Photophosphorylation and related properties of reaggregated vesicles from spinach Photosystem I particles

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylalation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH⁺₄ are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.     

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.     

Isolation and characterization of a glycerol auxotroph of Rhodopseudomonas capsulata: effect of lipid synthesis on the synthesis of photosynthetic pigments.

A glycerol auxotroph was isolated from Rhodopseudomonas capsulata for use as a system for studying membrane synthesis and function. When the mutant was deprived of glycerol, net phospholipid synthesis ceased immediately and a small amount of free fatty acids accumulated. A turnover of lipid occurred in both deprived and supplemented cultures. Deoxyribonucleic acid and protein synthesis continued for one doubling of cell massand then slowed down in deprived cells. Net ribonucleic acid synthesis slowed down more dramatically. Oxidative phosphorylation activity of membrane preparations from aerobically and semi-anaerobically grown cells appeared unaffected by glycerol deprivation, indicating that simultaneous lipid synthesis is not a requirement for new oxidative phosphorylating activity. In the absence of net phospholipid synthesis, bacteriochlorophyll and carotenoid syntheses were reduced to 30% of the activity of supplemented cultures. Delta-Aminolevulinic acid synthase, the first enzyme on the bacteriochlorophyll pathway that is subject to regulatory control, increased in activity in deprived cultures. Lascelles and Szilagyi (1965) showed an association between phospholipid synthesis and pigment production. They found an increased lipid content associated with pigmented cells. The present results indicate that not only is there an association between lipid and pigment synthesis, but also there is actually a dependence of bacteriochlorophyll synthesis on phospholipid synthesis.     

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.     

Newly synthesized norepinephrine accumulation in the readily releasable pool of a purified adrenergic vesicle fraction

A highly purified fraction of large dense core adrenergic vesicles was studied after isolation from bovine splenic nerve chilled within 10 to 12 minutes post mortem. In a standard medium containing 5 mM each of Mg⁺⁺ and ATP and 6 μM norepinephrine (NE), this vehicle fraction contained NE in a readily releasable and a more stable pool. When vesicle dopamine β-hydroxylase was activated with 1.33 mM ascorbic acid using 6 μM ¹⁴C-dopamine as substrate at 30°C, ¹⁴C-NE was synthesized at a linear rate during the 45 minute incubation. Net accumulation of NE (p < 0.01) and a proportional net retention of newly synthesized ¹⁴C-NE occurred only when the readily releasable pool could still be demonstrated. The halftime for the fast release pool was doubled from 3 to 6 minutes (p < 0.01) with no effect on the slower released, ATP-facilitated uptake pool. Thus, both during axoplasmic transport and induced NE synthesis $\text{in vitro}$, there is evidence that newly synthesized NE preferentially accumulates in the readily releasable pool, a property also characteristic of the physiologically active pool $\text{in vivo}$.     

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.