花生不同生育期小叶脉转移细胞形态变化和ATP酶活性定位的研究

迄今已在上千种高等植物中看到具有胞壁内突生长的转移细胞,认为它是一种输送溶质的细胞,在源库二端行使光合产物的垭距离运输。转移细胞质膜上具有较强的ATP酶活性,在发育成熟的转移细胞质膜上ATP酶活     

渗透胁迫引起玉米根冠处胞间连丝的ATP酶活性下降

利用光镜和电镜研究了短期渗透胁迫下玉米(Zea mays L.)根冠区超微结构和ATP酶活性变化。经历了质壁分离后,在根冠细胞仍然可以观察到许多从“撤退”的周质出发向细胞壁辐射的纤丝。利用ATP酶活性产物(磷酸铅)在原生质膜和细胞壁处沉积的特点,发现这些纤丝是质膜围绕原生质而形成的管状结构;在质壁分离过程中,这些纤丝依然与胞间连丝相连。与对照(未胁迫)相比,受渗透胁迫细胞胞间连丝处ATP酶活性明显下降。能量代谢在局部区段的抑制会影响胞间连丝的生理活性,可能包括胞间连丝的扩散调节能力和分子扩散上限的改变。     

ISOLATION AND PROPERTIES OF THE PLASMA MEMBRANE OF KB CELLS

Plasma membranes from KB cells were isolated by the method of latex bead ingestion and were compared with those obtained by the ZnCl₂ method. Optimal conditions for bead uptake and the isolation procedure employing discontinuous sucrose gradient centrifugation are described. All steps of preparative procedure were monitored by electron microscopy and specific enzyme activities. The plasma membrane fraction obtained by both methods is characterized by the presence of the Na⁺ + K⁺-activated ATPase and 5'-nucleotidase, and contains NADPH-cytochrome c reductase and cytochrome b₅. The latter two enzymes are also present in lower concentrations in the microsomal fraction. Unlike microsomes which are devoid of the Na⁺ + K⁺-activated ATPase and which contain only traces of 5'-nucleotidase activity, the plasma membrane fraction contains only trace amounts of the rotenone-insensitive NADH-cytochrome c reductase but no cytochrome P-450, both of which are mainly microsomal components. Morphologically the plasma membrane fraction isolated by the latex bead method is composed of vesicles of 0.1–0.3 µm in diameter. On the basis of the biochemical and morphological criteria presented, it is concluded that the plasma membrane fraction isolated by the above methods are of high degree of purity.     

百合花粉母细胞间细胞融合期间腺苷三磷酸酶活性的细胞化学定位及其与染色质胞间转移的关系

用标准的磷酸铅沉淀的细胞化学方法,对百合花粉母细胞间染色质穿壁运动期间及其前后三个时期中的腺苷三磷酸酶(ATP 酶)活性进行了超微结构的定位。结果表明:(1)在穿壁前,ATP 酶活性主要定位于质膜、胞间连丝及细胞间隙;在内质网、高尔基体、质体和某些局部的基质(groundplasm)中,也表现有 ATP 酶活性反应的产物;但在染色质和核仁中,一般都没有这种反应。(2)在穿壁时,染色质从一个细胞穿壁转移到另一个相邻细胞,同时看到染色质和核仁内出现密集的 ATP 酶活性反应产物;在内质网和高尔基体的腔内以及质体的片层上也产生明显的 ATP 酶活性反应;而在质膜、胞间连丝及细胞间隙内 ATP 酶活性明显降低,甚至看不到明显的活性反应。(3)在穿壁后,质膜及细胞间隙中又产生明显的 ATP 酶活性反应产物,但核内染色质上的 ATP 酶活性则显著降低,而核仁内则仍有较高的活性。同前二个时期一样,内质网、高尔基体和质体上的 ATP 酶仍表现明显的活性反应。最后讨论了三个不同发育时期 ATP 酶活性及其分布部位的改变与染色质胞间转移的关系。     

EFFECT OF CRY1A δ-ENDOTOXINS OF BACILLUS THURINGIENSIS ON Mg+-ATPASE IN THE COLUMNAR CELLS OF BOMBYX MORI MIDGUT

Abstract  The effects of Cry1A δendotoxins from Bacillus thringiensis subsp. kurstaki HD-1 on the activity of M²+-dependent ATPase in the columnar cells (CC) of Sombyx mori were examined. Cytochemical localization showed a strong M$ ' -ATPase activity on the apical brush border of CC and even on the peritrophic membrane, but not on the goblet cells except slight activity on their interdigitated valves. Although overdose toxins were imposed to the larvae and resulted in serious cellular pathological changes in the treated midgut in vim , no obvious reduction of the M²+-ATPase activity on the CC membrane was observed. This may exclude the M²+ ATPase on the CC brush border membrane to be the potential natural channel modulated by B-endotoxins of B. thuringiensis or a target of the toxin during larval intoxication.     

THE EFFECTS OF OUABAIN AND THE ACTIVATION OF NEURAL MEMBRANE ATPase BY BIOGENIC AMINES

Abstract— The effects of 10⁻⁵ m -noradrenaline (NA), 5-hydroxytryptamine (5-HT) and dopamine (DA) on the activities of Na⁺-K⁺ ATPase (EC 3.6.1.3) were studied in synaptic membranes from 6 regions of the rabbit brain. NA and 5-HT stimulated the synaptic membrane Na⁺-K⁺ ATPase from the cerebrum, but none of the amines influenced the activity of this enzyme in the other brain regions. The Na⁺-K⁺ ATPase activity of the cerebral synaptic membrane isolated at the 0.8/0.9 m & 0.9/1.0 m interphase of a sucrose density gradient was increased two-fold by 10⁻⁵ m -NA and 5-HT. The Na⁺-K ⁺ ATPase recovered at the 1.0/1.2 m interphase was not influenced by NA, DA or 5-HT. NA, DA and 5-HT did not activate the Mg ATPase of synaptic membranes from any of the 6 brain regions or whole brain synaptic vesicles. The cortex synaptic membrane (Na⁺-K⁺) ATPase is postulated to have a direct role in the uptake of the biogenic amines. An indirect role is proposed for this enzyme in amine uptake into brain stem.     

甘蔗叶不同部位ATP酶活性细胞化学定位

甘蔗叶片,叶鞘和肥厚带韧皮部 ATP 酶活性定位于筛管、伴胞的质膜、内质网和某些伴胞细胞基质、小囊泡和发育成熟的液泡上;叶片韧皮部薄壁细胞、厚壁细胞和厚壁通道细胞质膜及小囊泡中亦显示有 ATP 水解产物;维管束鞘细咆与厚壁细胞或厚壁通道细胞所构成的细胞间隙上也存在有 ATP 酶活性反应产物沉淀。甘蔗叶片大、中、小三种维管束,从小维管束到大维管束,面向细胞间隙的细胞表面上的 ATP 酶活性逐渐增强,而维管束鞘细胞质膜上的 ATP 酶活性则趋于减弱;同一维管束内则以韧皮部细胞的 ATP 酶活性最强。维管束鞘细胞与叶肉细胞之间存在很多的胞间连丝,并表现出高的 ATP 酶活性。讨论了 ATP 酶活性的分布状态与叶肉细胞的光合产物向韧皮部运输的关系。     

THE EFFECTS OF LITHIUM ON ATPase ACTIVITY IN SUBCELLULAR FRACTIONS FROM RAT BRAIN

Abstract— The effects of lithium chloride in vitro and in vivo were investigated on Na-K ATPase and Mg ATPase activities in synaptic plasma membrane, mitochondrial and synaptic vesicle fractions prepared from rat brain. In vitro , lithium chloride (10⁻³-10⁻⁸ m ) had no effect on ATPase activity in any of the fractions studied. Lithium chloride given chronically by i.p. injection (30 mg/rat/day) for 9 days had little effect on synaptic plasma membrane ATPases. Dietary administration of lithium chloride (60 mmol/kg food) produced a small but significant increase in synaptic plasma membrane Mg ATPase activity after 3 weeks administration and mitochondrial Mg ATPase activity after 1 week. There was no effect on synaptic plasma membrane Na-K ATPase activity. Salt supplementation reduced the toxic effects of lithium administration and it is suggested that toxicity may account for some of the previously reported changes in synaptic membrane ATPases produced by lithium.     

OBSERVATIONS ON THE MODE OF RELEASE OF HERPES VIRUS FROM INFECTED HELA CELLS

The development of a well adapted strain of herpes virus has been studied in HeLa cells using thin sectioning techniques for electron microscopy. Particular attention was directed to events in the cytoplasm and certain new features were observed. Profuse immature particles with a nucleoid and single limiting membrane were present in the nuclei of infected cells, often in crystalline array; morphologically indistinguishable immature particles were also found very frequently in the cytoplasm. Cells with such particles were intact and well preserved, and contained smooth vacuoles apparently derived from the Golgi component of the endoplasmic reticulum. The cytoplasmic particles escaped from the cells by bulging out as buds through the cell membrane or through that of the cytoplasmic vacuoles until they were attached only by a pedicle and then became free. During this process the particles were gradually enclosed by the membrane through which they passed and carried a coat of it with them as they matured. After permanganate fixation the triple-layered structure of the cell membrane and vacuolar membranes was evident and was identical with that of the outer coat of the mature virus. These findings are discussed both in relation to different types of virus structure and to function in the endoplasmic reticulum and cell membrane.     

EFFECT OF SOLUTION COMPOSITION AND PROTEOLYSIS ON THE CONFORMATION OF AXONEMAL COMPONENTS

The effect of solution composition and enzymic proteolysis on axonemes prepared from the sperm of sea urchins, Tripneustes gratilla, has been investigated. Aliquots of axonemes, prepared by treatment of sperm with Triton X-100 and differential centrifugation, were transferred to solutions of different composition with and without intervening tryptic proteolysis, and the particle conformations observed by dark-field and electron microscopy. In most solutions particles in partially digested preparations underwent conformational transformations to coiled or helix-like forms. Proteolysis was accompanied by an increase in the ATPase activity of the digest: by centrifuging down the insoluble digestion products it was shown that digestion resulted in the appearance of ATPase activity in the soluble phase with a concomitant decrease in ATPase activity in the pellet fraction. Gel electrophoresis showed this corresponded to the appearance of dynein in the supernatant and a decrease in dynein associated with the insoluble fraction. Supernatant dynein had a greater specific ATPase activity than dynein extracted from axonemes. Observations on specimens prepared for electron microscopy by thin sectioning allowed a rough correlation to be made between the dark-field observations, chemical analyses, and morphological alterations attendant with the proteolysis and solution conditions. It is concluded that in the intact axoneme the doublet tubules are under considerable tension and that proteolytic destruction of physical restraining elements allows spontaneous conformational alterations of the digestion products. In addition, proteolysis increases the specific ATPase activity of dynein and removes a portion of it from the axonemal structure.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.     

Immuno-Electron Microscopy of the Morphogenesis of Mumps Virus

The fine structure of mumps virus-infected chick embryo fibroblastic cells was examined sequentially after viral inoculation. Intracytoplasmic nucleoprotein strands, similar to those described for parainfluenza viruses, were detectable in small aggregates between 36 and 48 hr. The peripheral strands of this viral component lie beneath and along an antigenically altered bulging portion of the cell membrane. The outermost strands are consistently parallel to the differentiated segment of the plasma membrane, which is invariably associated with surface projections. As has been found with other myxoviruses, mumps virus replicates by budding from the cell surface. The virus particle, roughly spherical in shape, has a size ranging from 1,000 to 8,000 A. Filamentous forms are rarely observed in the present culture system. Ferritin-conjugated antibody specifically labels the cytoplasmic nucleoprotein, the modified cell membrane, and the virus particle. Intranuclear inclusions of low electron density and morphologically different from those described in measles virus-infected HeLa and amnion cells were observed in the nucleus of several infected cells. Immuno-electron microscopic observations suggest that the nucleoprotein synthesis rate exceeds that of cell membrane differentiation into viral envelope. This difference results in the accumulation of viral nucleoprotein in large intracytoplasmic masses which can be demonstrated by electron microscopy.     

PREPARATION AND CHARACTERIZATION OF A PLASMA MEMBRANE FRACTION FROM ISOLATED FAT CELLS

A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.     

枸杞体细胞胚发生中Ca2+和ATPase的超微结构定位研究

研究2,4-D诱导枸杞体细胞胚发生中的作用及其与Ca~(2+)含量和ATPase活性时空分布动态之间的关系,以探讨2,4-D诱导植物体细胞胚发生的作用机理。采用超微细胞化学定位的方法,跟踪分析了体细胞胚发生与发育的不同时期,Ca~(2+)和ATPase活性的时空分布动态。结果表明:2,4-D是诱导离体培养的枸杞体细胞进入胚胎状态的关键激素。在含有2,4-D和不含2,4-D的培养条件下,分别诱导枸杞体细胞脱分化后,再转入除去2,4-D的MS培养基上,进行分化培养,结果前者可分化形成体细胞胚,因而称为胚性愈伤组织。后者在相同条件却不能分化形成胚,故称为非胚性愈伤组织。在2,4-D诱导枸杞的胚性愈伤组织中,胚性细胞分化早期的细胞间隙和细胞壁上均有Ca~(2+)沉淀。随着胚性细胞的分化、分裂和多细胞原胚形成,这时Ca~(2+)在细胞内的分布主要集中在细胞膜和液泡膜上;球形胚期在细胞核中Ca~(2+)呈弥散性分布。在此过程中,ATPase活性时空分布与Ca~(2+)的定位变化具有高度一致性,仅仅稍滞后于Ca~(2+)出现的时间。而在胚性细胞分化早期,ATPase活性同样位于质膜上,随后在液泡和细胞核都可见ATPase活性分布。而在非胚性愈伤组织中,则未见Ca~(2+)和ATPase活性呈时空动态分布,而且随着非胚性细胞的液泡化,无论是Ca~(2+)含量,还是ATPase活性都呈逐渐降低的趋势。表明Ca~(2+)和ATPase活性变化与2,4-D诱导的胚性细胞分化和发育密切相关。并由此推测,Ca~(2+)和ATPase的时空分布对胚性细胞分化中的信息传递和调控相关基因表达起着关键性作用。     

RELATION OF PROTEIN SYNTHESIS TO THE DIVISION CYCLE IN MAMMALIAN CELL CULTURES

The distribution of the reaction product of a staining method for adenosine triphosphatase (ATPase) in rat small intestine, kidney, and liver was studied with electron microscopy. Several procedures were tried but the best results were obtained from tissue that had been quenched in liquid nitrogen, sectioned at 25 µ in a cryostat, fixed for 30 to 90 minutes at 4°C in formalin-sucrose buffered to pH 7.2, incubated with substrate, and then osmicated and prepared for electron microscopy in the usual way. This procedure enabled the localization of mitochondrial ATPase to be studied. In tissue fixed in small blocks in osmium tetroxide for 3 minutes prior to incubation with substrate, good preservation was noted, and the reaction product for ATPase was localized on the cell membrane and nuclei. The reaction product was present in abundant amount in the nuclei, and particularly within nucleoli, of all tissues studied. Because the histochemical localization of nuclear enzymes poses numerous interpretative problems at the present time, the significance of this nuclear localization is uncertain. Cell (plasma) membranes were the site of localization, especially at areas where it has been proposed that active transport mechanisms may occur, namely, on the microvilli of intestinal epithelium, endothelial lining of capillaries, glomerular epithelial cell membranes, basal infoldings of the cell membrane of renal tubules, on the microvilli of bile canaliculi, and on the microvilli of proximal convoluted tubular epithelial cells. ATPase localization on the cristae mitochondriales was also demonstrated.     

A STUDY OF THE FINE STRUCTURE OF THE EPIDERMIS OF RANA PIPIENS

The epidermis of adult Rana pipiens has been studied by electron microscopy and histological and histochemical methods. It was found that the epidermis is engaged in the production of both keratin and mucus. The basal cells are mainly filled with tonofilaments, whereas the cells located in the mid-portion of the epidermis contain both tonofilaments and mucous granules. Golgi vesicles and endoplasmic reticulum are found in relative abundance in the mucus-producing cells and seem to be involved in the production of mucous granules. The mucus seen was partly retained within the cells and partly secreted into the intercellular spaces. The outermost keratinized cells contain mainly filaments and a few remnants of cell constituents.     

Association of the Transformation-Specific Protein pp60src with the Membrane of an Avian Sarcoma Virus

The transformation-specific protein pp60(src) coded for by avian sarcoma viruses and its associated protein kinase activity is present in virus particles of Rous sarcoma virus, Schmidt-Ruppin strain, subgroup D. Quantitative comparison of the immunoglobulin G-phosphorylating activity in Schmidt-Ruppin D virus and Schmidt-Ruppin D virus-transformed fibroblasts indicated that there was two- to fourfold less activity in the virus particles. Disruption of virus particles with nonionic detergent demonstrated that the protein kinase activity fractionated together with the viral membrane protein gp85. Therefore, viral membranes were isolated by floating detergent-disrupted virus through a discontinuous sucrose density gradient. At a characteristic density corresponding to 26% sucrose, viral membranes were identified by the radioactively labeled viral glycoprotein and furthermore by the membrane marker enzyme Na(+)-K(+)-stimulated, Mg(2+)-activated ATPase and were visualized by electron microscopy. Contamination by cell membranes could be ruled out, since (i) the virus preparation was free of cell membrane contaminants as judged from electron microscopy, (ii) floating of intact virus did not release membraneous material, and (iii) virus-free tissue culture fluid from Schmidt-Ruppin D virus-transformed nonproducer cells (which potentially contain cell membranes) did not contribute any immunoglobulin G-phosphorylating activity after mixing with nontransforming virus and pelleting it. Both pp60(src) and the protein kinase activity were found to be associated with the viral membrane. Solubilization of virus by detergent released two phosphoproteins, with molecular weights of 42,000 and 45,000 which reacted with sera specific for pp60(src) and revealed protein kinase activity but which were not membrane bound and may have represented degradation products of pp60(src). Surface iodination of intact virus particles (harvested at 3-h intervals) did not result in radioactive labeling of pp60(src), whereas collection at 24-h intervals allowed iodination of pp60(src). In contrast to the viral glycoprotein gp85, the iodinated virion-associated pp60(src) was insensitive to mild proteolytic treatment. Binding to tumorbearing-rabbit serum, immunoglobulin G phosphorylation, and endogenous phosphorylation of 60,000-, 45,000-and 42,000-dalton proteins required lysed virus and were not possible with intact virus. These results indicated that pp60(src) was embedded within the viral membrane. Membrane proteins phosphorylated in vitro were analyzed for their phosphoamino acid composition. Eight polypeptides exhibited phosphorylation in tyrosine and were absent in nontransforming viral controls.     

Adenosinetriphosphatase and 5-Nucleotidase Activities in the Plasma Membrane of Liver Cells as Revealed by Electron Microscopy

The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.     

The ultrastructure and ATPase nature of polar membrane in Campylobacter jejuni

Polar membrane in Campylobacter jejuni has been visualized on membrane vesicles. It was composed of doughnut-shaped particles 5-6 nm in diameter, with stalks, arranged in a hexagonal array. This structure was stabilized on the membrane by a high ionic strength buffer in the presence of 2-mercaptoethanol. Histochemical staining indicated localized ATPase activity at the poles of the cells. An ATPase with distinctive properties has been isolated and purified from this organism; it gives a specific activity of approximately 0.3 units/mg of protein. Electron microscopy showed doughnut-shaped particles 5-6 nm in diameter. Nondissociating and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed, respectively, a single band with ATPase activity and a molecular weight of ca. 75,000 Da. The enzyme was cold labile and activity was abolished by trypsin. Dicyclohexylcarbodiimide inhibited the membrane-bound form of the enzyme, but did not inhibit the soluble form. Oligomycin had no inhibitory activity on either form of the enzyme. The enzyme specifically hydrolysed ATP, but other nucleotide substrates were not degraded. The enzyme was activated by Mg2+ and inhibited by Ca2+, whereas other ions had no effect on activity. Antibodies prepared to this enzyme bound to the polar regions of whole cells as shown by protein A - colloidal gold immunoelectron microscopy. The antibodies to this ATPase cross reacted (shown by Western blotting) with four proteins from a whole-cell extract of this organism, two proteins in Aquaspirillum serpens MW5, and three proteins from Escherichia coli K12. They did not cross-react with any proteins from Spirillum volutans, Methanococcus voltae, Vibrio cholerae, or rat liver mitochondria. Antibodies raised against the F1-ATPase of E. coli K12 cross reacted with six proteins in a whole-cell extract of this organism, and one protein species in each of the whole-cell extracts of V. cholera, A. serpens MW5, S. volutans, and rat liver mitochondria. These antibodies did not recognize any whole cell proteins from either C. jejuni or M. voltae. These results along with the ATPase activity localized by histochemical staining suggest that polar membrane is an assembly of ATPase molecules at the poles of the cell and that the ATPase isolated from C. jejuni is serologically and structurally unusual.