Krueger J M, Cagan R H
J Biol Chem. 1976 Jan 10;251(1):88-97.
Large numbers of taste buds are distributed over the body surface of the channel catfish ictalurus punctatus, with the barbels having an especially high density. L-Alanine, as well as certain other amino acids, are taste stimuli in this animal. Epithelial tissue obtained by gentle scraping of the barbel surface was fractionated by differential centrifugation. A sedimentable fraction (P2) was prepared that was enriched in L[OH]alanine binding activity, the plasma membrane marker enzyme 5'-nucleotidase, and the mitochondrial marker succinate cytochrome c reductase, but not the microsomal marker NADH cytochrome c redu.ctase. Binding of L-[OH]alanine was measured using a Millipore filter method in which correction for non-specific binding was also determined. Time, temperature, and pH for measuring binding activity were established. At the optimal pH of 7.8, the KD for L-alanine is 4.8 X 10(-6) M. The first order dissociation rate constant at 6 degrees is 3.8 X 10(-4) s-1 and at 24 degrees it is 12.1 X 10(-4) s-1. The second order rate constant for association is between 10(2) and 10(3) M-1 S-1. Reversibility of the binding interaction was also demonstrates by the rapid displacement of bound L-[3H]alanine by a large excess of unlabeled L-alanine. That the binding does not represent incorporation into protein was confirmed by the lack of effect of puromycin. The amounts bound of several other chemostimulatory amino acids werealso determined.
大量味蕾分布在斑点叉尾鮰的体表，触须上的味蕾密度尤其高。L-丙氨酸以及某些其他氨基酸是这种动物的味觉刺激物。通过轻轻刮擦触须表面获得的上皮组织经差速离心进行分级分离。制备了一个可沉淀级分（P2），其富含L-[OH]丙氨酸结合活性、质膜标记酶5'-核苷酸酶和线粒体标记琥珀酸细胞色素c还原酶，但不含微粒体标记NADH细胞色素c还原酶。使用微孔滤膜法测量L-[OH]丙氨酸的结合，其中还测定了非特异性结合的校正值。确定了测量结合活性的时间、温度和pH。在最佳pH 7.8时，L-丙氨酸的解离常数KD为4.8×10⁻⁶ M。6℃时的一级解离速率常数为3.8×10⁻⁴ s⁻¹，24℃时为12.1×10⁻⁴ s⁻¹。缔合的二级速率常数在10²和10³ M⁻¹ s⁻¹之间。大量未标记的L-丙氨酸能快速置换结合的L-[³H]丙氨酸，这也证明了结合相互作用的可逆性。嘌呤霉素无作用，证实了这种结合并不代表掺入蛋白质。还测定了其他几种化学刺激氨基酸的结合量。
