k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

The function of tonoplast ATPase in intact vacuoles of red beet is governed by direct and indirect ion effects

The pH gradient and the electric potential across the tonoplast in mechanically isolated beetroot vacuoles has been studied by following the uptake of [¹⁴C]methylamine and [¹⁴C]triphenyl-methylphosphoniumchloride. In response to Mg-ATP, the vacuolar interior is acidified by 0.8 units. This strong acidification is accompanied by a slight hyperpolarization of the membrane potential, which is probably caused by a proton diffusion potential. In preparations where only a small acidification (0.4 units) occurred, the membrane potential was depolarized by the addition of Mg-ATP. Different monovalent cations and anions were tested concerning their effect on the pH gradient and ATPase activity in proton-conducting tonoplasts. Chloride stimulation and NO ₃ ^- inhibition were clearly present. The observed decline of the pH gradient upon the addition of Na⁺ salts is probably caused by an Na⁺/H⁺ antiport system.Abbreviations and symbol CCCP carbonylcyanide-m-chlorophenylhydrazone - Mes 2(N-morpholino)ethanesulfonic acid - TPMP⁺ triphenylmethylphosphoniumchloride - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - membrane potential Dedicated to Professor A. Betz on the occasion of his 65th birthday     

Central actions of calcitonin on body temperature and intestinal motility in rats: evidence for different mediations

The effects of intracerebroventricular (i.c.v.) administration of calcitonin and PGE2 on intestinal motility and body temperature were examined in conscious rats chronically fitted with intraparietal electrodes in the small intestine, a cannula in a cerebral lateral ventricle and a subcutaneous thermistor probe. Both calcitonin and PGE2 restored the fasted pattern of intestinal motility in fed rats and induced an increase in body temperature. Indomethacin, an inhibitor of the cyclooxygenase with calcium antagonistic properties, and TMB-8, an intracellular calcium antagonist, blocked the effects of calcitonin on intestinal motility and body temperature. Piroxicam, an inhibitor of the cyclooxygenase which does not affect calcium uptake blocked the thermic but not the intestinal effects of calcitonin. TMB-8 but not indomethacin or piroxicam partially blocked the effects of PGE2 on both intestinal motility and body temperature. It is concluded that the central hyperthermic effect of calcitonin is mediated through the formation and the release of prostaglandins whereas the central action of calcitonin on digestive motility results from intracerebral effects on calcium fluxes.     

Occurrence of oscillations in ATP synthesis and hydrolysis in chromatophores of Rhodospirillum rubrum

The conditions under which an oscillatory behaviour is observed during net hydrolysis or synthesis of ATP in chromatophores of Rhodospirillum rubrum FR1 are described. In the case of ATPase the oscillations are observed at low temperature (ca. 11°C) in the dark after an initial transient behaviour. These oscillations are attenuated or disappear by the addition of an uncoupler.Oscillations are also observed during ATP synthesis. At 3°C the oscillations appear spontaneously if photophosphorylation is measured during a sufficiently long time. At 30°C the mere intercalation of a dark period also at 30°C is sufficient to trigger the oscillations in the following light period.Abbreviations Bchl Bacteriochlorophyll - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - PMS phenazine methosulfate - TMPD, N,N,N,N tetramethyl-1,4-phenylenediamine Dedicated to Prof. Dr. Gerhart Drews as a homage for his permanent example as hard worker and careful scientist and also for his remarkable human quality     

Binding of 5-methoxypsoralen to human serum low density lipoproteins

5-methoxypsoralen (5-MOP) binds to human serum low density lipoproteins (LDL) according to a two-step process. Scatchard analysis of the first step yields K = 1.4 × 10⁵ M^?1 and 4 binding sites. It involves the LDL apoprotein. The second step corresponds to a solubilization, in the lipidic core, of ? 45 molecules of 5MOP per LDL molecule. It is accompanied by a large blue shift of the 5MOP fluorescence. The ability of LDL to bind 5MOP and to carry it into various cells may explain some biological effects sometimes encountered during PUVA therapy.     

Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells

In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells.     

Condensed tannins: co-occurrence of procyanidins,prodelphinidins and profisetinidins in the heartwood of Acacia baileyana

The flavonoids and condensed tannins of the heartwood of Acacia baileyana var. purpurea are described. In conformity with other Acacia species, the hydroxylation pattern of the flavonoids is of the resorcinol type but, in sharp contrast, the tannins are heterogeneous consisting of a mixture of the resorcinol and phtoroglucinol series. Dimeric proanthocyanidins of the phloroglucinol type were absent and this exception to the general observation that they invariably co-occur with the polymers may be explained by the relative nucleophilicity of the aromatic A-rings.     

Uptake of Magnesium by Chromaffin Granules In Vitro: Role of the Proton Electrochemical Gradient

Abstract: The chromaffin granule membrane in vitro is impermeable to protons as well as to Mg²⁺; however, when granules are incubated in the presence of the proton ionophore carbonyl cyanide p -trifluoromethoxy-phenylhydrazone or an inhibitor of the granule membrane Mg²⁺-dependent ATPase, the metal ion is accumulated inside the granules. This accumulation is dependent upon the granule transmembrane potential. The simultaneous presence of the ATPase inhibitor and the proton ionophore markedly increases metal ion incorporation. Mg²⁺ incorporation is also promoted by nigericin in the presence of potassium or sodium ions, indicating that Mg²⁺ accumulation is also dependent upon the transmembrane pH gradient. Concomitant with the Mg²⁺ accumulation, there is a significant loss of endogenous catecholamines. It is concluded that Mg²⁺ accumulation is determined by the electrochemical gradient maintained across the membrane. Once the metal ion has accumulated into the granules it displaces catecholamines from their storage sites.     

Characterization of Cyclic AMP-Dependent Phosphorylation of Neuronal Membrane Proteins

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.     

Sequence of Guinea Pig Myelin Basic Protein

This paper proposes a tentative amino acid sequence of guinea pig myelin basic protein obtained by comparison of peptide fragments of the guinea pig and bovine proteins. Analyses of the tryptic peptides confirmed the known sequence differences in the NH2-terminal half of the molecule and showed that in the COOH-terminal half of the guinea pig protein Ser131 was missing, Ala136 - His137 was deleted, Leu140 was replaced by Phe, and an extra Ala was inserted somewhere within sequence 142-151 (tryptic peptide T23 ). Sequence determination of guinea pig tryptic peptides corresponding to residues 130-134 ( T20 ), 135-138 ( T21 ), and 142-151 ( T23 ) of the bovine protein confirmed the above sequence changes and placed the extra Ala between Gly142 and His143 . The sequence of the region corresponding to bovine residues 130-143 is thus Ala-Asp-Tyr-Lys-Ser-Lys-Gly-Phe-Lys-Gly-Ala-His. No species differences were observed in the amino acid compositions of the remaining tryptic peptides obtained from the COOH-terminal half of the molecule. Based upon these results, the guinea pig basic protein contains 167 amino acid residues and has a molecular weight of 18,256.