Graduate Thesis Or Dissertation
 

A comparative study of the in vitro localization of mercury from phenylmercuric acetate and mercuric salt in rat kidney and liver subcellular fractions and their effect on alkaline phosphatase

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  • Studies were undertaken to determine the extent and rate of binding of Hg-203 labeled phenylmercuric acetate and mercuric ace tate in rat kidney and liver slices and their subcellular fractions after 37°C incubation of the slices in Krebs-Ringer -phosphate solutions containing the mercurials at 10⁻⁴ molar. A fast and parallel rate of uptake of both mercurials in kidney slices between 1 to 3 hour periods was observed. The uptake of the two mercurials into liver slices was much less than that found in kidney slices, The binding rate of phenylmercuric acetate was almost double the rate of inorganic mercury. The subcellular fractions (nuclear, mitochondrial, microsomal, and soluble) of the tissue slices were prepared by homogenizing in 0.25 molar sucrose with subsequent differential centrifugation. Even though the two mercurials showed similar binding in kidney slices, it was found that phenylmercuric acetate was bound to almost twice the extent that of inorganic mercury in the mitochondria, micro-somal, and soluble fractions, with the preponderate of the inorganic mercury being bound in the nuclear. Phenylmercuric acetate was also bound to twice the extent of inorganic mercury in the mitochondrial, microsomal, and soluble fractions from incubated liver slices; however, the binding of the two mercurials in the liver nuclear fraction was similar during the first hours. Also, there was a decrease of the binding of inorganic mercury in the soluble fraction from incubated liver slices as the incubation time increased. Sephadex G-100 elution patterns of the soluble protein fractions from incubated kidney and liver slices were determined. The mercury binding patterns in the elution fractions were also determined. There was found to be three main peaks in the elution pattern from liver and kidney soluble proteins. The first peak represents proteins with molecular weights of 100,000 or greater. The second peak consists of 15,000 to 30,000 molecular weight proteins followed by a trough or dip in the pattern representing large polypeptides (molecular weights of 2,000 to 3,000). The last peak consists of small polypeptides. The specific binding of phenylmercuric acetate in the proteins of the elution pattern corresponding to a molecular weight of 100,000 or larger is greater than inorganic mercury by as much as two-fold. The 15,000 to 30,000 molecular weight proteins in the Sephadex G-100 elution patterns show 2.5 to five times as much specific binding of phenylmercuric acetate as compared to mercuric ion. There was no other area in the patterns in which the mercurials were bound to any significant extent. The binding patterns of the two mercury compounds in the soluble proteins of incubated kidney slices filtered through Sephadex G-100 columns, were similar to those of the liver soluble proteins, except for a very high specific binding of both in the region of the elution pattern corresponding to large polypeptides (2,000 to 3,000 molecular weight). Sephadex G-100 filtration of the incubation soluble proteins leached from liver slices indicated that phenylmercuric acetate caused a greater loss of large molecular weight proteins as compared to the control or inorganic mercury incubated slices. The migration characteristics of soluble proteins from kidney slices with and without mercurial treatment were measured by disc electrophorosis on polyacrylamide gel columns. The results indicated that phenylmercuric acetate caused a possible loss of large molecular weight proteins from the soluble fraction as compared to control or inorganic mercury incubated kidney slices. There was found to be no correlation between the specific binding of the two mercurials with the enzymatic activity of alkaline phosphatase in the soluble and microsomal fractions. It was found that kidney slices incubated in solutions of inorganic mercury resulted in approximately 83 percent enhancement of alkaline phosphatase activity in the soluble fraction as compared to control. Phenylmercuric acetate caused about a 40 percent increase. The microsomal fractions from inorganic mercury treated kidney slices showed a greater decrease in the alkaline phosphatase activity than did phenyl-mercuric acetate as compared to that of control. Twenty to 30 percent of the alkaline phosphatase activity of the 35,000 X G soluble fraction was removed by ultracentrifugation at 150,000 X G. It is possible that the apparent activation of alkaline phosphatase in the soluble fraction was due to a solubilization of the enzyme, with its genesis possibly being in the microsomes. Addition of phenylmercuric acetate to the reaction mixture at concentrations up to 10⁻³ molar caused no significant decrease in the activity of alkaline phosphatase from kidney soluble proteins, while inorganic mercury showed 20 percent inhibition at 10⁻⁵ molar and almost 75 percent inhibition at 5 X 10⁻⁵ molar. There was no significant stimulation of the enzyme when either of the mercurials was added to the reaction mixture.
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