence of vesicles of the subset of peptides without proline followed their SOHHI values. Peptides with multiple prolyl residues had circular dichroism spectra with strong signals, but since they did not have altered spectra in the presence of vesicles relative to phosphate buffer alone, the hydrophobic surface of the vesicle did not appear to stabilize those structures.