Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional Kinetic Constants for Blood–Brain Barrier Pyruvic Acid Transport in Conscious Rats by the Monocarboxylic Acid Carrier

The present investigation using labeled pyruvate describes the regional distribution and kinetics of the monocarboxylic acid carrier at the blood-brain barrier of conscious rats. The experimental procedure involved the arterial injection of a single bolus of 200 microliter containing [1-14C]pyruvate, [3H]water, and varying concentrations of unlabeled pyruvate into the common carotid via an indwelling externalized catheter. The hemisphere ipsi-lateral to the injection and rostral to the midbrain was removed and dissected into five regions. A kinetic analysis revealed no significant regional differences in Km values with an overall average of 1.37 mM. However, there was regional variation in the density of the monocarboxylic acid carrier as indicated by varied levels of the kinetic constant Vmax. The cortex showed the highest Vmax value of 0.42 +/- 0.08 mumol/min/g whereas values for the caudate/putamen, thalamus/hypothalamus, and remaining portion of hemisphere ranged significantly lower at 0.22-0.27 mumol/min/g. The Vmax for the hippocampus was intermediate at 0.37 +/- 0.12 mumol/min/g. The nonsaturable carrier described kinetically by KD had an overall average of 0.034 ml/min/g. The present study confirms quantitatively previous results suggesting a variable regional distribution of the monocarboxylic acid carrier.     

Cationic probes: Specificity of distribution of metal-binding sites in bovine sperm

Salts of transition elements that alter the rate of sperm cell movement act at or near calcium-binding sites. After living bull sperm cells had been preincubated in VO₄^3?, Ni²⁺, Zn²⁺, Mn²⁺, and also La³⁺, they were then fixed. Crisply defined organelles and the absence of particulate deposits in the morphological controls contrasted sharply with the treated specimens; the latter contained regions of increased electron density, the nature and distribution of which depended on the test substance, reflecting the differential affinities of the specific ions. La³⁺ formed fine dense areas, mainly at the exocytic surface of the plasma membrane. VO₄^3? marks the cell surface but also left particulate densities within the cell. Ni²⁺ caused a nearly uniformly dense deposit at the surface and on the satellite fibers and axonemal microtubules. Zn²⁺ formed less uniform but coarser deposits, while in Mn²⁺ the distribution was similar to that in Zn²⁺ but much denser in the axonemal matrix and on the satellite fibers. Verapamil restricted the size and number of the opacities, while procaine permitted a similar distribution of slightly larger size reaction product. The differences in size and distribution of the enhanced densities were consistent and replicable for the individual assay substances. Vanadate, which specifically inhibits Na, K-ATPase, bound to ouabain-sensitive enzyme loci, however, completely disrupting the axonemal complex. This suggests that an important role of dynein in flagellar motion may relate to intracellular transport of Ca²⁺.     

Cellular retinoic acid-binding protein and its relationship to the biological activity of four synthetic retinoids in hamster tracheal organ culture

Summary The mechanism of action of retinoid in reversing keratinization in hamster trachea is yet unknown. The purpose of this study was to determine if cellular retinoic acid binding protein (CRABP) is present in tracheal epithelium following incubation in serum-free, vitamin A-deficient culture medium for 10 days, and if the effectiveness of a retinoid in reversing keratinization in organ culture is correlated with its ability to compete for CRABP sites. The cytosol prepared from tracheal cultures contained CRABP at a concentration of 2.61 pmoles per mg protein. Of the four retinoids with carboxyl end group selected for the study, two of the biological active retinoids competed for the CRABP sites. However, no correlation was observed between the biological activity of the inactive retinoids and their ability to associate with the CRABP sites. These results indicate that even though the action of retinoid may be mediated by retinoid binding protein, it cannot be used as a sole predicator of retinoid response in hamster trachea. This investigation was supported by Contract N01-CP-31012 and U. S. P. H. Grants CA30512 and CA32428, which were awarded by the Division of Cancer Etiology, National Cancer Institute, DHHS. Editor's Statement Tracheal organ cultures provide a useful model for the study of epithelial differentiation and carcinogenesis. Much attention has been given to the action of retinoids in this process. Mehta et al. demonstrate a lack of correlation between biological activity and specific cytosolic binding of members of this class of compounds, pointing out the need for a more complete biochemical understanding of the mechanism of action and active forms of retinoids in this and other systems in vivo and in vitro. David W. Barnes     

Dissociation of the receptor for immunoglobulin E in mild detergents

We previously showed that, in the absence of phospholipids, exposure of the tetrameric receptor for immunoglobulin E to mild detergents dissociates the intact beta chain and two gamma chains from the alpha chains. Having developed a practical method for assaying the dissociation, we have now explored a variety of different detergents, detergent concentrations, temperatures, times, salts, pHs, and other factors that influence the detergent-induced dissociation. Our findings should be useful for optimizing the stability of the receptor and for future studies on recombination of the subunits. The data suggest the following: (1) The critical perturbant is micellar detergent. (2) Unlike solubilization of membranes, where a molar ratio of micellar detergent:lipid of 2 is adequate, dissociation of the receptor is incomplete even at molar ratios of micellar detergent:receptor of greater than 10(5) and may be limited by a reversible component. (3) Detergents that are best for solubilizing membranes are also best for dissociating the receptors. (4) The latter observation and other data implicate bound lipid as stabilizing the receptor. Our findings may be applicable to the study of interactions between membrane proteins in general.