Modification of arginine residues in ovine prolactin by 1,2-cyclohexanedione. Effect on binding capacity to lactogenic receptors.

The reactivity of arginine residues in ovine prolactin was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit with two simultaneous pseudo-first-order equations with apparent velocity constants of 0.28 and 1.2 x 10(-2) min-1, corresponding to 1.8 'fast' and 8.7 'slow' residues, respectively. Modification led to a decrease in binding capacity to lactogenic rat liver receptors, and apparently the modification of the two 'fast' reacting arginine residues is responsible for the rapid loss of this capacity. The presence of a non-reacting arginine has been described in human and bovine growth hormones, and it is located near the carboxy-terminus. This lack of reactivity is probably due to the formation of a salt bridge, since the arginine residue becomes susceptible to modification once the peptide is separated from the rest of the molecule. This salt bridge is absent in ovine prolactin, since the homologous arginine residue is reactive with cyclohexanedione. This result suggests that there could be a difference between the three-dimensional structure of ovine prolactin and of the growth hormones, at least near the carboxy-terminal region of the molecule.

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5'-azido-salicylamido)-undecanoic acid derivatives.

Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5'-azido-salicylamido)-undecanoic acid (5' ASU) and its acetyl ester (Ac5' ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5' ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34 x 10(-7) M, evidenced a slightly higher affinity than that reported for C16-C20 fatty acids. Consistent with the binding curve, 5' ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 microM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of 125I-5' ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5' ASU and Ac5' ASU respectively. In turn, irradiation of L-FABP incubated with 5' ASU or Ac5' ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat brain fatty acid-binding protein during development.

Cytosolic fatty acid-binding proteins (FABPs) have been described in rat and bovine whole brain. In the present study we investigated the distribution of FABP among white matter and gray matter as well as its changes during development. Fatty acid binding activity was similar in white and gray matter up to 40 days of age. In white matter it showed an age dependent increase thereafter, while in gray matter it remained constant throughout. Gel filtration (Sephadex G-75) of white matter cytosol of adult female rats resolved the fatty acid-binding activity in two peaks: A (Vo) and B (12-14 KDa; FABP). The specific binding activity in the FABP fraction was 10.4 pmol/micrograms of protein. The activity in peak A showed an age-dependent increase which paralleled myelin deposition. In contrast, the activity in the FABP fraction (peak B) remained undetectable up to 40 days of age, increasing thereafter. The differential distribution of cellular brain proteins with the capacity to bind fatty acids in gray matter and white matter suggests that this activity could be related to glial cells or to cell related structures such as myelin.

Modification of arginine residues in human growth hormone by 1,2-cyclohexanedione: effects on the binding capacity to lactogenic and somatogenic receptors.

Reactivity of arginine residues in human growth hormone was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit to a pseudo first order curve, with an apparent velocity constant k = 1.26 x 10(-2) min-1 and a maximum modification of 9.6 out of the 11 arginines of the molecule. Modification led to a decrease in binding capacity to both lactogenic and somatogenic rat liver receptors. In either case Tsou plots suggest that the modification of two arginine residues is responsible for this behavior, although it cannot be ascertained whether the two relevant residues are the same for both receptor types. Circular dichroism studies indicated no apparent changes in protein conformation in the modified hormone. Binding capacity was restored upon regeneration of arginines by incubation with Tris-HCl buffer. Only the carboxy-terminal peptide was isolated by HPLC from a tryptic digest of succinylated Arg-modified hGH, indicating that 183 is the nonreacting arginine residue.