P-Hydroxymercuribenzoate

Aqueous solutions of aequorin also emit light upon the addition of various thiol-modification reagents, such as p-quinone, Br2, I2, N-bromosuccinimide, N-ethylmaleimide, iodoacetic acid, and p-hydroxymercuribenzoate (Shimomura et al., 1974b). The luminescence is weak and long-lasting ( 1 hour). The quantum yield varies with the conditions, but seldom exceeds 0.02 at 23-25°C. The luminescence is presumably due to destabilization of the functional moiety caused by the modification of thiol and other groups on the aequorin molecule. 

Cysteine Iodoacetamide, maleimides, Ellman s reagent, p-hydroxymercuribenzoate... 

Studies on the active agent of the yeast extract have shown it to be nondialyzable, heat labile, and inactivated by trypsin and chymotrypsin as well as silver ions and p-hydroxymercuribenzoate.(7l) These and other properties suggest that the active agent is an enzyme. Sedimentation studies have shown that the yeast photoreactivating enzyme combines with UV-irradiated DNA, in which condition it is more resistant to heat inactivation and inactivation due to silver ions and p-hydroxymercuribenzoate.<75) The... 

Other chemicals which inhibit milk lipase include hydrogen peroxide, animal cephalin, sodium arsenite, diisopropyl fluorophosphate, 2,4 din-itro-l-fluorobenzene, p-hydroxymercuribenzoate, potassium dichromate, lauryl dimethyl benzyl ammonium chloride, aureomycin, penicillin, streptomycin, and terramycin (Schwartz 1974). 

The behavior of aspartate carbamoylase changed dramatically when the enzyme was treated with the organic mercurial compound p-hydroxymercuribenzoate (see fig. 9.14). The binding of aspartate no longer showed positive cooperativity, and ATP or CTP were without effect. Exposure to mercurials was found to cause the enzyme to dissociate into two types of fragments, one of which retained the enzymatic activity but was no longer affected by CTP or... 

The activity of [i-galactosidasc (P-Gal) was studied on a quartz chip using a static micromixer to mix the enzyme and substrate on the ms time scale. Inhibition by phenylethyl-P-D-thio-galactoside was also studied [1048]. In another report, the enzyme P-Gal was assayed on a chip in which P-Gal would convert a substrate, resoruhn-P-D-galactopyranoside (RBG), to resoruhn to be detected fluorescently [1049]. By varying the substrate concentrations and monitoring the amount of resoruhn by LIF, Michaelis-Menten constants could be determined. In addition, the inhibition constants of phenylethyl-P-D-thiogalactoside, lactose, and p-hydroxymercuribenzoic acid to the enzyme P-Gal were determined [1049]. 

Sulfhydryl agents, e.g., p-hydroxymercuribenzoate, both inactivate and activate the Pseudomonas enzyme, depending on the presence of oxidized and reduced substrates, respectively (17). Inactivated protein may be reactivated by mercaptoethanol suggesting that p-hydroxymercuribenzo-ate acts on sulfhydryl groups near or at the active site. Reversible effects of sulfhydryl agents were also observed with the Chromatium enzyme (16). Proteolytic enzymes such as trypsin did not inactivate the Pseudomonas transhydrogenase (8). 

Since cells contain a variety of proteases with different modes of action, it is necessary to add a cocktail of protease inhibitors to deal with them all e.g., phenylmethyl-sulphonyl fluoride (PMSF) to inhibit serine proteases EGTA to inhibit Ca2+-acti-vated proteases and p-hydroxymercuribenzoate (PHMB) to inhibit cysteine proteases. Maintaining pH neutrality offers protection against aspartate proteases. 

The importance of the changes in quaternary structure in determining the sigmoidal curve is illustrated nicely by studies of the isolated catalytic trimer, freed by p-hydroxymercuribenzoate treatment. The catalytic subunit shows Michaelis-Menten kinetics with kinetic parameters that are indistinguishable from those deduced for the R state. Thus, the term tense is apt in the T state, the regulatory dimers hold the two catalytic trimers sufficiently close to one another that key loops on their surfaces collide and interfere with conformational adjustments necessary for high-affinity substrate binding and catalysis. 

Figure 10.3. Modification of Cysteine Residues. p-Hydroxymercuribenzoate reacts with crucial cysteine residues in aspartate transcarbamoylase.

The only cofactor required for this condensation is a divalent cation, either Mg or Mn ", and the activity can be inhibited by iodoacetamide, N-ethylmaleimide and p-hydroxymercuribenzoate. Cell-free extracts catalyzing phytoene formation have also been isolated from pea fruits spinach leaves Mycobacterium, Halobacterium cutirub-... 

An easy method for the detection of abnormal a or y8 chains in hemoglobin variants is based on the observation that treatment of the hemoglobin with p-hydroxymercuribenzoate (PMB) produces monomers and dimers that can be separated by electrophoresis. Each test requires four small test tubes a control tube and a reaction tube for each of two molar concentrations of PMB. 

Fohr KJ, Scott J, Ahnert-Hilger G et ol. (1989) Characterization of the inositol 1,4,5,-trisphosphate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate and p-hydroxymercuribenzoate. Biochem. J. 262 83-89. 

Although lipophilic and able to diffuse across the cell membrane TH can enter cells more rapidly by facilitated transport systems that tend to be specific for T4 or T3. Based on studies on isolated hepatocytes and RBCs, TH uptake in fish can be blocked by inhibitors of protein binding (phloretin, bromosulphothalein, 5,5 -di phenylhydan-toin and 8-anilino-1-naphthalene sulphonic acid) and by certain sulphydryl blockers (p-hydroxymercuribenzoate and /V-cthylmaleimide)59,63,64. 

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