Cytoplasmic alkalinization

Na+,H+ antiporters (NHE) occur in synaptosomes, glia and neuroblastoma cells [60] (Fig. 5-8B). They are relatively inactive at neutral pH but with a decrease in intracellular pH they produce an efflux of protons at the expense of the Na+ gradient. The NHE transport stoichiometry is 1 1. Activation by an internal pH decrement apparently results from protonation of a cytoplasmic site, which allosterically increases the affinity of the proton ionophoric site. In some cells, the NHE is under additional control by receptor mechanisms. Several growth factors and hormones produce transient cytoplasmic alkalinization, probably by mediating a protein kinase... 

Shen SS, Buck WR. 1990. A synthetic peptide of the pseudosubstrate domain of protein kinase C blocks cytoplasmic alkalinization during activation of the sea urchin egg. Dev Biol 140(2) 272-280. 

Extraction of proteia requires breaking the cell wall to release the cytoplasmic contents. This can be achieved by high speed ball or coUoid mills or by high pressure (50—60 Mpa) extmsion. Proteia is extracted by alkaline treatment followed by precipitation after enzymatic hydrolysis of nucleic acids. Although the proteia can be spun iato fibers or texturized, such products are more expensive than those derived from soybean and there is no market for them. 

Of the 20 residues that react with A-ethylmaleimide in the non-reduced denatured Ca -ATPase at least 15 are available for reaction with various SH reagents in the native enzyme [75,239,310]. These residues are all exposed on the cytoplasmic surface. After reaction of these SH groups with Hg-phenyl azoferritin, tightly packed ferritin particles can be seen by electron microscopy only on the outer surface of the sarcoplasmic reticulum vesicles [143,311-314]. Even after the vesicles were ruptured by sonication, aging, or exposure to distilled water, alkaline solutions or oleate, the asymmetric localization of the ferritin particles on the outer surface was preserved [311,313,314]. 

During the electron tranters at the three classic sites of phosphorylation (marked I, II, and III), protons are pumped out of the mitochondria into the cytoplasm. The exact number of protons pumped at each site is somewhat controversial however, this proton pumping makes the interior of the mitochondria alkaline. 

ATP is made by the FiFo ATPase. This enzyme allows the protons back into the mitochondria. Since the interior is alkaline, the reaction is favorable—favorable enough to drive the synthesis of ATP by letting protons back into the mitochondria. Exactly how the FiFq ATPase couples the flow of protons down their concentration gradient to the formation of ATP is not known in molecular detail. The proton flow through the FiFo ATPase is required to release ATP from the active site where it was synthesized from ADP and Pj. The ATP is made in the interior of the mitochondria and must be exchanged for ADP outside the mitochondria to keep the cytosol supplied with ATP. The exchange of mitochondrial ATP for cytoplasmic ADP is catalyzed by the ATP/ADP translocase. 

An alkaline pyrophosphatase from rat liver cytoplasm has been partially purified and characterized (24) the corresponding enzyme from mice is inhibited by Mg J+-ADP and free PPj, and free Mg2+ has been implicated as an allosteric activator (23). Partial heat inactivation results in loss of the apparent allosteric effects. Rat liver mitochondrial pyrophosphatase, which is inhibited by adenine nucleotides (36), appears to be bound to the inside of the inner mitochondrial membrane (37). This enzyme, after solubilization, has been separated into two fractions which have somewhat different specificity (24, 38). A pyrophosphatase strongly simulated by sulfhydryl reagents (39) has been partially purified from brain tissue (40). The mono-magnesium PPj complex appears to be the true substrate for this enzyme (41). Pynes and Younathan have purified a pyrophosphatase 1800-fold from human erythrocytes (43). The properties of this enzyme are strikingly similar to those of the yeast enzyme the major difference appears to be the more rigid substrate specificity of the erythrocyte enzyme in the presence of Znz. ... 

Racker and Schroeder (85) questioned the importance of the alkaline FDPase in photosynthesis because of its lack of activity at neutral pH, its apparent cytoplasmic localization, and the presence of a second enzyme or enzymes which appeared to be associated with the chloroplasts and which hydrolyzed both FDP and SDP. Later work, however, has clearly established the function of this enzyme in the photosynthetic carbon cycle. Smillie has shown that the alkaline FDPase is associated with photosynthetic tissues in higher plants and Euglena (101, 102). The enzyme was also shown to be localized in the chloroplasts and to be absent in nonphotosynthetic tissue or bleached algae. It was the only FDPase detected in the photosynthetic bacterium Chromatium grown under autotrophic conditions (102). Preiss et al. (103) have pointed... 

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

The term nucleoside was originally proposed by Levene and Jacobs in 1909 for the carbohydrate derivatives of purines (and, later, of pyrimidines) isolated from the alkaline hydrolyzates of yeast nucleic acid. The phosphate esters of nucleosides are the nucleotides, which, in polymerized forms, constitute the nucleic acids of all cells.2 The sugar moieties of nucleosides derived from the nucleic acids have been shown, thus far, to be either D-ribose or 2-deoxy-D-eri/fAro-pentose ( 2-deoxy-D-ribose ). The ribo-nucleosides are constituents of ribonucleic acids, which occur mainly in the cell cytoplasm whereas 2-deoxyribo -nucleosides are components of deoxypentonucleic acids, which are localized in the cell nucleus.3 The nucleic acids are not limited (in occurrence) to cellular components. They have also been found to be important constituents of plant and animal viruses. 

The infection cycle is shown in Figure 19.2. When the polyhedra are dispersed in the environment, the viral particles inside them (ODVs) are deposited on the plant leaves. When the caterpillars feed on the virus-contaminated foliage, they ingest the polyhedra. The alkaline environment in the caterpillar medium intestine causes breakdown of the polyhedra and the viral particles are released from the polyhedra. The infection of the cells occurs via a receptor-mediated fusion process. Once in the cytoplasm, the nucleocapsids without membrane are transported to the nucleus of the cell, where gene expression and genome replication begins. 

For eukaryotic microorganisms, the involvement of PolyPs in biochemical regulation under stress has also been observed. For example, the involvement of vacuolar PolyP in survival under osmotic or alkaline stress has been shown in algae and fungi. In the alga Dunaliella salina, alkalinization of the cytoplasm results in a massive hydrolysis of PolyP, resulting in pH stat. Various authors have suggested that the hydrolysis of PolyP provides the pH-stat mechanism to counterbalance the alkaline stress (Bental et al, 1990 Pick et al, 1990 Pick and Weis, 1991). 

The ammonium-induced cytoplasmic alkalization in the unicellular algae Dunaliella salina resulted in degradation of long-chain PolyPs to PolyP3 (Pick etal, 1990 Bental etal, 1990 Pick and Wess, 1991). The hydrolysis was shown to correlate with the recovery of cytoplasmic pH and might provide the pH-stat mechanism to counterbalance the alkaline stress. 

Following such treatment, the cultured cells in monolayer are washed with phosphate-buffered saline and extracted in 4 ml 0.5% Triton X-100 in saline/EDTA (100 mM NaCl, 10 mM EDTA, pH 8.0) for 2 min at room temperature. This releases most of the cytoplasmic material whilst the nuclei remain attached to the culture dish. 0.5% sodium dodecyl sulphate and 40 //g/ml pancreatic RNAse (preincubated at 80°C for 10 min, to inactivate DNAse) in saline/EDTA (above) is then added and the mixture incubated for 20 min at 37°C. One volume of chloroform/isoamyl alcohol (20 5, v/v) is then added and the phases mixed gently. The aqueous phase is separated by centrifugation and extracted again with chloroform/isoamyl alcohol. DNA is precipitated from the aqueous phase with 2 vol 95% ethanol and resuspended in 0.01 M Tris, pH 7.5. Alkaline sucrose density gradients (5-20%) are prepared in 0.1 M NaCl, 0.1 M NaOH with a final volume of 4.1 ml. Samples of DNA (max 3 fig) are layered on the top of these gradients and spun at 32 000 rpm at 20°C in a SW.50.1 Beckman rotor for 120 min. Fractions are collected and the [3H]DNA precipitated... 

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