Alkaline phosphatase enzyme protein synthesis

Horiuchi et al. (2), and Torriani (S) that orthophosphate repressed the formation of a nonspecific phosphomonoesterase in E. coli that research on this enzyme began. This work (2, 3) showed a maximum rate of synthesis of the enzyme occurred only when the phosphate concentration became low enough to limit cell growth. With sufficient phosphate, the amount of active enzyme is negligible. Under conditions of limiting phosphate, alkaline phosphatase accounts for about 6% of the total protein synthesized by the cell (4). 

The assays for the enzyme synthesis of sulfogalactosyl-glycerolipid, sulfatides and galactocerebrosides were carried out as previously described respectively by Subba Rao, et al. (28) Sarlieve, et al. 05, 29), and Neskovic, et al. (30). The assay for 2, 3 cyclic nucleotide phosphohydrolase was performed according to the method of Prohaska, et al. (31). EL coli. alkaline phosphatase type III-S, 2, 3 -cAMP, and sodium deoxycholate were obtained from Sigma (St. Louis, Mo.). Protein was determined by the method of Lowry, et al. (32) with crystalline bovine serum albumin as the standard. 

Boyer s studies (B39) appear to indicate a lower degree of organ enzyme specificity than that observed by Nisselbaum et al., in that antihuman intestine alkaline phosphatase sera cross-reacted with kidney and placenta. Also, the antihuman bone preparation precipitated alkaline phosphatase from spleen, liver, kidney, and intestine. Boyer considers liver, bone, spleen, and kidney 3-phosphatases to be closely related proteins, followed by intestine and placenta, the enzyme from these latter tissues representing a second and third class of alkaline phosphatase proteins. Intestine and placenta partially cross-react with one another and with a minor kidney component. Three genetic loci are considered by Boyer, therefore, to control the synthesis of alkaline phosphatase. The most recent study in this area is reported by Birkett et al. (B19). 

There appears to be no research that traces the path of synthesis of alkaline phosphatase to its final location on the absorptive surfaces of cell-wall membranes. The usual sequence of specific enzyme synthesis envisions the collection of newly synthesized protein in secretion vacuoles that eventually leave the cell of origin as part of a secretion process. The sites of synthesis are the ribosomes of the rough-surfaced endoplasmic reticulum. 

On the other hand, the Golgi apparatus is implicated in the synthesis of cell-wall membranes. It is possible that the alkaline phosphatase is a catalytically inactive, structural protein of such cell membranes when they are still in the cytoplasm, which may explain many failures to demonstrate its presence in the Golgi apparatus. Upon becoming part of the outer surface, say of the brush border of the intestinal villus, the enzyme may be activated by the alkaline contents of the duodenal lumen. The concept of catalytically inactive membrane enzyme proteins is developed in a recent publication with regard to 8-glucuronidase (FIO). 

The nephrotoxicity of 16 continues to generate considerable interest. Orellanine was highly toxic to mice (LD50 = 12.5 mg/kg i.p.)[101] and caused interstitial nephritis and tubular necrosis in mouse kidney [102], A summary of 16-induced changes in renal function and morphology has been reported [103]. In LLC-PKi renal epithelial cell cultures, 16 decreased the activity of alkaline phosphatase and lactate dehydrogenase, and decreased the incorporation of H-leucine and H-thymidine [104]. Orellanine was a noncompetitive inhibitor of renal alkaline phosphatase, but a competitive inhibitor of the intestinal and placental enzymes [105]. In canine kidney MDCK cell cultures, 16, or a metabolite of 16, inhibited protein, RNA and DNA synthesis [106]. 

After a 40-hour continuous exposure to a low acrolein concentration (2.1 ppm), an increased activity of the alkaline phosphatase was observed. The exposure to 4 ppm for 4, 8 and 20 hours resulted in increased values of the alkaline phosphates of the liver as compared to controls — 135, 222 and 253%, respectively. The activity of the liver alkaline phosphatases and tyrosine-ketoglutanate transaminases were increased in rats 5 to 10 hours after an injection or inhalation of acrolein. The results of research indicate the irritating effect of acrolein in stimulating the pituitary-adrenal system, which induces a hypersecretion of glucocorticoids conditioning the induction or stimulation of the synthesis of larger amounts of protein enzymes of the liver. 

Bacterial alkaline phosphatase is the gene product of phoA, a member of the pho regu-lon (Table 9.1). When the pho regulon is induced by low external quantities of phosphate, synthesis of this alkaline phosphatase can represent as much as 6 mole% of total protein synthesis, and enzyme activity per cell can increase 1000-fold (Coleman and Gettins, 1983). The enzyme is synthesized as 43,000 Da monomers, which are transported to the periplasmic space and become active only after dimerization. As with many alkaline phosphatases, this enzyme accepts a broad range of substrates, which it hydrolyses at similar rates (Fernley and Walker, 1967 Reid and Wilson, 1971). Substrates are compounds with the general formula... 

Synthesis of triazine haptens and hapten-protein conjugates. Simazine and atrazine were derivatized with mercaptopropionic add (mpa) at Rl, or aminohexanoic acid (aha) at R2, and these haptens were covalently linked to keyhole limpet hemoyanin (KLH), conalbumin (CON), or bovine serum albumin (BSA), by forming active esters with N-hydroxysucdnimide (g) (Figure 1, structures III and IV). This technique was also used to couple simazine-aminohexanoic add to calf intestine alkaline phosphatase (Figure 1, structure V), for use as the %aptenated enzyme in the EIA format described below. 

The glucocorticoid induction of HeLa cell alkaline phosphatase requires concomitant protein synthesis [100], but immunoprecipitation and radioimmunoprecipitation studies strongly suggest that the quantities of alkaline phosphatase antigen and its rate of synthesis are not altered by the steroid inducer [101]. Physical and biochemical differences have been observed between the uninduced or basal enzyme and the induced form [101]. Thus, the induction does not seem to be due to an increased rate of de novo phosphatase synthesis but more likely is due to the conversion of an antigenic precursor to a catalytically active (or more active) molecule. The conversion process apparently is inhibited... 

Some physiological or pathological stimuli induce the liver cell to synthesize or break down some protein selectively. Even during starvation the content of all liver protein does not drop simultaneously. For example, while the activities of catalase, xanthine oxidase, alkaline phosphatase, and acid phosphatase drop at various rates as starvation progresses, that of glucose-6-phosphatase increases. Hydrocortisone and tryptophan administration induces a massive increase in tryptophan peroxidase activity. In either case, at least part of the increase in enzyme activity results from de novo enzyme synthesis. If tryptophan administration is interrupted, the activity of the peroxidase returns to normal. During the induction, turnover rates of other proteins do not change. 

It has been found that 1,25-dihydroxycholecalciferol stimulates calcium and phosphate transport in the small intestine and calcium accumulation within the cells by means that do not require RNA or protein synthesis. Yet, many of the other changes in protein and enzyme activity require the induction of protein synthesis, which is compatible with the activation of DNA-dependent RNA synthesis in the nucleus by 1,25-dihydroxycholecalciferol. The stimulation of the brush border enzyme alkaline phosphatase by inhibitors of DNA-dependent RNA synthesis is puzzling. These various results suggest, but do not prove, that 1,25-dihydroxycholecalciferol has extranuclear actions (Bikle et al., 1979). 

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