Stromal alkalinization

Furthermore, to nitrite reduction as a source of stromal alkalinity, must be added the rate of ammonium uptake by chloroplasts in photorespiratory cycling and turnover of amino acids independent of photorespiration for reassimilation by glutamine synthetase and glutamate synthase. 

Ishijima, S. Uchibori, A. Takagi, H. Maki, R. Ohnishi, M. Light-induced increase in free Mg concentration in spinach chloroplasts measurement of free Mg by using a fluorescent probe and necessity of stromal alkalinization. Arch. Biochem. Biophys. 2003, 412, 126-132. 

As discussed in Section 22.7, illumination of chloroplasts leads to light-driven pumping of protons into the thylakoid lumen, which causes pH changes in both the stroma and the thylakoid lumen (Figure 22.27). The stromal pH rises, typically to pH 8. Because rubisco and rubisco activase are more active at pH 8, COg fixation is activated as stromal pH rises. Fructose-1,6-bisphosphatase, ribulose-5-phosphate kinase, and glyceraldehyde-3-phosphate dehydrogenase all have alkaline pH optima. Thus, their activities increase as a result of the light-induced pH increase in the stroma. 

Electron-transferring molecules in the chain of carriers connecting PSII and PSI are oriented asymmetrically in the thylakoid membrane, so photoinduced electron flow results in the net movement of protons across the membrane, from the stromal side to the thylakoid lumen (Fig. 19-57). In 1966 Andre Jagendorf showed that a pH gradient across the thylakoid membrane (alkaline outside) could furnish the driving force to generate ATP. 

Electron microscopy of sectioned chloroplasts shows ATP synthase complexes as knoblike projections on the outside (stromal or N) surface of thylalcoid membranes these complexes correspond to the ATP synthase complexes seen to project on the inside (matrix or N) surface of the inner mitochondrial membrane. Thus the relationship between the orientation of the ATP synthase and the direction of proton pumping is the same in chloroplasts and mitochondria. In both cases, the Fl portion of ATP synthase is located on the more alkaline (N) side of the membrane through which protons flow down their concentration gradient the direction of proton flow relative to Fi is the same in both cases P to N (Fig. 19-58). 

The reductive assimilation of C02 requires a lot of ATP and NADPH, and their stromal concentrations increase when chloroplasts are illuminated (Fig. 20-17). The light-induced transport of protons across the thylakoid membrane (Chapter 19) also increases the stromal pH from about 7 to about 8, and it is accompanied by a flow of Mg2+ from the thylakoid compartment into the stroma, raising the [Mg2+] from 1 to 3 ira to 3 to 6 ulm. Several stromal enzymes have evolved to take advantage of these light-induced conditions, which signal the availability of ATP and NADPH the enzymes are more active in an alkaline environment and at high [Mg2+], For example, activation of rubisco by formation of the... 

FIGURE 20-17 Source of ATP and NADPH. ATP and NADPH produced by the light reactions are essential substrates for the reduction of C02. The photosynthetic reactions that produce ATP and NADPH are accompanied by movement of protons (red) from the stroma into the thylakoid, creating alkaline conditions in the stroma. Magnesium ions pass from the thylakoid into the stroma, increasing the stromal [Mg2+],... 

A physiologic phosphate concentration is required for bone mineralization. Lowering the concentration prevents mineralization, but raising it does not ensure precipitation because pyrophosphate is present to inhibit precipitation. The concentration of PPi in cartilage and bone is controlled by three enzymes, two on the outer surface of matrix vesicles (Fig. 9.5b). One is tissue-nonspecific alkaline phosphatase (TNAP), which decreases stromal pyrophosphate and the other is NTP-PPi hydrolase (also called plasma cell membrane glycoprotein-1), which increases it. The progressive ankylosis gene product (ANK protein) is expressed by osteoblasts to add to the pyrophosphate of the osteoid matrix from osteoblast cytosol. 

Figure 9.8 outlines how matrix vesicles increase and decrease the concentration of pyrophosphate. NTP-PPi hydrolase synthesizes pyrophosphate from stromal fluid nucleotides, mostly ATP (ATP —> AMP + PPi). Many cells secrete ATP into the extracellular fluid and it passes into the blood plasma where it affects a variety of cells independently of its function in intracellular energy metabolism. In mice, a nonfunctional ANK protein or a deletion of NTP-PPi hydrolase decreases the extracellular pyrophosphate concentration and the phenotype exhibits extensive mineralization. Thus, the hydrolysis of pyrophosphate appears to be a major function of alkaline phosphatase (TNAP) after the calcium phosphate precipitate has raptured the matrix vesicles. Rapid mineralization of collagen and the rest of the osteoid matrix ensue without a need to transport any more Ca2+ or Pi to the region. 

Osteoblasts secrete osteoid, a matrix rich in type I collagen fibers and vesicles. Precipitation of calcium phosphate is inhibited by a high concentration of pyrophosphate in stromal interstitial fluids, and a high concentration also of albumin and citrate in blood plasma. Pyrophosphate is derived from (1) transport out of the cytosol, and (2) synthesis from nucleoside triphosphates in the stromal interstitial fluid that permeates the osteoid matrix. Precipitation occurs only when calcium and phosphate ions are taken up into vesicles whose inner membrane is composed of phosphatidylserine. The high concentration of calcium and phosphate ions in the vesicle is mediated by annexin and type HI Pi Na-dependent transporters. This overwhelms the pyrophosphate and nucleation occurs. As the precipitate grows and ruptures the membrane, tissue-nonspecific alkaline phosphatase is activated to remove pyrophosphate from the osteoid matrix fluid so that calcium phosphate precipitates around phosphorylated serine residues within the collagen fibers. 

To check whether the PPase found in the thylakoids is peripherally or integrally bound to the thylakoid membranes or simply adsorbed stromal PPase, thylakoids were washed at different pH. As the extrinsic proteins are easily released at alkaline pH (10), our osmotically shocked thylakoids were washed several times in buffer with pH 6.5 and 8.5. The total PPase activity was then determined in all subsequent supernatants and in the last pellet. As shown in Table 2 the washings release PPase into the aqueous solutions, but the extent of release of PPase from the membrane is about the same whether the washings are performed at pH 6.5 or pH 8.5. About 2% of the PPase activity remained in the final thylakoid pellet. 

Prins, H.-J., Braat, A.K., Gawlitta, D., Dhert, W.J.A., Egan, D.A., Tijssen-Slump, E., et al., 2014. In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells. Stem Cell Res. 12, 428-440. http //dx.doi. org/10.1016/j.scr.2013.12.001. 

After bone marrow stromal cells were seeded and cultured on PHBHHx, their proliferation was investigated by MTT. Differentiation of the cells was assessed by measuring alkaline phosphatase activity and by histochemical assay. The wettability and thermal properties of PHBHHx films were also studied. The... 

---