Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment.

Mutational studies were previously carried out at the omega site intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the omega + 1 and omega + 2 sites in a cell-free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidyl-inositol (GPI)-anchored form. We have now mutated the omega + 1 and omega + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the omega + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the omega + 1 site was promiscuous, with only proline being inactive. These and the previous studies indicate that the omega and omega + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.

Decrease in adrenal tyrosine hydroxylase and increase in norepinephrine synthesis in rats given L-dopa.

When large doses of L-dopa (1000 milligrams per kilogram, given subcutaneously) were administered to rats, the rate of catecholamine synthesis was increased in both the heart and adrenal gland. It is likely that increases also occur in other sympathetically innervated tissues. When the same dose of L-dopa was given daily for 4 or 7 days, the levels of tyrosine hydroxylase in the adrenal were lowered in comparison to controls. These findings may be a further indication of the existence of a regulatory mechanism which modifies endogenous levels of tyrosine hydroxylase in response to changes in the biosynthetic demand for norepinephrine.

Collagen proline hydroxylase in wound healing, granuloma formation, scurvy, and growth.

Compared to homologous tissues from adult animals, rapidly growing embryonic and fetal tissues contain large amounts of collagen proline hydroxylase. Lung and skin from scorbutic guinea pigs contain one-third as much enzyme as normal tissues do. A rapid increase in proline hydroxylase occurs on the 2nd day of formation of granuloma after injection of carrageenan and on the 4th day of wound healing. The increase in enzyme activity is associated with the onset of collagen biosynthesis and deposition.

p-Chlorophenylalanine-induced chemical manifestations of phenylketonuria in rats.

p-Chlorophenylalanine, a potent inhibitor of phenylalanine hydroxylase in vivo, has been used to simulate phenylketonuria in rats. This inhibitor, when administered with phenylalanine, produces marked elevation of blood and tissue phenylalanine without an increase in tyrosine. Disproportionate ratios of phenylalanine to tyrosine are characteristic of phenylketonuria in humans. The use of p-chlorophenylalanine permits the production of this characteristics amino acid imbalance in experimental animals.

Hypertension: increase of collagen biosynthesis in arteries but not in veins.

In two models of hypertension in rats, it was shown that collagen synthesis and deposition are increased in arteries where blood pressure is elevated. By contrast, there were no alterations in any of the markers of collagen synthesis in veins, where blood pressure was only slightly elevated. It would appear that the stimulus for vascular collagen synthesis is provided by a direct effect of the increased pressure on the arterial cells rather than by a humoral factor released into the general circulation.

An about 50,000-dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin.

A protein that may be an enkephalin precursor has been identified in extracts of bovine adrenal medulla. This protein (about 50,000 daltons) appears to contain seven copies of [Met]enkephalin and one copy of [Leu]enkephalin. Digestion with trypsin and carboxypeptidase B yields [Met]enkephalin and [Leu]enkephalin in a ratio of almost 7 to 1. The enkephalins were identified by chromatography and by their binding to opiate receptors. Some characteristics of several other adrenal peptides that may serve as intermediates in the biosynthesis of the enkephalins are presented.

Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range.

Fluorescamine is a new reagent for the detection of primary amines in the picomole range. Its reaction with amines is almost instantaneous at room temperature in aqueous media. The products are highly fluorescent, whereas the reagent and its degradation products are nonfluorescent. Applications are discussed.

Metabolism of alpha-methyltyrosine in man: relationship to its potency as an inhibitor of catecholamine biosynthesis.

The metabolic fate of the tyrosine hydroxylase inhibitor, alpha-methyl-para-tyrosine (alpha-MPT), was studied after oral administration of single and multiple doses to patients with pheochromocytoma and essential hypertension. No major urinary excretion product was found other than the drug itself, which accounted for 44-88% of the fate of single or repeated oral doses. Though less than 1% of the administered drug could be recovered in the urine as catechol metabolites, it was possible to identify alpha-methyldopa, alpha-methyldopamine, and alpha-methylnorepinephrine and to quantify the excretion of the first two of these compounds. This minor route of metabolism required revision of methodology (presented herein) for measuring urinary catecholamines during alpha-MPT treatment since these compounds produce spurious fluorescence in routine methods of assay for catecholamines. The catechol metabolites probably are not present in sufficient amounts to contribute to the biochemical effects of the drug. Determination of plasma concentrations of alpha-MPT during maintenance therapy and considerations of the kinetics of enzyme inhibition enabled a calculation to be made of the degree of inhibition of catecholamine synthesis to be expected in the patient. This was calculated to be about 75% for the highest doses employed and is similar in magnitude to experimentally determined values.

On betaH-Leu5-endorphin and schizophrenia.

A previously unknown peptide, betaH-Leu5-endorphin, has been reported in the dialysates of schizophrenic patients. Accordingly, hemofiltrates from two schizophrenic and two control patients were examined for the presence of betaH-Leu5-endorphin. The opioid peptides were detected by a radioreceptor assay after separation and identification by gel filtration and high-performance liquid chromatography. With a detection limit of 30 pmole/L of hemofiltrate, no betaH-Leu5-endorphin or Met5-endorphin was found in controls or in patients. Whatever the possible involvement of endorphins in schizophrenic behavior, they are not present at detectable levels in the hemofiltrates of two well-characterized schizophrenic patients, thereby casting doubt on a general relationship of Leu-endorphin and schizophrenia.

Cloning and sequencing of bovine kidney alkaline phosphatase cDNA.

The molecular cloning and nucleotide sequencing of bovine kidney alkaline phosphatase is reported. The homology with the human enzyme is about 90% at both the nucleotide and amino acid levels. The only significant sequence differences occur at the respective C termini. The high degree of homology also extends into the 5' and 3' untranslated regions of the two cDNAs.

Immunocytochemical localization of the renal neutral and basic amino acid transporter in rat adrenal gland, brainstem, and spinal cord.

A neutral and basic amino acid transporter (NBAT) cloned from rat kidney was recently localized to enteroendocrine cells and enteric neurons. We used an antibody directed against a synthetic peptide representing a putative extracellular domain of NBAT to determine whether this transporter was also present in other endocrine and neural tissues, including rat adrenal gland, brainstem, and spinal cord. Abundant, highly granular labeling for NBAT was observed in the cytoplasm of chromaffin and ganglion cells in the adrenal medulla. A small population of intensely labeled varicose processes was also seen in both the cortex and the medulla of the adrenal gland. More numerous, intensely labeled varicose processes were detected in brainstem and spinal cord nuclei, including the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, dorsal motor nucleus of the vagus, and intermediolateral cell column of the thoracic spinal cord. Significant perikaryal labeling for NBAT was only detected in brainstem and spinal cord following intraventricular colchicine treatment, which increased the number, distribution, and intensity of NBAT-immunolabeled cells. These NBAT-immunoreactive perikarya were most numerous in the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, and raphe nuclei. Ultrastructural examination of the nuclei of the solitary tract of normal rats showed that NBAT was localized predominantly to axon terminals. Within these labeled terminals, NBAT was associated with large dense core vesicles and discrete segments of plasma membrane. The observed localization of NBAT suggests that this renal specific amino acid transporter subserves a role as a vesicular or plasmalemmal transporter in monoamine-containing cells, including chromaffin cells and autonomic neurons.

Binding assay for opioid peptides with neuroblastoma x glioma hybrid cells: specificity of the receptor site.

A sensitive and rapid radioreceptor assay has been developed to monitor opioid peptides in column effluents. It is based on competitive binding to NG 108-15 cells using [3H-Tyr]Leu-enkephalin as the displaced ligand. The specificity of binding to the cells of naturally occurring opioid peptides and synthetic analogs has been shown to be similar to that found with synaptic plasma membranes.

Prolongation of procaine's and procainamide's actions by binding to acryloyl polymers.

Procaine and procainamide were covalently bound to acryloyl monomers and polymers. The dose-response and time-action parameters of the cardiac antiarrhythmic protection afforded by the prototype drugs and their acryloyl derivatives against chloroform-hypoxia-induced cardiac arrhythmias in unanesthetized mice and epinephrine-induced arrhythmias in alpha-chloralose anesthetized cats were determined. Similarly, the pharmacological parameters which characterized their acute toxic responses in unanesthetized male albino mice were also determined. The similar pharmacological spectra of their activity and the parallelism of their lethal dose-response curves indicate that the active constituents of the polymer derivatives are the local anesthetic moieties. Compared to the prototype drugs, the polymer derivatives were more potent on a molar basis and their pharmacological effects were prolonged. The increased potency and duration of action reinforce the idea that the local anesthetic moieties are pharmacologically active while still bound to the polymer backbones.