Enzymes subcellular localization

The estrogens are a family of hormones synthesized in a variety of tissues. 17P-Estradiol is the primary estrogen of ovarian origin. In some species, estrone, synthesized in numerous tissues, is more abundant. In pregnancy, relatively more estriol is produced, and this comes from the placenta. The general pathway and the subcellular localization of the enzymes involved in the early steps of estradiol synthesis are the same as those involved in androgen biosynthesis. Features unique to the ovary are illustrated in Figure 42-7. 

From overexpression studies, it can be inferred that individual isoforms of PKC are precisely directed to distinct subcellular locations (e.g. PKCa to the endoplasmic reticulum and PKCS to the Golgi). Directing PKC isozymes to specific subcellular loci appears to occur via interaction of the enzyme with localized intracellular binding proteins. Such proteins may or may not be substrates for PKC. An example of the latter category would be RACK (receptors for activated C kinase) 1. RACKs are thought to interact only with activated PKCs and to direct translocated PKCs to specific loci. 

Statham, C.N., Szyjka, S.P., Menahan, L.A. and Lech, J.J. Fractionation and subcellular localization of marker enzymes in rainbow trout liver. Biochem. Pharmacol. (1977) 26 1395-1400. 

Ghersi-Egea JF, Perrin R, Leininger-Muller B, Grassiot MC, Jeandel C, et al. 1993. Subcellular localization of cytochrome P450, and activities of several enzymes responsible for drug metabolism in the human brain. Biochem Pharmacol 45 647-658. 

The extent and specificity of the reactions of protein kinases and protein phosphatases are extremely dependent on the degree to which substrate and enzyme are localized at the same place in the cell. Many substrates of protein kinases occur either as membrane associated or particle associated forms (see 7.6.1, enzymes of glycogen metabolism). For protein kinases or protein phosphatases to perform their physiological function in a signal transduction process, they must be transported to the location of then-substrate in many cases (review Hubbard and Cohen, 1992 Mochly-Rosen, 1995). This is vahd both for the Ser/Tbr-specific protein kinases as well as for many Tyr-speci-fic protein kinases. In the course of activation of signal transduction pathways, com-partmentahzation of protein kinases, redistributed to new subcellular locations, is often observed. 

Yarlett N, Lindmark DG, Goldberg B, Moharrami MA, Bacchi CJ (1994) Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Tritrichomonas foetus. J Eukaryot Microbiol 41 554-559... 

The enzymes involved in bio transformation also have a particular subcellular localization many are found in the smooth endoplasmic reticulum (SER). Some are located in the cytosol, and a few are found in other organelles such as the mitochrondria. These subcellular localizations may have important implications for the mechanism of toxicity of compounds in some cases. 

Very helpful review of the subcellular localization of glycolytic enzymes and the regulation of glycolysis in plants. 

The enzyme was originally found to be membrane bound and resisted solubilization and purification (26). Lundblad and Moore (27), however, have reported solubilizing it using dilute (5 mM) sodium borate buffer at pH 9 after 16 hr at 37°. Studies on regional and subcellular distribution using density gradient techniques have revealed that the 2, 3 -cyclic phosphate diesterase concentrates in those fractions containing myelin (28, 29), and the conclusion has been reached that the enzyme is localized in the myelin sheath or intimately associated structures. Kurihara and... 

Cell walls, total membrane-bound components, and ribosomes were separated and assayed for cellulase activity to study the subcellular localization of the enzymes as follows. Segments (approx. 5 g fresh wt) were ground in two volumes of extraction medium containing 0.4M sucrose (ribonuclease-free), 5mM Mg acetate, lOmM Tris-HCl (pH 7.5 at 22°C), 20mM KC1 and 5mM / -mercaptoethanol. The brei was filtered and the filtrate centrifuged at 500 Xg for 20 min. The post-500 Xg supernatant was fractionated essentially as previously described (28). Aliquots (7 mL) of the supernatant were layered on a discontinuous gradient composed of 2 mL 70% (w/v) sucrose and 3 mL 15% (w/v) sucrose both in lOmM Tris-HCl (pH 7.5 at 22°C), lOmM KC1, 2.5mM Mg acetate and ImM / -mercaptoethanol. The tubes were centrifuged at... 

Hrazdina G, Wagner GJ, Siegelman HW. 1978. Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts. Phytochem 17 53-56. 

Immunolocalization is a technique for identifying the presence of a protein within the cell, its relative abundance and its subcellular localization. After suitable preparation of the cells, they are treated with an antibody (the primary antibody) that binds to the protein of interest. An antibody that binds to the primary antibody (the secondary antibody) is then allowed to bind and form an antigen—primary antibody—secondary antibody complex. The detection system generally consists of the formation of a colored insoluble product of an enzymatic reaction, the enzyme, such as alkaline phosphatase or horseradish peroxidase, being covalently linked to the secondary antibody. 

DE LUCA, V., CUTLER, A.J., Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol., 1987, 85, 1099-1102. 

Subcellular localization studies have identified P-450-dependent monooxygenase activity in adult hairless mice sebaceous glands. Phase II conjugation pathways have also been identified in skin. Extracellular enzymes including esterases are present in skin, which has been utilized to formulate lipid-soluble ester prodrugs which penetrate the stratum corneum and then are cleaved to release active drug into the systemic circulation. Finally, co-administration of enzyme inducers and inhibitors modulate cutaneous biotransformation and thus alter the systemic toxicity profile. These metabolic interactions that occur in skin have attracted a great deal of research attention and clearly illustrate that skin is more than a passive barrier to toxin absorption. 

Okita, T. W., Greenberg, E., Kuhn, D. N., and Preiss, J. 1979. Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves. Plant Physiol. 64,187-192. 

---