Allosteric interactions of the membrane-bound acetylcholine reception: kinetic studies with alpha-bungarotoxin.

The specific and irreversible reaction of a snake neurotoxin, α-bungarotoxin, with the acetylcholine receptor of electroplax membrane preparations from $\text{Electrophorus electricus}$ proceeds by an initial fast phase followed by a slower one. The fraction of the reaction in the fast phase increases with increasing initial toxin concentrations, while the fraction going slowly decreases correspondingly. Both phases are affected by compounds which initiate or inhibit nerve impulse transmissions. The time course of the reaction can be fitted to the sum of two exponentials. The dependence on initial toxin concentration of the two exponentials, and of the fraction of reaction governed by the exponentials, can be fitted to a minimum reaction mechanism which involves at least two types of toxin binding sites with different dissociation constants and ligand-induced conversion of one type of site into the other. The mechanism is consistent with our previous data which showed that activators and inhibitors of membrane electrical potential changes occupy separate sites, only half of which interact. This type of mechanism has been seen in a number of allosteric regulatory enzymes.     

Linkage of Six Genes in Chlamydomonas reinhardtii and the Construction of Linkage Test Strains

Linkage relationships were determined for six genes in Chlamydomonas reinhardtii: pyr, act, can-la, ey, y-1, and nic-7. Four of these were combined with five other genes to form linkage test strains.     

N6-(Δ2-Isopentenyl)adenosine,an inhibitor of cellular transport of uridine and cytidine

N⁶(Δ²-Isopentenyl)adenosine (IPAR) inhibited severely the incorporation of uridine and cytidine into S-180 cells in culture. When IPAR and the nucleosides were simultaneously present in the medium the inhibition was competitive (Kⁱ 3.4 m̈M) and indicated inhibition of transport. However, the inhibition occurred even in the absence of extracellular IPAR if the cells had been preincubated with IPAR. Since 5′-IPAMP was the product which accumulated in large quantities in S-180 cells when incubated with IPAR, the effects of this AMP analog on the intracellular metabolism of uridine had to be considered. No direct correlation between the amount of intracellular IPAMP and the degree of inhibition of uridine utilization was observed and the relative distribution of uridine nucleotides in the acid soluble pool of the cells was unaltered in cells treated with IPAR. Also, IPAMP was not an inhibitor of uridine kinase in a cell free system nor was the activity of this enzyme affected by treatment of cells with IPAR. In addition, a profound inhibition of uridine utilization was also observed in a resistant subline of S-180 cells, which is unable to form IPAMP. These data suggest that IPAMP was not the inhibitory agent. Furthermore, the observation that the inhibition in both sensitive and resistant cells was caused even by a 15-second exposure to 100 m̈M IPAR, followed by rinsing, suggests that IPAR itself is the effective agent. It is concluded that IPAR exerts its inhibitory effect on uridine and cytidine utilization by becoming lodged in the cell membrane and thereby preventing the passage of these nucleosides into the cells. It is also shown that the inhibition of uridine and cytidine utilization by IPAR and by other potent nucleoside uptake inhibitors is unrelated to inhibition of growth or of RNA-synthesis when the cells do not depend on an extracellular source of a nucleoside for growth.     

Renal cyclic GMP and cholinergic mechanisms in erythropoietin production.

Cobalt treatment in rats produced sequential elevations in both renal cyclic GMP concentration and lysosomal enzyme activity in plasma. These effects of cobalt were significantly inhibited by atropine pretreatment, as was cobalt-mediated erythropoietin (ESF) production. Physostigmine, an inhibitor of acetylcholinesterase, potentiated the erythropoietic effect of cobalt. These data are consistent with the hypothesis that the erythropoietic effect of cobalt is associated with a cholinergic mechanism involving cyclic GMP-mediated lysosomal enzyme release. These cholinergic events may precede a previously described cyclic AMP activation of a renal erythropoietic factor.     

Lithium transport across isolated frog skin epithelium

Summary Transepithelial Li⁺ influx was studied in the isolated epithelium from abdominal skin ofRana catesbeiana. With Na⁺-Ringer's as inside medium and Li⁺-Ringer's as outside medium, the Li⁺ influx across the epithelium was 15.6 A/cm². This influx was considerably reduced by removal of either Na⁺ or K⁺ from the inside bath or by the addition of ouabain or amiloride. Epithelial K⁺ or Na⁺ concentration was respectively lower in epithelia bathed in K⁺-free Ringer's or Na⁺-free Ringer's. In conditions of negligible Na⁺ transport, a 20mm Li⁺ gradient (outin) produced across the short-circuited epithelium a Li⁺ influx of 11.8 A/cm² and a mean short-circuit current of 10.2 A/cm². The same Li⁺ gradient in the opposite direction produced a Li⁺ outflux of only 1.9 A/cm². With equal Li⁺ concentration (10.3 and 20.6mm) on both sides of the epithelium, plus Na⁺ in the inside solution only, a stable Li⁺-dependent short-circuit current was observed. Net Li⁺ movement (outin) was also indirectly determined in the presence of an opposing Li⁺ gradient. Although Li⁺ does not substitute for Na⁺ as an activator of the (Na⁺+K⁺)-ATPase from frog skin epithelium, Li⁺ influx appears to be related to Na⁺–K⁺ pump activity. It is proposed that the permeability of the outer barrier to Na⁺ and Li⁺ is regulated by the electrical gradient produced by electrogenic Na⁺–K⁺ pumps located in the membrane of the deeper epithelial cells.     

The architecture of 5S rRNA and its relation to function

An extensive comparative analysis of the available primary sequence data on 5S rRNA has been made. A universal secondary structure is presented for procaryotic 5S rRNA which contains four helical regions. Eucaryotic 5S rRNAs are found to have only three of these helices and thus have a somewhat different architecture. In addition, a highly conserved segment of more than thirty nucleotides is identified in the 5' half of the procaryotic molecule. This segment includes the oligonucleotide -CGAAC- which presumably binds to the t-RNA "common" sequence -GTpsiCG-. Among the eucaryotes, the plants display a procaryotic nature in this region, but no eucaryote has the sequence -CGAAC- in this segment. A functional role for the procaryotic 5S rRNA molecule is discussed in which it is envisioned to undergo conformational change, i.e., coiling and uncoiling of one of the helices, which can result in a cyclic interaction of the 5S rRNA molecule with two t-RNA molecules. A general principle also emerges: the natural rotational motion inherent in coiling and uncoiling of nucleic acid helices can be converted quite simply to linear mechanical motion.