Adrenomedullary Secretion of DOPA, Catecholamines, Catechol Metabolites, and Neuropeptides

Abstract: Catecholamines and their metabolites have been proposed as markers of sympathetic nervous system stimulation. However, the adrenal medulla is a rich source of catecholamines and catecholamine metabolites and may play a significant role in plasma levels of these compounds. In addition to adrenal catecholamine metabolite efflux, the role of the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) has not been fully evaluated. The simultaneous effluxes of catecholamines, metabolites, DOPA, and neuropeptides were measured in perfusates from isolated dog adrenals. The relative abundance of compounds detected consistently during unstimulated conditions was epinephrine ≫ norepinephrine > 3,4-dihydroxyphenylglycol > metanephrine > normetanephrine > dopamine > 3,4-dihydroxyphenylacetic acid > 3-methoxy-4-hydroxyphenylglycol ≥ DOPA ≫ [Met]enkephalin ≫ neuropeptide Y. Effluxes of analytes were not affected by cocaine and the ratios of catecholamines to metabolites increased dramatically with carbachol stimulation, consistent with negligible reuptake into adrenal cells. Thus, most of the 3,4-dihydroxyphenylglycol is expected to be derived from epinephrine and norepinephrine subsequent to translocation from chromaffin vesicles into the cytosol. The efflux of DOPA increased dramatically during stimulation with 30 µ M carbachol in a calcium-dependent manner. Efflux of DOPA during the initial stabilization period of the perfusion preparation declined exponentially, in parallel with the effluxes of the catecholamines and neuropeptides but not with metabolites. Evoked release of DOPA was Ca²⁺-dependent. These data suggest that DOPA can be stored and released exocytotically from chromaffin granules.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Evolution of methionine initiator and phenylalanine transfer RNAs

Summary Sequence data from methionine initiator and phenylalanine transfer RNAs were used to construct phylogenetic trees by the maximum parsimony method. Although eukaryotes, prokaryotes and chloroplasts appear related to a common ancestor, no firm conclusion can be drawn at this time about mitochondrial-coded transfer RNAs. tRNA evolution is not appropriately described by random hit models, since the various regions of the molecule differ sharply in their mutational fixation rates. Hot mutational spots are identified in the TC, the amino acceptor and the upper anticodon stems; the D arm and the loop areas on the other hand are highly conserved. Crucial tertiary interactions are thus essentially preserved while most of the double helical domain undergoes base pair interchange. Transitions are about half as costly as transversions, suggesting that base pair interchanges proceed mostly through G-U and A -C intermediates. There is a preponderance of replacements starting from G and C but this bias appears to follow the high G + C content of the easily mutated base paired regions.     

Stimulation of the adenylate cyclase of A B16 melanoma cell line by pro-opiocortin-related peptides--a structure-activity study

The ability of alpha-melanotrophin (alpha-MSH or ACTH 1-acetyl-13 amide) and other structurally related peptides derived from the common precursor, pro-opiocortin, to stimulate adenylate cyclase activity in a pigmented B16 mouse melanoma was investigated. The peptides ACTH 1-39, ACTH 1-24, alpha-MSH, ACTH 1-13 amide and beta-MSH all stimulated the enzyme to a similar maximal extent and with similar potency (ED50 = 1.3 . 10(-6) M) except that ACTH 1-39 was slightly less potent (ED50 = 5 . 10(-6) M). ACTH 4-10 (ED50 = 4 . 10(-5) M) and gamma-MSH (ED50 = 5 . 10(-6) M) were partial agonists. ACTH 1-10 was no more effective than ACTH 4-10 in stimulating the enzyme whereas ACTH 1-13 amide was a full agonist. The peptides beta-endorphin and its derivatives, Met-enkephalin and melanotrophin potentiating factor (MPF), failed to stimulate the enzyme. We suggest that the B16 melanoma requires not only the sequence ACTH 4-10 but also some part of the sequence ACTH 11-13, or a similar sequence in the terminal portion of beta-MSH, for full activation of the receptor-linked enzyme.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

Transport and utilization of methionine sulfoxide in the rabbit

Methionine sulfoxide is transported into purified intestinal and renal brush border membrane vesicles from rabbit by an Na⁺-dependent mechanism and is accumulated inside the vesicles against the concentration gradient. Both in intestine and kidney, the rate of transport is enhanced with increasing concentrations of Na⁺ in the external medium. Increasing the Na⁺ gradient reduces the apparent K_t for methionine sulfoxide without causing any change in V_max. With an outward K⁺ gradient (vesicle > medium), valinomycin stimulates the Na⁺-gradient-dependent transport of methionine sulfoxide in the kidney, showing the electrogenicity of the transport process. A number of amino acids inhibit methionine sulfoxide transport in both the intestine and kidney. An enzymatic activity capable of reducing methionine sulfoxide to methionine is present in the intestinal mucosa, renal cortex and liver. The activity is highest in renal cortex and lowest in intestine. The methionine sulfoxide-reducing activity is stimulated by NADH, NADPH, glutathione and dithiothreitol and the potency of the stimulation is in the order: dithiothreitol > NADPH > glutathione > NADH.     

Methionine adenosyltransferase activity in cultured cells and in human tissues

We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitt's lymphoma) and EL4 (mouse, T cell lymphoma) cell showed MAT activity near 300 pmol/mg per min. Daudi (human, Burkitt's lymphoma) and almost all monolayer cells had MAT activity below 100 pmol/mg per min. Human peripheral blood lymphocytes had MAT activity of 36 pmol/mg permin. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 pmol/mg per min, while in normal lung it was 2.4 pmol/mg per min.     

The assay of Met-enkephalin aminopeptidase with [125I]Met-enkephalin

Presented here are procedural modifications whic permit the utilization of ¹²⁵I-labeled Met-enkephalin as substrate in the assay of rat brain enkephalin amipeptidase. The hydrolysis of enkephalin is monitored by the release of [¹²⁵I]tyrosine separated on Porapak Q. The release of tyrosine is proportionate with both increasing time and tissue concentration. The estimated K_m is near 10^?4 M and the enzyme activity can be inhibited more than 95% with puromycin. The majority of the enzyme activity remains in the 100 000 × g supernatant following differential centrifugation.     

beta-Endorphin: characteristics of binding sites in the rat brain.

Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using $\text{[}{}^3\text{H-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the ligand. The binding is time dependent and saturable with respect to β_h-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of β_h-endorphin, is presented.     

Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine