Enzymes, intracellular

The main drug targets are enzymes, intracellular receptors, and extracellular (cell surface) receptors. Drugs are normally designed to interact with these entities either as agonists or antagonists to achieve control over the disease pathway. 

Early studies by Stuehr and Nathan and colleagues demonstrated a speciflc stimulation of NOS activity in crude subcellular fractions by BH4, which is true for all purifled NOS enzymes to date. BH4 is important both as a stabilizer of protein conformation and also as an electron transfer component.The activity of the enzyme intracellularly can be affected by modulations in cellular BH4 levels, and this may have important consequences under physiological and pathophysiological conditions. " ... 

Small nuclear ribonucleoprotein particle Soil organic carbon store-operated channel Suppressors of cytokine signaling Superoxide dismutase CuZn-SOD enzyme (intracellular)... 

The plasma membranes of many types of eukaryotic cells also contain receptor proteins that bind specific signaling molecules (e.g., hormones, growth factors, neurotransmlt-ters), leading to various cellular responses. These proteins, which are critical for cell development and functioning, are described in several later chapters. Finally, peripheral cytosolic proteins that are recruited to the membrane surface function as enzymes. Intracellular signal transducers, and structural proteins for stabilizing the membrane. 

Two strains of DKP-assimilating bacteria which degrade cyclo(Gly-Gly) or cyclo(Gly-L-Tyr) were isolated. Both strains produced DKP-hydrolyzing enzymes intracellularly in contrast to an cyclo(Gly-Gly) hydrolase from Bacillus sp. [2], the only DKP-hydrolyzing enzyme that was localized extracellularly. Furthermore, the resting-cells of both strains isolated in the present study exhibited a broad spectrum for DKP substrates, while the cyclo(Gly-Gly) hydrolase catalyzed the hydrolysis of only cyclo(Gly-Gly) among a variety of DKPs tested. Therefore,... 

In cfl-l-12-lC-112 the membrane-associated polypeptides (36 and 21 kD) were induced only weakly, although the soluble 45 to 50 kD polypeptides and periplasmic CA were induced normally (Fig. 2). Weak induction of these polypeptides also was observed with two other cn-1 alleles (data not shown), so it is unlikely that the altered induction resulted from a second site mutation. In spite of the apparent lack of induction of the membrane-associated polypeptides in cn-1 mutants during the first 4 h, the polypeptides were observed in stained SDS-PAGE gels after 24 h of induction (data not shown). Mutants in the ca-l locus appear to be deficient in an intracellular CA (9, 12), so a missing polypeptide could represent this enzyme. Intracellular CA has been reported to be present in both C02 enriched and air-adapted cells (12), and the functional defect in the ca-l mutants is expressed in both (see above). Weak induction of the intracellular CA is inconsistent with these findings. Therefore, the altered polypeptide induction is associated with cn-1 mutations but probably not as the primary lesion. 

The biodegradation of PHA can occur inside the cell, by microorganisms that produce PHA, or outside the cell by microorganisms that do not produce PHA, but can also use it as carbon and energy sources. However, in both cases the hydrolysis is catalyzed by specific enzymes, intracellular, or extracellular PHA depolymerases. 

When stimulation of the normal activity of the target tissue requires increased synthesis of key enzymes, intracellular effects of cyclic 3, 5 -AMP have been suggested to take place either at the translation level, as in the stimulation by ACTH of the synthesis of enzymes involved in pregnenolone formation from cholesterol, or at the transcription level, as in the stimulation by TSH of iodide transport in the thyroid. The main evidence for stimulation at the transcription level is the inhibition of the effect by both inhibitors of RNA synthesis (as actinomydn) and of protein synthesis (as puromycin), while stimulation at the translation level is suggested by inhibition with protein-synthesis inhibitors, but not with RNA-syn-tbesis inhibitors. Effects of 3, S -AMP on amino-acid incorporation in acellular systems, and on peptide release by polysomes have been reported, but their relevance to the activation at the translation level of protein synthesis in the intact cell is not known. 

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

Biosynthesis ofS(— )-M llc Acid. Aqueous fumaric acid is converted to levorotatory malic acid by the intracellular enzyme, fumarase, which is produced by various microorganisms. A Japanese process for continuous commercial production of S(—)-mahc acid from fumaric acid is based on the use of immobilized Brevibacteriumflavum cells in carrageenan (32). The yield of pyrogen-free S(—)-mahc acid that is suitable for pharmaceutical use is ca 70% of the theoretical. 

Active Transport. Maintenance of the appropriate concentrations of K" and Na" in the intra- and extracellular fluids involves active transport, ie, a process requiring energy (53). Sodium ion in the extracellular fluid (0.136—0.145 AfNa" ) diffuses passively and continuously into the intracellular fluid (<0.01 M Na" ) and must be removed. This sodium ion is pumped from the intracellular to the extracellular fluid, while K" is pumped from the extracellular (ca 0.004 M K" ) to the intracellular fluid (ca 0.14 M K" ) (53—55). The energy for these processes is provided by hydrolysis of adenosine triphosphate (ATP) and requires the enzyme Na" -K" ATPase, a membrane-bound enzyme which is widely distributed in the body. In some cells, eg, brain and kidney, 60—70 wt % of the ATP is used to maintain the required Na" -K" distribution. 

Adenosine. Adenosine [58-61-7] (Ado), (29), a purine nucleoside, is an intracellular constituent acting as both an enzyme cofactor... 

Two types of immobilization are used for immobilizing glucose isomerase. The intracellular enzyme is either immobilized within the bacterial cells to produce a whole-ceU product, or the enzyme is released from the cells, recovered, and immobilized onto an inert carrier. An example of the whole-ceU process is one in which cells are dismpted by homogenization, cross-linked with glutaraldehyde, flocculated using a cationic flocculent, and extmded (42). 

Immobilization. Enzymes, as individual water-soluble molecules, are generally efficient catalysts. In biological systems they are predorninandy intracellular or associated with cell membranes, ie, in a type of immobilized state. This enables them to perform their activity in a specific environment, be stored and protected in stable form, take part in multi-enzyme reactions, acquire cofactors, etc. Unfortunately, this optimization of enzyme use and performance in nature may not be directiy transferable to the laboratory. 

There are five known classes of enzyme-linked receptors (1) receptor tyrosine kinases, which phosphorylate specific tyrosine residues on intracellular signaling proteins (2) tyrosine kinase-associated receptors, such as the prolactin and growth hormone receptors we have already discussed, which... 

Endonuclease-catalyzed hydrolysis of DNA at the internucleosomal linker regions into multimers of 180 base pairs which are visualized by electrophoresis as a ladder of nuclear DNA fragments. Access of the endonuclease to DNA is facilitated by depletion of polyamines, and the activity of the enzyme is mcrea.sed by and decreased by ADP-tibosylation. Thus, agents that increase intracellular Ca " or inhibit l>oly(ADP-ribose) polymerase can induce apoptosi.s. ... 

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