Showing papers on "Cellular compartment published in 1982"

Characteristics of Membrane-Bound Lectin in Developing Phaseolus vulgaris Cotyledons.

Abstract: Cotyledons of developing Phaseolus vulgaris L. cv Greensleeves seeds were labeled for 2 to 3 hours with 3H-amino acids, and newly synthesized phytohemagglutinin (PHA) was isolated by affinity chromatography with thyroglobulin-Sepharose. The presence of 1% Tween in the homogenate increased the yield of radioactive PHA by 50 to 100%. Isopycnic sucrose gradients were used to show that this detergent-released PHA was associated with the endoplasmic reticulum (ER), and pulse-chase experiments showed that the half-life of the PHA in the ER was 90 to 120 minutes. Since PHA is transiently associated with the ER and accumulates in protein bodies, we postulate that this rapidly turning over pool of PHA in the ER represents protein en route to the protein bodies. The PHA in the ER has the same sedimentation constant as mature PHA and is capable of agglutinating red blood cells. The observations substantiate earlier claims that plant cells contain membrane-bound lectins. However, they also indicate that these lectins are not necessarily functional components of the membranes with which they are associated, but may represent transport pools of lectin normally localized in other cellular compartments.

Sequestration of tetanus toxin in developing neuronal cell cultures.

Abstract: Cultivation of pure populations of cerebral cells and the unambiguous identification of the neuronal and glial elements is a prerequisite for studying biochemical parameters associated with the developing nervous system under in vitro controlled conditions. We now report on sequestration of tetanus toxin, an exclusive marker for the neuronal cell surface, into a cellular compartment which prevents its release by neuraminidase. This compartment is associated with cellular elements remaining after detergent extraction of neuronal cells grown in monolayer cultures. Toxin sequestration into this compartment is preceded by a neuraminidase-sensitive receptor mediated binding process which can be correlated with the appearance of lipid-bound sialic acid in developing neuronal cultures.

Studies on calcium efflux in the yeast Saccharomyces cerevisiae.

Abstract: The properties of the 45Ca efflux systems in Saccharomyces cerevisiae were investigated in yeast cells grown overnight in medium containing 45Ca. Efflux was measured in medium containing glucose and Tris-Hepes buffer adjusted to the required pH. In the absence of permeable cations in the medium, at pH 5.2, 20% of the cellular Ca was extruded from the cells during the first 2 h. There was no further decrease in the amount of cellular Ca during an additional 24 h of incubation. The initial rate of Ca efflux was markedly reduced with the increase in the pH of the medium. On the other hand the efflux during the second phase (2-24 h) increased with the increase in medium-pH up to pH 7.5. It is suggested that the initial rapid phase of Ca efflux, in the absence of permeable cations, represents transport across the plasma membrane and is mediated via a Ca2+/H+ antiport. The second phase represents the release of Ca sequestered in some cellular organelles, probably the vacuoles, and is mediated via a different mechanism. Addition of Ca or Mg to the medium markedly stimulated the rate of Ca efflux from both cellular compartments. At the same time a predominant influx of divalent cations was observed. This exchange between intracellular Ca and extracellular divalent cations was not affected by the pH of the medium between pH 5.2 and 7.0. Both processes, Ca efflux and Ca-Mg exchange, required cellular energy; they were almost completely inhibited in the absence of glucose and the presence of antimycin A, a respiratory inhibitor.

The membrane association among the rough-and smooth-surfaced endoplasmic reticulum and Golgi apparatus in rat hepatocytes at certain circadian stages.

Abstract: UCHIYAMA, Y. The Membrane Association among the Rough- and Smooth-Surfaced Endoplasmic Reticulum and Golgi Apparatus in Rat Hepatocytes at Certain Circadian Stages. Tohoku J. exp. Med., 1982, 136 (3), 299-302-Subcellular structures of the cytoplasmic components in perilobular hepatocytes were examined at 0500h and 1700h in 6 adult male Wistar rats per time period. At both time points, the endoplasmic reticulum (ER) having smooth and rough parts could clearly be observed. Corresponding to the decrease and increase of the smooth-surfaced endoplasmic reticulum (sER) during the 24-hr span, it is thought that the membrane transformation from the sER to rough-surfaced endoplasmic reticulum (rER) at 0500h and from the rER to sER at 1700h occurs in the flowing-off of membranes which is accompanied by attachment or detachment of ribosomes. Furthermore, at these time points, the direct continuity of membranes between the rER and Golgi apparatus is discerned. From these findings, it may be elicited that the membrane association among the cytoplasmic components takes an important role in the reorganization of the cytoplasmic components in hepatocytes during the 24-hr span.

Estrone Receptor Formation During the Processing of Estradiol-Receptor Complex in MCF-7 Cells

Abstract: Human breast cancer cells (MCF-7, maintained in long-term culture) contain separate estrogen receptors specific for either 17 beta-estradiol or estrone. Utilizing optimum conditions for the protamine sulfate assay, it has been possible to demonstrate both receptors in the 0.6 M KCl extract of nuclei and in the cytosol. Similarly, in the exchange assay, high-affinity low-capacity binding sites for 17 beta-estradiol and estrone have been found in the salt-extracted nuclear residue. Dissociation constants and binding capacities were determined for either receptor in the absence of the other [e.g., estrone receptor (E1R) in the cytosol or nuclear residue from 17 beta-estradiol-stimulated cells] or, when both receptors were present, a saturating amount of the other estrogen (unlabeled) was added to the assay mixture (e.g., the salt-extractable nuclear receptors). Specificity was demonstrated by the inability of estrone to compete with 17 beta-[2,4,6,7-3H]estradiol for the 17 beta-estradiol receptor (E2R) at molar excesses less than 10-fold. Likewise, there was no inhibition of [6,7-3H]estrone binding to its receptor by molar excesses of 17 beta-estradiol below 100-fold. Other steroid hormones were very weak competitors of [6,7-3H]estrone, even at 1000-fold molar excesses. The quantitative relationships of these two estrogen receptors were shown to fluctuate in the various cellular compartments following incubation (37 degrees) of MCF-7 cells with 10(-8) M 17 beta-estradiol. This level of 17 beta-estradiol elicited the translocation of all detectable cytosolic E2R to the nucleus, where, after an incubation of 1 hr, the salt-resistant 17 beta-estradiol disappeared and 40% of the extractable 17 beta-estradiol-binding capacity was lost (processed). Simultaneously, the E1R which remained in the nuclear residue appeared in the nuclear extract, and ultimately this receptor accumulated in the cytosol. The estrone-binding capacity (0.78 pmol/mg DNA) which appeared following the processing of E2R nearly equalled the loss of 17 beta-estradiol binding sites per cell (0.85 pmol/mg DNA). Concentrations of 17 beta-estradiol which elicited the greatest processing of E2R in these incubations brought about the appearance of maximum levels of E1R in MCF-7 cells. Considering these results in the light of data previously reported from this laboratory concerning the metabolic and ligand fate of 17 beta-[3H]estradiol in MCF-7 cells, processing would appear to involve the formation of E1R in the salt-resistant nuclear compartment followed by the accumulation of E1R in the cytosol.

Subcellular fractionation of bone marrow-derived macrophages: localization of phospholipase A1 and A2 and acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase.

Abstract: Analysis of the subcellular distribution of lipid-metabolizing enzymes was carried out in bone marrow-derived macrophages with special respect to a comparison of the subcellular localisation of phospholipase A1 and A2 and to acyl-CoA:1-1-acylglycero-3-phosphorylcholine-0-acetyltransferase. After cell disruption differential centrifugation was followed by additional sucrose gradient purification of three main fractions. Satisfactory enrichment factors were obtained by this method for the following marker enzymes. The plasma-membrane enzyme alkaline phosphodiesterase I was enriched up to 25-fold and the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase was enriched up to 30-fold. The marker enzyme for the endoplasmic reticulum, NADPH-cytochrome c reductase showed a similar enrichment and distribution as the acyltransferase. Therefore it was concluded that the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase of bone marrow-derived macrophages is mainly located in the endoplasmic reticulum. Phospholipase A1 and A2 occurred in a high proportion together with the lysosomal marker enzyme N-acetyl-beta-glucosaminidase in the soluble supernatant and in the gradient fractions. In the endoplasmic reticulum phospholipase A2 occurred only in trace activities whereas phospholipase A1 was maximally enriched in this subcellular fraction. No subcellular fraction could be obtained where phospholipase A2 was enriched exclusively. However, it can be concluded that the two enzymes which are responsible for the balance of fatty acid liberation and re-acylation are located in two different cellular compartments. Furthermore it can be claimed that in the cell there has to exist an exchange of substrates and products between these compartments to achieve a complete metabolic cycle of the de- and re-acylation reaction of phospholipids in bone marrow-derived macrophages.

A study of calcium compartments in rat brain cortex thin slices: effects of veratridine, lithium and of a mitochondrial uncoupler.

Abstract: The efflux kinetics of45Ca from rat brain cortex thin slices previously equilibrated with it, was studied in a superfusion system. Two first order kinetic components of efflux from the tissue were found: k2=0.0667 min−1, that was unchanged by lowering the temperature from 37°C to 15°, and k3=0.0167 min−1 at 37°C, that was reduced to 0.0897 min−1 at 15°C. This suggests that k2 represents efflux from the extracellular space, and k3 that from the cellular compartment. Addition of the mitochondrial uncoupler carbonyl cyanide,m-chlorophenylhydrazone (CCCP) (10−5 M) increased the efflux fractional rate constant of45Ca by 35%, while no change in efflux was induced by 10 mM caffeine. Veratridine (10−5M) drastically reduced45Ca efflux if superfusion was with physiological salt solution (150 mM sodium present), but not if 50 mM lithium replaced an equivalent amount of sodium in the superfusion fluid. This lithium-containing solution did not affect45Ca efflux in the absence of veratridine. These results indicate that mitochondria accumulate only a minor fraction of intracellular45Ca; that45Ca possibly turns over very rapidly in the endoplasmic reticulum, and that most of45Ca is present in a different, non-mitochondrial, non endoplasmic reticular compartment, the nature of which can be only conjectured.

Deoxyribonucleic Acid Synthesis in Isolated Chloroplasts and Chloroplast Extracts of Maize

Abstract: Isolated chloroplasts are capable of synthesizing chloroplast DNA in the presence of Mg2+ and deoxynucleoside triphosphates. The in vitro reaction proceeds for at least 60 min and is inhibited by KCl and N-ethylmaleimide. Stretches of several hundred nucleotides in length are synthesized within an hour. Little or no inhibition is shown by aphidicolin (an inhibitor of eukaryotic DNA polymerase a), dideoxythymidine triphosphate (an inhibitor of eukaryotic DNA polymerases P and y), nalidixic acid, or rifampicin. Ethidium bromide is a moderate inhibitor of DNA synthesis in the isolated chloro- Eukaryotic cells are characterized by compartmentalization of those cellular components which contain their own genetic apparatus. In animal cells, the nuclear compartment has a genomic organization and DNA replication machinery which is apparently quite different from that in the other cellular compartment, the mitochondria (Kasamatsu et al., 1974). Higher plant cells contain three, rather than two, cellular compartments (Olson, 198 1) since they contain another au-