When Glu-325 in the lac permease of Escherichia coli is replaced with Ala, lactose/H+ symport is abolished. Thus, the altered permease catalyzes neither uphill lactose accumulation nor efflux. Remarkably, however, permease with Ala-325 catalyzes exchange and counterflow at completely normal rates. Taken together with the results presented in the accompanying paper [Püttner, I. B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry (preceding paper in this issue)], the findings suggest that the His-322 and Glu-325 may be components of a charge-relay system that plays an important role in the coupled translocation of lactose and H+.

The lacY gene of Escherichia coil, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue. Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity. In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a normal complement of permease molecules, as determined by immunoadsorption assays. Interestingly, although substitution of histidine-205 or histidine-322 by arginine results in the loss of ability to catalyze active lactose transport, permease molecules with arginine at residue 322 appear to facilitate downhill lactose movements at high concentrations of the disaccharide. The results provide strong support for the contention that histidine residues in the lac permease play an important role in the coupling between lactose and proton translocation.The lac permease ofEscherichia coli is an intrinsic membrane protein encoded by the lacY gene that mediates symport (co-transport) of ,-galactosides with H' (cf. refs. 1-3 for recent reviews). Thus, in the presence of a H' electrochemical gradient (AAH+-interior negative and/or alkaline), the permease catalyzes accumulation of lactose against a concentration gradient. Conversely, under nonenergized conditions, downhill movement of /3-galactosides along a concentration gradient drives uphill translocation of H' with generation of A/.H+-The lacY gene has been cloned (4) and sequenced (5), and the permease has been purified to homogeneity in a completely functional state (6-14). Since proteoliposomes containing purified permease catalyze all of the reactions typical of (-galactoside with a high degree of specificity, and the approach has been used recently (27,28) to evaluate the role of cysteine-148 in the permease. We have now utilized the technique to systematically replace the four histidine residues in the permease with arginine, and the results are described herein.

The lac permease of Escherichia coli was modified by site-directed mutagenesis such that His-205 or His-322 is replaced with either Asn or Gln. Permease with Asn or Gln in place of His-205 exhibits normal activity, while permease with Asn or Gln in place of His-322 exhibits no activity. The results are consistent with the interpretation that His-205 and His-322 play different roles in lactose/H+ symport, the former involving hydrogen bonding of the imidazole nitrogens and the latter requiring positive charge in the imidazole ring. In addition, it is demonstrated that permease with Arg in place of His-322 does not catalyze efflux, exchange, or counterflow. The observations, in conjunction with those in the accompanying paper [Carrasco, N., Antes, L. M., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry (following paper in this issue)], suggest that His-322 plays an important role in H+ translocation, possibly as a component of a charge-relay system with Glu-325, a neighboring residue in helix 10.

No abstract

A cDNA probe was prepared to investigate the regulation of proenkephalin biosynthesis in the rat. This was necessary because human and bovine proenkephalin cDNA were not sensitive enough for the accurate detection of preproenkephalin mRNA in tissues that contain low copy numbers of this message, such as the adrenal gland. The rat probe was prepared in the following manner. Preproenkephalin mRNA was enriched by sucrose gradient centrifugation of poly(A)-containing mRNA from rat brain and was used as a template for double-stranded cDNA synthesis. The resulting cDNA was inserted into the plasmid pBR322, and recombinant plasmids were used to transform Escherichia coli RR1 cells. A synthetic oligodeoxyribonucleotide (30 bases long) with a sequence that had previously been shown to be identical in bovine and human preproenkephalin cDNA was prepared to screen the clone bank. The plasmid with the longest cDNA insert (about 1200 bases) from the positive clones was isolated, and the sequence of the entire protein coding region was determined. Like the bovine and human gene products, rat preproenkephalin contains four [Met]enkephalin sequences and one copy each of [Leujenkephalin, [Met]enkephalin-Arg6-Gly7-Leu8, and [Met]enkephalin-Arg6-Phe7. Rat preproenkephalin is 80% and 83% homologous to the bovine and human forms, respectively, at the nucleotide level and is 82% homologous to both species at the amino acid level. Rat preproenkephalin contains 269 amino acid residues, making it larger than the human (267 residues) and bovine (263 residues) precursors. The sensitivity for detection of rat preproenkephalin mRNA with the rat cDNA was several times greater than with the corresponding cDNAs from bovine and human sources.Since the discovery of the enkephalin pentapeptides nearly a decade ago (1), much has been learned about the biosynthesis of [Met]-and [Leu]enkephalin. In addition to the isolation and sequence determination of many enkephalin-containing peptides from bovine adrenal medulla (see ref. 2 for review), the mRNA from bovine adrenal medulla and human pheochromocytoma, which encodes the precursor protein preproenkephalin, has been cloned and sequenced (3-5).The exact function of the enkephalin-containing peptides is still not known even after many years of intensive investigation. It is likely that knowledge concerning the mechanism of action of these peptides and the regulation of their metabolism will come from research on the rat, the animal that has provided most of our knowledge concerning opiate pharmacology. Studies in our laboratory on the stimulation of proenkephalin synthesis in the rat adrenal gland following denervation (6-8) have been hampered because human and bovine cDNA were found not to be sufficiently sensitive as probes for detecting preproenkephalin mRNA in this rat tissue, which has a low copy number of the message under normal conditions. Rat preproenkephalin cDNA was cloned to provide a homologous and, quite likely, a more sensitive probe for monitoring transcriptional changes in rat ...