Protein turnover in photoreceptor cells of isolated Limulus lateral eyes

Abstract— A system was devised for the study of protein turnover in isolated lateral eyes of Limulus . In eyes incubated in a medium containing L-[³H]leucine, radioactivity of the fraction soluble in trichloroacetic acid increased steadily during 8 h. Amino-acid incorporation into proteins was investigated by scintillation counting of trichloroacetic-acid precipitates and by radioautography. At least 89% of the incorporation was inhibited by puromycin and 86% by emetine, an inhibitor of cytoplasmic protein synthesis. Label accumulated preferentially in the rhabdomes, which contain the visual pigment. Radioautography indicated that labeled protein, probably synthesized in the cytoplasm, was distributed in the microvilli of the photoreceptor cells in a diffuse pattern. In contrast to in vivo results, the photoreceptor cells in isolated eyes were labeled to a greater extent in light than in the dark. These different kinetics of labeling were both explained by the hypothesis that light increased the rate of degradation of proteins (particularly opsin) in the visual cells.     

Synthesis and degradation of protein of visual receptor membranes in lateral eyes of Limulus.

—The spatial and temporal distribution of labelled protein in lateral eyes of Limulus polyphemus has been followed after injection of l -[³H]leucine into the pericardial sinus. Labelled protein in the eyes rises for 3 h, for the first hour more rapidly in the cytoplasm but thereafter preferentially in the rhabdomal membranes of the visual receptor cells. In light, the label in the membrane reaches a peak at 3 h whereas in dark the peak is at 6 h. The subsequent decrease in label in light has a half life of about 2 h, but in dark is biphasic with halflives of 2 h and 8 days. These results can be interpreted on the basis that the major labelled protein in the rhabdomal membrane is rhodopsin, the degradation rate of which is enhanced when the membranes are depolarized by light.     

PRECURSOR INCORPORATION INTO CORTICAL PROTEIN DURING FIRST EXPOSURE OF RATS TO LIGHT; CELLULAR LOCALIZATION OF EFFECTS

—The rate of incorporation of [³H]lysine into acid-insoluble material in vivo was determined in neurons and neuropil from the visual cortex of dark-reared rats, littermates exposed to the light for varying lengths of time and normally reared controls. Following onset of light exposure, the elevation of incorporation was confined to the neuronal fraction. On continuous exposure for up to 96 h, the level of incorporation in the neuronal fraction dropped to that of the dark control value. In dark-reared animals, the rate of incorporation in the neuronal fraction was 68 per cent of that in neuropil, in normals it was 150 per cent. On onset of exposure, the ratio in light exposed animals approached the normal level, but on prolonged continuous exposure both light exposed and normal ratios dropped to the dark control value once more. This drop did not occur if the animals were exposed to a 12 h light/dark cycle. These results are taken as suggesting that part of the protein synthesis of the visual cortex is functionally controlled, and that neuronal and neuropil fractions show a metabolic relationship which can be affected by environmental changes. The failure to show a depression of incorporation in prolonged exposure, by comparison with earlier results under somewhat different behavioural conditions, was taken as further evidence for the ‘state-dependence’ of a number of brain biochemical parameters.     

The effect of red and far red light on the desaturation of Fatty acids in barley leaves

Barley (Hordeum vulgare) leaf tissue was either (a) exposed to continuous red light or (b) exposed to red, far red, or red followed by far red light. The fatty acid composition and incorporation of acetate-2-¹⁴C into linolenate were determined. Changes occurred in the fatty acid composition of dark-grown barley leaves regardless of whether the plants were subsequently exposed to red light or whether the tissue remained in the dark. Measurements were also made of the fatty acids of the coleoptile. Red light treatment did not reduce the lag period for the synthesis of linolenate when chlorophyll synthesis was inhibited. It appears that the desaturation process per se in the synthesis of linolenate is not phytochrome-mediated but may appear to be phytochrome mediated if, possibly, galactolipid and chlorophyll syntheses occur concomitantly.     

蓝光对绿豆下胚轴愈伤组织形成和生长过程中蛋白质代谢的影响

蓝光、白光和黑暗对绿豆下胚轴愈伤组织形成和生长过程中蛋白质代谢的影响不同。培养后３～１８ ｄ ,蓝光处理材料的可溶性蛋白质含量明显高于白光处理,更高于黑暗培养的材料。蓝光和白光明显促进３Ｈ亮氨酸掺入蛋白质,而蓝光和白光处理后游离氨基酸含量与黑暗对照相比,下降时间早,幅度大。在培养过程中,蛋白酶活性的变化与游离氨基酸相似。蛋白质合成抑制剂环己酰亚胺（ＣＨＭ） 抑制愈伤组织生长,其中以蓝光最大,白光次之,黑暗最小。在培养基中加入ＣＨＭ 愈早,抑制程度愈大。实验表明,ＣＨＭ 抑制愈伤组织蛋白质合成,也是以蓝光最甚。由此可见,蓝光促进绿豆下胚轴愈伤组织的形成、生长和蛋白质合成。     

Light Effects on the Synthesis of Ribulose-1,5-Bisphosphate Carboxylase in Lemna gibba L. G-3

Placing light-grown Lemna gibba L. G-3 into the dark results in a changed pattern of protein synthesis. Although the amount of protein in the tissue and the over-all rate of incorporation of [³⁵S]methionine into protein does not significantly decline during four days of darkness, the rate of synthesis of three polypeptides declines dramatically. One of these polypeptides is the chlorophyll a/b-binding protein and the two others are the large and small subunits of ribulose-1,5-bisphosphate carboxylase. The changed rates of synthesis of the two subunits were examined after transitions of plants from light to dark and dark to light. The in vivo synthesis of both subunits, while declining to a low level during four days of darkness, increases rapidly upon returning the plants to white light. In addition, the level of poly(A) mRNA coding for the precursor polypeptide of the small subunit of the enzyme falls to a low level in the dark and increases rapidly in response to white light. The increase in translatable mRNA for the small subunit is rapid enough to account for a major part of the increased synthesis of this subunit.     

Development of Stigmatella aurantiaca: effects of light and gene expression.

Stigmatella aurantiaca, a gliding, gram-negative bacterium, exhibits complex developmental changes upon starvation. In the light the cells aggregate and develop multicellular fruiting bodies with stalks and sporangia within 20 h. Between 23 and 27 h, sonication-resistant myxospores are synchronously formed inside the sporangia. On the other hand, in the dark, the cells aggregate and differentiate into myxospores between 13 and 27 h without forming stalks and sporangia. The pattern of protein synthesis during development in the light as well as in the dark was investigated. Three periods of synthesis, characterized by sharp increases and decreases in the rate of isotope incorporation into certain proteins, were distinguished. In the light these periods corresponded approximately to an early stage before the formation of aggregates, a middle period during which aggregates appeared and developed into fruiting bodies, and a late stage that corresponded to the appearance of myxospores. The pattern of protein synthesis in the dark could also be divided into three stages, but the middle stage was considerably shorter than in the light and showed diminished synthesis of certain proteins that were actively synthesized in the light. In particular, the synthesis of one protein was detected only in samples that developed in the light.     

Influence of ethylene on nucleic acid synthesis in etiolated Pisum sativum

Ethylene applied to intact etiolated seedlings of Pisum sativumcv. Alaska inhibits incorporation of ³H-thymidine into DNA insubsequently excised plumular and subapical tissue segmentsbut has no influence on incorporation of ³H-uridine into RNA.The effect on DNA synthesis begins about 2 hr after ethyleneis applied, and intensifies progressively. A similar inhibitionof DNA synthesis occurs when ethylene is applied directly toplumular sections cut from control plants, but not with subapicalsegments under these conditions. Inhibition of DNA synthesisby ethylene is reversed by benzyl adenine in plumular sections.Brief exposure of dark grown seedlings to red light causes asubsequent increase in DNA synthesis in plumular tissue. Thechanges in DNA synthesis in tissues exposed to ethylene, benzyladenine and red light are correlated with the effects of thesetreatments on the mitotic index. (Received March 12, 1973; )     

Identification and Function of Octopamine and Tyramine Conjugates in the Limulus Visual System

Major metabolites of octopamine and tyramine in the Limulus nervous system are identified here as gamma-glutamyl octopamine and gamma-glutamyl tyramine. We show that these conjugates are normal products of amine metabolism in Limulus, and that they are normally present in octopamine-rich Limulus tissues. The synthesis of these conjugates is not restricted to nervous tissue, but the highest activity of gamma-glutamyl amine synthetase was measured in the CNS. Our interest in these molecules stems from our previous observations which showed that they were synthesized and stored in, and released from, the efferent fibers to Limulus eyes which modulate the sensitivity of the eyes to light. Here we provide direct evidence for the release of the conjugates from Limulus eyes in response to depolarization, and that gamma-glutamyl octopamine can increase the sensitivity of the lateral eye to light. Our observations lend support to the hypothesis that gamma-glutamyl octopamine may serve as an intercellular messenger in the Limulus visual system.     

Protein synthesis in isolated etioplasts after light stimulation

Grains of Zea mayswere germinated in the dark for 5 days. Etioplasts were then isolated in the dark and exposed to light in the presence of labelled amino acids and various inhibitors. After periods of incorporation, either in the dark or light, proteins were isolated and then examined with the aid of polyacrylamide gel electrophoresis. The highest specific activity of incorporation was found in the lamellar protein fraction. The use of inhibitors enabled the specific products of etioplast incorporation to be identified on the gels. Analyses of radioactivity in protein bands indicate that the plastid is capable of responding to light in vitro in at least two ways: (1) by an increase in the rate of protein synthesis; and (2) by a reproducible control of the various proteins synthesized either in the dark or light, which resulted in the ‘turning off’ of some proteins synthesized in the dark, and the subsequent initiation of the synthesis of others, in response to light. The results presented inthis study indicate that the plastid in vitro is capable of a rather complex response mechanism when subjected to environmental change, such as light stimulation. This suggests that the plastid is capable of a great degree of autonomy, at least when necessary, and is possibly more independent of nuclear control than heretofore suggested in the literature.     

The effect of light-dark adaptation on the ultrastructure ofLimulus lateral eye retinular cells

Summary The fine structure of retinular cells within lateral eyes ofLimulus polyphemus which had been dark or light adapted for 12 h in vivo was studied via electron microscopy. The ommatidium to ommatidium and retinular cell to retinular cell variability observed in light microscope studies was confirmed. The rhabdomeric microvilli were longer and narrower, the area of contiguous microvillar membranes greater, the endoplasmic reticulum less abundant and the mitochondrial granules (? calcium containing) more numerous in well dark adapted than in well light adapted retinular cells (Figs. 1, 3, 4, 7, 8) and membrane whorls or vacuoles were present in the peripheral cytoplasm of very well light adapted retinular cells (Fig. 6). Phagocytotic vesicles, multivesicular bodies and lysosomes were present in the interrhabdomeral cytoplasm of partially light adapted retinular cells (Figs. 1, 2, 3, 10). The number of retinular cell microvilli in contact with the eccentric cell dendrite was smaller in very well light adapted than in well dark adapted ommatidia (Fig. 9). The possible functional significance of these light-dependent structural changes is discussed.This investigation was supported in part by Grant 2 RO1 EY 00236 National Eye Institute, National Institutes of HealthMember of the SFB 160 of the Deutsche Forschungsgemeinschaft     

Light Exposue Activates Retina Ganglion Cell Lysophosphatidic Acid Acyl Transferase and Phosphatidic Acid Phosphatase by a c-fos-Dependent Mechanism

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in ganglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in vitro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light; no increase was observed in preparations obtained from light-exposed animals treated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.     

Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle.

Phospholipid incorporation of 32P by primary myotube cultures and the tissue activity of sarcolemmal Na+/K(+)-transporting ATPase were studied to determine whether the absence of dystrophin from dystrophic (mdx) muscle would affect membrane lipid synthesis and membrane function. The incorporation of 32P by phospholipid as a ratio with total protein was greater in cultured dystrophic cells compared with control cells. The mdx cells also incorporated more 32P than control cells into phosphatidylethanolamine, which is thought to increase prior to myoblast fusion, and less into phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. There was no difference in total protein content or [3H]leucine or 32P incorporation into the aqueous fraction of dystrophic and control cells, although dystrophic cells incorporated less [35S]methionine into protein than controls. Isolated sarcolemma from mdx skeletal muscle tissue demonstrated a consistently greater specific activity of ouabain-sensitive Na+/K(+)-transporting ATPase than sarcolemmal preparations from control skeletal muscle. These observations suggest that cytoskeletal changes such as dystrophin deficiency may alter the differentiation of membrane composition and function.     

Changes in Average Cell Concentrations of Various Constituents During Synchronous Division of Platymonas striata Butcher (Prasinophyceae)

The changes in the average cell composition of the green algaPlatymonas striata Butcher have been determined during a singlecell cycle in synchronous culture induced by an alternatinglight/dark regime. The cells divided into two at the onset ofdarkness, but remained attached until exposed to light 10 hlater. There appeared to be virtually no net synthesis of constituentsduring the dark period. On exposure to light most components(apart from DNA) showed some continuing net synthesis, but inthe majority of cases there was a short part of this light syntheticperiod in which there was very active net synthesis. The activesynthetic period was frequently immediately prior to the onsetof division. DNA synthesis occurred only in the 6 h precedingdivision. The major period of net protein synthesis occurredwhilst the divided cells were separating, at the commencementof the light period. The other factors studied were RNA, carbohydrate,chlorophyll a, chlorophyll b, carotenoids, phospholipids, acid-solublecompounds, and phosphorus uptake. The results are discussed.     

Fatty acid synthesis in liver and adipose tissue of normal and genetically obese (ob/ob) mice during the 24-hour cycle.

1. The synthesis of long-chain fatty acids de novo was measured in the liver and in regions of adipose tissue in intact normal and genetically obses mice throughout the daily 24h cycle. 2. The total rate of synthesis, as measured by the rate of incorporation of 3H from 3H2O into fatty acid, was highest during the dark period, in liver and adipose tissue of lean or obese mice. 3. The rate of incorporation of 14C from [U-14C]glucose into fatty acid was also followed (in the same mice). The 14C/3H ratios were higher by a factor of 5-20 in parametrial and scapular fat than that in liver. This difference was less marked during the dark period (of maximum fatty acid synthesis). 4. In normal mice, the total rate of fatty acid synthesis in the liver was about twofold greater than that in all adipose tissue regions combined. 5. In obese mice, the rate of fatty acid synthesis was more rapid than in lean mice, in both liver and adipose tissue. Most of the extra lipogenesis occurred in adipose tissue. The extra hepatic fatty acids synthesized in obese mice were located in triglyceride rather than phospholipid. 6. In adipose tissue of normal mice, the rate of fatty acid synthesis was most rapid in the intra-abdominal areas and in brown fat. In obese mice, all regions exhibited rapid rates of fatty acid synthesis. 7. These results shed light on the relative significance of liver and adipose tissue (i.e. the adipose 'organ') in fatty acid synthesis in mice, on the mino importance of glucose in hepatic lipogenesis, and on the alterations in the rate of fatty acid synthesis in genetically obese mice.     

Effect of phaseolotoxin on the synthesis of arginine and protein

Mesophyll cells in discs cut from primary leaves of Phaseolus vulgaris L. were exposed to a concentration of phaseolotoxin that inhibited ornithine carbamoyltransferase (OCTase) measured in an extract of the tissue. This treatment also blocked incorporation of exogenous [¹⁴C] ornithine into protein-arginine of the mesophyll cells. By contrast more than 80% of the [¹⁴C]ornithine supplied to untreated tissue was incorporated into protein-arginine in 565 minutes. Protein synthesis in mesophyll cells was unaffected by phaseolotoxin because treated tissue continued to incorporate [¹⁴C]leucine into protein at the same rate as the untreated control. The phaseolotoxin-treated tissue should therefore remain metabolically competent and this prediction was reinforced by the finding that the rate of photosynthetic O₂ evolution per unit chlorophyll was similar for tissue from the phaseolotoxin-induced chlorosis and from green healthy tissue. Phaseolotoxin also blocked OCTase but not protein synthesis in exponentially growing cell suspension cultures. Phaseolotoxin rapidly inhibited growth of Escherichia coli and this effect was rapidly reversed by arginine. Thus, the toxic effects of phaseolotoxin may be attributed to the inhibition of OCTase which, in turn, blocks arginine synthesis. Protein accumulation is blocked as a consequence, but protein synthesis is unaffected. Chlorosis is due to reduced chlorophyll synthesis and this is presumably a consequence of the lower protein level in affected tissue.     

The rate of protein synthesis in needles of Norway spruce (Picea abies): Light stimulation, regulation through photophosphorylation, stress enhancement

The incorporation of ¹⁴C-leucine into the total-protein fraction of needles of Norway spruce (Picea abies [L.] Karst.) during short time incubation was used as a measure of protein synthesis in the light and in the dark. Light saturation curves, obtained for needles of different ages (new flush and 1 and 2 years old) or at different seasons (summer-winter) followed the Michaelis-Menten algorithm, exhibiting marked differences with regard to light saturation (V_max) and the half-saturation constant (K_{5. 2}). The light saturation curves of ATP level (mg g^?1 fresh weight) and of leucine incorporation into protein (nmol mg^?1 h^?1) matched each other, suggesting that photophosphorylation may be decisive for the rate of protein synthesis in the light. This is confirmed by the action spectrum of leucine incorporation. which resembled an action spectrum of leaf photosynthesis, and also by partial inhibition of protein synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of non-cyclic photophosphorylation. Light stimulated protein synthesis showed pronounced seasonal fluctuations with a summer maximum. Furmigation of 5 years old spruce trees for 3 months with SO₂ in combination with O₃ and/or NO₂ caused a distinct enhancement of the protein synthesis rate in the light and, at a reduced absolute level, also in the dark. A similar result was obtained for 40 to 70 years old spruce stands when healthy and sick trees were compared: the latter being afflicted by the novel type of forest decline, which is characterized by yellowish bronze discolouration of sun-exposed older needles and partial loss of older needle generations (3 to 4 years old). The 1 year old needles of the unhealthy trees showed a markedly increased ¹⁴C-leucine incorporation rate which, in the dark, was even more pronounced than in the light. Stress-physiological mechanisms, which could possibly explain this stimulation, are discussed.     

Leishmania tarentolae: streptomycin and chloramphenicol resistance of promastigotes.

Stable mutant strains of Leishmania tarentolae promastigotes resistant to chloramphenicol (CAP) were isolated by replica-plating techniques. In addition, cell lines stress-adapted to streptomycin and to high culture temperature (33 C) were obtained. Drug resistance was influenced by temperature, culture media, and plating technique. Inhibition of amino acid incorporation into protein occurs in CAP-resistant cells when exposed to 600 μg CAP/ml but this inhibition was 50–80% lower than that found in wild type sensitive cells. The primary site of CAP action appears to be inhibition of protein synthesis. CAP also adversely affected proline oxidation.