Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with L-[3H]adenosine, our objectives here were to determine (1) if L-[3H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological D-adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. L-[3H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. L-[3H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 microM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with L-[3H]adenosine alone or I-[3H]adenosine plus unlabeled L-adenosine, D-adenosine, or uridine, L-[3H]adenosine release was inhibited 42% by L-adenosine, 69% by uridine, and 81% by D-adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)
미성숙 상태의 녹색밀을 식품가공소재로 이용하기 위한 영양적 기초자료를 얻고자 밀이 성숙과정 중에 일반성분, 유리당, 유리아미노산, β-glucan, 무기물 및 색도를 측정하였다. 공시품종인 금강밀, 그루밀, 청계밀의 수분, 조단백, 조지방, 회분은 완숙에 가까워질수록 감소하는 경향을 보여 주었고 조섬유의 함량은 금강밀은 3.0%에서 3.26%로, 그루밀은 3.35%에서 4.59%로, 청계밀은 4.25%에서 6.11%로 각각 증가하였다. 출수 25일 경에 밀의 유리당 함량은 maltose, maltotriose가 높았고 금강밀, 그루밀, 청계밀 모두 완숙에 가까워질수록 감소하였다. 유리 아미노산 함량은 금강밀, 그루밀, 청계밀 모두 출수 후 25일경에는 alanine, GABA, glycine 순으로 많았고 완숙밀에 가까워질수록 각각의 유리 아미노산은 감소하는 경향을 보였으나 glutamic acid만이 증가하는 경향을 보여 성숙한 밀에서는 alanine, glutamic acid, GABA가 많았다. 셋 품종 중 그루밀이 유리아미노산 함량이 비교적 높았다. 모든 공시재료에서 완숙에 가까워질수록 β-glucan함량은 감소하는 경향을 보여 주었다. 금강밀의 출수 후 26일 경 β-glucan 함량은 1.5%에서 출수 후 43일에는 0.28%로 셋 품종 중 가장 크게 감소하였으며 모든 품종에서 같은 경향이었다. 출수 후 완숙에 가까워질수록 공시밀의 무기물 함량은 전반적으로 증가하였고 특히 K, Mg의 증가하는 경향은 뚜렷하였다. 공시품종 모두에서 완숙에 가까워질수록 L값은 감소하였으며 a값과 b값은 증가하였으며 출수 후 33일 경까지 녹색을 유지하고 증미성 물질이 다량 존재하여 가공식품의 소재를 이용하는 것이 바람직하다.
When the cattail pollen was identified by using fibrinolytic agents, we found that the fibrinolytic activity was controlled by an enzyme. Therefore, for determining the fibrinolytic activity of cattail pollen, the fibrinolytic enzyme in cattail pollen was purified by gel filtration using DEAE-cellulose, Sephadex G-150 and HPLC. Also, its purity was certified by polyacrylamide gel electrophoresis, and its physico-chemical properties, such as pH and temperature stabilities and effects of metal, inhibitors and substrates, were examined. The specific activity, purification fold, and molecular weight of the enzyme were 38U/mg, 86.4,and 75kDa, respectively. The optimum pH for the purified enzyme was at 4.0 and it was stable at pH 4.0-6.0. The optimum temperature was and it was stable at . But the enzyme began to be inactivated at and its activity was totally lost at temperatures above . As for substrate specificity, the enzyme was most effective in dissolving fibrin, followed by whole casein, -casein, -casein, -casein, and BSA. With casein as the substrate, Km value was found to be 0.44mM and the enzyme showed a high affinity for casein. As for the metal ions affecting enzyme activity, , , and had no effect on enzyme reaction while and showed potent inhibitory activity. Judging from the fact that the purified enzyme was also strongly inhibited by PMSF, iodoacetic acid, and SDA, it assumed to be a serine protease.
Abstract: The stereoenantiomers D‐[ 3 H]adenosine and L‐[ 3 H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L‐Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a K i value of 385 μ M . It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L‐[ 3 H]adenosine accumulation measured in assays where transport was stopped either with inhibitor‐stop centrifugation or with rapid filtration methods were 82 ± 14 and 75 ± 10, respectively. Using the filtration method, the rates of L‐[ 3 H]adenosine accumulation were not significantly different from the value of 105 ± 15 fmol/mg of protein/s measured for D‐[ 3 H]adenosine transport. Unlabeled D‐adenosine and nitrobenzylthioinosine, both at a concentration of 100 μ M , reduced the levels and rates of L‐[ 3 H]adenosine accumulation by >44%. These findings suggest that L‐adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D‐adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L‐adenosine may represent an important new probe with which adenosine uptake may be studied.
Abstract: Abstract: [ 3 H]Adenosine transport was characterized in cerebral cortical synaptoneurosomes prepared from postmortem human brain using an inhibitor‐stop/centrifugation method. The adenosine transport inhibitors dipyridamole and dilazep completely and rapidly blocked transmembrane fluxes of [ 3 H]adenosine. For 5‐s incubations, two kinetically distinguishable processes were identified, i.e., a high‐affinity adenosine transport system with K t and V max values of 89 μ M and 0.98 nmol/min/mg of protein, respectively, and a low‐affinity adenosine transport system that did not appear to be saturable. For incubations with 1 μ M [ 3 H]adenosine as substrate, intrasynaptoneurosomal concentrations of [ 3 H]adenosine were 0.26 μ M at 5 s and 1 μ M at 600 s. Metabolism of accumulated [ 3 H]adenosine to adenine nucleotides was 15% for 5‐s, 23% for 15‐s, 34% for 30‐s, 43% for 60‐s, and 80% for 600‐s incubations. The concentrations (μ M ) of total accumulated 3 H‐purines ([ 3 H]‐adenosine plus metabolites) at these times were 0.3, 0.5, 1.0, 1.3 and 5.6, respectively. These results indicate that in the presence of extensive metabolism, the intrasynaptoneurosomal accumulation of 3 H‐purines was higher than the initial concentration of 1 μ M [ 3 H]adenosine in the reaction medium. For 5‐, 15‐, 30‐, 60‐, and 600‐s incubations in the presence of the adenosine deaminase inhibitor EHNA and the adenosine kinase inhibitor 5′‐iodotubercidin, metabolism of the transported [ 3 H]adenosine was 14, 14, 16, 14, and 38%, respectively. During these times, total 3 H‐purine accumulation was 0.3, 0.5, 0.5, 0.7, and 1.8 μ M , respectively. Thus, the apparently “concentrative'’accumulation of 3 H‐purines can be prevented by inhibition of adenosine metabolism and, taken together, these results suggest that adenosine transport in at least synaptoneurosomes prepared from postmortem human brain is via a nonconcentrative and equilibrative system.
Abstract: The kinetic characteristics of [ 3 H]adenosine uptake, the extent to which accumulated [ 3 H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis‐Menten kinetic values of K T and V max , and the sensitivities with which nucleoside transport inhibitors blocked [ 3 H]adenosine accumulations were determined in cultured human fetal astrocytes. K T and V max values for accumulations of [ 3 H]‐labeled purines using 15‐s incubations in the absence of the adenosine deaminase inhibitor erythro ‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5′‐iodotubercidin (ITU) were 6.2 µ M and 0.15 nmol/min/mg of protein for the high‐affinity and 2.6 m M and 21 nmol/min/mg of protein for the low‐affinity components respectively. In the presence of EHNA and ITU, where <4% of accumulated [ 3 H]adenosine was metabolized, transport per se was measured, and kinetic values for K T and V max were 179 µ M and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [ 3 H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of “concentrative” uptake in that the intracellular levels of [ 3 H]‐labeled purines (adenosine plus its metabolites) were 1.4‐fold higher than in the medium. No apparent concentrative accumulations of [ 3 H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [ 3 H]adenosine transport; for the inhibitor‐sensitive components the IC 50 values were 0.7 n M for NBI, 1.3 n M for DPR, and 3.3 n M for dilazep, and for the inhibitor‐resistant component the IC 50 values were 2.5 µ M for NBI, 5.1 µ M for dilazep, and 39.0 µ M for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor‐sensitive and ‐resistant adenosine transporters in nontransformed human cells.
Abstract: The relationship between transport and metabolism in synaptoneurosomes was examined to determine the metabolic stability of rapidly accumulated D‐[ 3 H]adenosine and L‐[ 3 H]adenosine and the degree to which metabolism of the accumulated purines affected measurements of apparent K T and V max values for adenosine transport. For D‐[ 3 H]adenosine, high‐ and low‐affinity accumulation processes were present. For the high‐affinity system an inverse relationship was found between transport reaction times and K T and V max values. For incubations of 5, 15, and 600 s, which corresponded to 24, 32, and 76% phosphorylation of accumulated D‐[ 3 H]adenosine to nucleotides, apparent K T values were 9.4, 8.4, and 4.5 μ M , respectively, and V max values were 850, 70, and 12 pmol/min/mg of protein, respectively. Pretreatment with 10 μ M erythro ‐9‐(2‐hydroxy‐3‐nonyl)adenine, an adenosine deaminase inhibitor, and 5′‐iodotubercidin, an adenosine kinase inhibitor, decreased the phosphorylation of accumulated D‐[ 3 H]adenosine to 6% with 5‐s and 9% with 15‐s incubations. This resulted in significantly higher K T values: 36 μ M at 5 s and 44 μ M at 5 s. At 10‐min incubations in the presence of these inhibitors, metabolism of accumulated D‐[ 3 H]adenosine was 32%, and apparent K T and V max values at this time were not significantly different from those obtained without inhibitors. For L‐[ 3 H]adenosine, apparent K T and V max values for 20‐s incubations were 38.7 μ M and 330 pmol/min/mg of protein, respectively. Metabolism (mainly phosphorylation) of accumulated L‐[ 3 H]adenosine was observed only at incubations of >30 s. Taken together, these results demonstrate that adenosine transport is significantly faster than subsequent metabolism; that accumulated D‐adenosine is rapidly incorporated into and trapped intracellularly as adenine nucleotides, thereby affecting measured kinetic parameters for adenosine transport and giving an “appearance” of concentrative accumulations; and that the apparent K T T value of 39 μ M for D‐adenosine transport conducted in the presence of the enzyme inhibitors was the same as the apparent K T value for L‐adenosine transport.
3-Chloro-L-tyrosine (3CT) is an inhibitor of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis. In vivo inhibition of tyrosine hydroxylase results in lower catecholamine levels. 3CT (0.5 mg/kg), administered as a bolus i.v. to anesthetized uninephrectomized rats, elicited increases of 72% and 44% in urinary sodium concentration and volume, respectively, whereas a dose of 1 mg/kg caused increases of 27% and 29%. 3CT, 1 mg/kg, resulted in a 2-fold increase in plasma aldosterone (ALD); 0.5 mg/kg was without significant effect. At a dose of 1 mg/kg 3CT significantly antagonized the renal effects of atrial natriuretic peptide (ANP) (1.5 micrograms kg-1 min-1 by intrarenal infusion), expressed as an enhanced excretion of urine volume (102 +/- 14 vs. 70 +/- 11 microliters/min) and sodium (16.1 +/- 1.8 vs. 11.5 +/- 1.7 microEq/min) and increased osmolar clearance (171 +/- 12 vs. 144 +/- 13 microliters/min). A dose of 0.5 mg/kg of 3CT did not produce these same responses to ANP. The increased urine flow caused by 3CT may reflect reduced norepinephrine synthesis. The inverse dose-effect relationship of 3CT on urine flow rate may result from concomitant depletion of dopamine (DA) and elevated circulating ALD. The antagonism of 3CT on responses to ANP is not at the receptor level, because 3CT did not compete for [125I] ANP binding or inhibit ANP-stimulated guanylate cyclase in kidney cell membranes. It was proposed that the reduced basal sympathetic and renal DA tone, together with the elevated ALD level, account for this antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)
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