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• 1. [Article] Sulfhydryl and disulfide changes in young wheat seedlings during vernalization

  A study of the SH and SS content levels during vernalization of wheat was made. In order to carry out the study, a rapid analytical technique was worked out which allowed fast sequential analyses of the ...

  Citation × Citation Citation Abstract Copy Title: Sulfhydryl and disulfide changes in young wheat seedlings during vernalization Author: Ellis, Robert William A study of the SH and SS content levels during vernalization of wheat was made. In order to carry out the study, a rapid analytical technique was worked out which allowed fast sequential analyses of the samples of wheat tissue. Two techniques were used: a) an amperometric titration method and b) a fluorescence quenching method. The amperometric titration method consisted of adding aliquots of samples of wheat tissue homogenate to the system and noting the decrease in current, due to a tie-up of Ag⁺ by the SH groups of the sample. The system consisted of a rotating platinum electrode connected to a mercury-mercuric oxide reference cell. The supporting electrolyte was a 0.14M solution of tris at pH 7.4. The fluorescence quenching method consisted of adding aliquots of sample to a solution of fluorescien mercuric acetate (FMA) in a 1N NaOH media. The SH and SS groups reacted with FMA in a one:one ratio. The reacted FMA was unable to fluoresce, thus making possible a quantitative assay for SH and SS groups in solution. Red Bobs, a spring wheat, and Elgin, a winter wheat, were grown in the dark at 3° C. and at 20° C. The SH and SS contents of the shoots were measured throughout a period of five weeks in the group grown at 3° C. and a period of 48 hours in the group grown at 20° C. The results of the analyses showed that the SH, SS, and SH+SS contents based on fresh weights were present in greater quantities in the tissues of Red Bobs grown at 20° than in the Elgin tissues grown at 20°. The high SH and SS concentrations present in the warm-grown Red Bobs tissues were due to a high concentration of protein extracted from that tissue. The SH, SS, and SH+SS contents per fresh weight were quite similar in the cold-grown tissues of both varieties during the growth period. These constituents present in the cold-grown tissues occurred in quantities that were less than those found in warm-grown Red Bobs tissues and greater than those present in the warm-grown Elgin tissues. Both varieties grown at both temperatures showed a similar decrease in SH and SS contents based on fresh weights as maturation progressed. The analyses indicated that SH, SS, and SH+SS contents based on protein levels were similar in Elgin and Red Bobs tissues grown at 20°. Both varieties grown at 3° contained greater concentrations of SH, SS, and SH+SS groups based on protein levels than those present in the tissues grown at 20°. The Elgin tissues at 3° underwent a fluctuation of these values as growth progressed, while the Red Bobs tissues at 3° maintained relatively constant levels of SH, SS, and SH+SS contents based on protein levels present during the growth period. It was concluded that the levels or patterns of SH and SS groups in young spring and winter wheat shoots were not correlated with the capacity of these plants to flower. Title: Sulfhydryl and disulfide changes in young wheat seedlings during vernalization Author: Ellis, Robert William A study of the SH and SS content levels during vernalization of wheat was made. In order to carry out the study, a rapid analytical technique was worked out which allowed fast sequential analyses of the samples of wheat tissue. Two techniques were used: a) an amperometric titration method and b) a fluorescence quenching method. The amperometric titration method consisted of adding aliquots of samples of wheat tissue homogenate to the system and noting the decrease in current, due to a tie-up of Ag⁺ by the SH groups of the sample. The system consisted of a rotating platinum electrode connected to a mercury-mercuric oxide reference cell. The supporting electrolyte was a 0.14M solution of tris at pH 7.4. The fluorescence quenching method consisted of adding aliquots of sample to a solution of fluorescien mercuric acetate (FMA) in a 1N NaOH media. The SH and SS groups reacted with FMA in a one:one ratio. The reacted FMA was unable to fluoresce, thus making possible a quantitative assay for SH and SS groups in solution. Red Bobs, a spring wheat, and Elgin, a winter wheat, were grown in the dark at 3° C. and at 20° C. The SH and SS contents of the shoots were measured throughout a period of five weeks in the group grown at 3° C. and a period of 48 hours in the group grown at 20° C. The results of the analyses showed that the SH, SS, and SH+SS contents based on fresh weights were present in greater quantities in the tissues of Red Bobs grown at 20° than in the Elgin tissues grown at 20°. The high SH and SS concentrations present in the warm-grown Red Bobs tissues were due to a high concentration of protein extracted from that tissue. The SH, SS, and SH+SS contents per fresh weight were quite similar in the cold-grown tissues of both varieties during the growth period. These constituents present in the cold-grown tissues occurred in quantities that were less than those found in warm-grown Red Bobs tissues and greater than those present in the warm-grown Elgin tissues. Both varieties grown at both temperatures showed a similar decrease in SH and SS contents based on fresh weights as maturation progressed. The analyses indicated that SH, SS, and SH+SS contents based on protein levels were similar in Elgin and Red Bobs tissues grown at 20°. Both varieties grown at 3° contained greater concentrations of SH, SS, and SH+SS groups based on protein levels than those present in the tissues grown at 20°. The Elgin tissues at 3° underwent a fluctuation of these values as growth progressed, while the Red Bobs tissues at 3° maintained relatively constant levels of SH, SS, and SH+SS contents based on protein levels present during the growth period. It was concluded that the levels or patterns of SH and SS groups in young spring and winter wheat shoots were not correlated with the capacity of these plants to flower. Close

• 2. [Article] Some biochemical studies of phenylmercuric acetate and mercuric acetate toxicity in rats

  Biochemical studies of phenylmercuric acetate (PMA) and mercuric acetate toxicity in rats carried out during this investigation were (1) the metabolism of ²⁰³Hg(Ac)₂ and ²⁰³Hg-PMA within kidney and liver ...

  Citation × Citation Citation Abstract Copy Title: Some biochemical studies of phenylmercuric acetate and mercuric acetate toxicity in rats Author: Ellis, Robert William Biochemical studies of phenylmercuric acetate (PMA) and mercuric acetate toxicity in rats carried out during this investigation were (1) the metabolism of ²⁰³Hg(Ac)₂ and ²⁰³Hg-PMA within kidney and liver tissues following oral ingestion, (2) the effect on some soluble enzyme activities and determinations of ²⁰³Hg binding in the enzyme fractions, and (3) the metabolism of ¹⁴C-labeled substrates by kidney slices from control animals and animals receiving PMA and Hg(Ac)₂. In vitro ²⁰³Hg binding of the kidney soluble proteins was determined after incubating the soluble fraction with ²⁰³mercury labeled Hg(Ac)₂ and PMA. The proteins were separated by Sephadex G-100 gel filtration and the ²⁰³Hg and O.D.₂₆₀[subscript]mμ were determined in the eluate. Hg-binding depended on the type of mercurial and its concentration, and Hg did not seem to combine selectively with any particular group of proteins. Incubation of kidney slices in PMA solution resulted in mercury binding to groups of proteins of the soluble fraction in the 100,000 (peak I), 40,000 to 60,000 (peak II) and the 8,000 to 13,000 (peak III) molecular weight ranges, and incubation of Hg(Ac)₂ showed mercury binding to the peak I and III proteins. These binding patterns were different from those obtained with in vitro incubation of the soluble fraction. In vivo metabolism of ²⁰³Hg from a single dose of PMA or Hg(Ac)₂ resulted in a very large portion of ²⁰³Hg being bound to the peak III proteins, especially after 48 hours. PMA and Hg(Ac)₂ treatment resulted in different ²⁰³Hg-binding patterns the first 48 hours, especially in peaks I and II, and gradually became similar after that. Rats receiving multiple doses of PMA or Hg(Ac)₂ also showed similar labeling patterns in the kidney soluble proteins. However, the O. D. profile was definitely altered, with an increase of O. D. in the area of peak III. Single dose of PMA or Hg(Ac)₂, slightly increased the level of kidney soluble lactic acid dehydrogenase (LDH). Multiple doses of PMA or Hg(Ac)₂ decreased the LDH activity levels in the kidney soluble fraction. The maximum bindings were around 0.32 and 0.50 mole Hg/mole protein in the LDH fraction for Hg(Ac)₂ and PMA respectively. When the kidney soluble fraction was incubated in 1 x 10⁻⁴M Hg(Ac)₂, about 2-4 moles of Hg were bound per mole of protein, and the LDH activity was not affected. LDH in the rat kidney did not return to a normal value with a significant decrease in mercury binding when a 2 week period of time was allowed to lapse following Hg(Ac)₂ dosage. It is highly possible that mercury binding at the active site of LDH enzyme is not the prime factor involved for the reduction of activity. It may be that the synthesis of enzyme is affected. Single or multiple PMA dosage did not affect malic acid dehydrogenase (MDH) activity. A single dose of Hg(Ac)₂ showed no effect on MDH activity, but multiple doses showed a decrease. The Hg-binding was less than one mole of Hg/mole of protein in the MDH fraction. It seems doubtful that Hg binding at the active site of the MDH molecule is the main reason for the decrease of MDH activity following in vivo treatment. When kidney slices were incubated with lactate-1-¹⁴C, -2-¹⁴C, or glutamate-3,4-¹⁴C, a marked difference between those taken from control rats and from rats receiving oral doses of PMA (Hg accumulation; 198 μg²⁰³Hg/gr. fr. wt. kidney) or Hg(Ac)₂ (98 μg²⁰³Hg/gr. fr. wt. kidney) was observed. The effects were not due to an inhibition of lactate or glutamate uptake by the slices. Complete metabolism of labeled lactate or glutamate to CCO₂ and lactate-l- or -2-¹⁴C incorporation into glutamate were definitely affected more by PMA, and to a lesser degree by Hg(Ac)₂. It seems possible that in vivo mercurial treatment induced an inhibition of decarboxyla se activity in the kidney. Due to a reduction of lactate-¹⁴C conversion through the decarboxylating pathway, a greater amount of lactate would become available for its metabolism through another pathway (a combination of assimilation followed by a degradation). A larger second peak which was found in all runs with kidney slices from mercurial treated rats illustrates this alteration. A delay of 2 or 3 hours for the appearance of the second peak may be explained by the overall depression of metabolic reactions. A decrease in respiration of isolated kidney tissues following toxic doses of both inorganic and organic mercurials has been reported. Title: Some biochemical studies of phenylmercuric acetate and mercuric acetate toxicity in rats Author: Ellis, Robert William Biochemical studies of phenylmercuric acetate (PMA) and mercuric acetate toxicity in rats carried out during this investigation were (1) the metabolism of ²⁰³Hg(Ac)₂ and ²⁰³Hg-PMA within kidney and liver tissues following oral ingestion, (2) the effect on some soluble enzyme activities and determinations of ²⁰³Hg binding in the enzyme fractions, and (3) the metabolism of ¹⁴C-labeled substrates by kidney slices from control animals and animals receiving PMA and Hg(Ac)₂. In vitro ²⁰³Hg binding of the kidney soluble proteins was determined after incubating the soluble fraction with ²⁰³mercury labeled Hg(Ac)₂ and PMA. The proteins were separated by Sephadex G-100 gel filtration and the ²⁰³Hg and O.D.₂₆₀[subscript]mμ were determined in the eluate. Hg-binding depended on the type of mercurial and its concentration, and Hg did not seem to combine selectively with any particular group of proteins. Incubation of kidney slices in PMA solution resulted in mercury binding to groups of proteins of the soluble fraction in the 100,000 (peak I), 40,000 to 60,000 (peak II) and the 8,000 to 13,000 (peak III) molecular weight ranges, and incubation of Hg(Ac)₂ showed mercury binding to the peak I and III proteins. These binding patterns were different from those obtained with in vitro incubation of the soluble fraction. In vivo metabolism of ²⁰³Hg from a single dose of PMA or Hg(Ac)₂ resulted in a very large portion of ²⁰³Hg being bound to the peak III proteins, especially after 48 hours. PMA and Hg(Ac)₂ treatment resulted in different ²⁰³Hg-binding patterns the first 48 hours, especially in peaks I and II, and gradually became similar after that. Rats receiving multiple doses of PMA or Hg(Ac)₂ also showed similar labeling patterns in the kidney soluble proteins. However, the O. D. profile was definitely altered, with an increase of O. D. in the area of peak III. Single dose of PMA or Hg(Ac)₂, slightly increased the level of kidney soluble lactic acid dehydrogenase (LDH). Multiple doses of PMA or Hg(Ac)₂ decreased the LDH activity levels in the kidney soluble fraction. The maximum bindings were around 0.32 and 0.50 mole Hg/mole protein in the LDH fraction for Hg(Ac)₂ and PMA respectively. When the kidney soluble fraction was incubated in 1 x 10⁻⁴M Hg(Ac)₂, about 2-4 moles of Hg were bound per mole of protein, and the LDH activity was not affected. LDH in the rat kidney did not return to a normal value with a significant decrease in mercury binding when a 2 week period of time was allowed to lapse following Hg(Ac)₂ dosage. It is highly possible that mercury binding at the active site of LDH enzyme is not the prime factor involved for the reduction of activity. It may be that the synthesis of enzyme is affected. Single or multiple PMA dosage did not affect malic acid dehydrogenase (MDH) activity. A single dose of Hg(Ac)₂ showed no effect on MDH activity, but multiple doses showed a decrease. The Hg-binding was less than one mole of Hg/mole of protein in the MDH fraction. It seems doubtful that Hg binding at the active site of the MDH molecule is the main reason for the decrease of MDH activity following in vivo treatment. When kidney slices were incubated with lactate-1-¹⁴C, -2-¹⁴C, or glutamate-3,4-¹⁴C, a marked difference between those taken from control rats and from rats receiving oral doses of PMA (Hg accumulation; 198 μg²⁰³Hg/gr. fr. wt. kidney) or Hg(Ac)₂ (98 μg²⁰³Hg/gr. fr. wt. kidney) was observed. The effects were not due to an inhibition of lactate or glutamate uptake by the slices. Complete metabolism of labeled lactate or glutamate to CCO₂ and lactate-l- or -2-¹⁴C incorporation into glutamate were definitely affected more by PMA, and to a lesser degree by Hg(Ac)₂. It seems possible that in vivo mercurial treatment induced an inhibition of decarboxyla se activity in the kidney. Due to a reduction of lactate-¹⁴C conversion through the decarboxylating pathway, a greater amount of lactate would become available for its metabolism through another pathway (a combination of assimilation followed by a degradation). A larger second peak which was found in all runs with kidney slices from mercurial treated rats illustrates this alteration. A delay of 2 or 3 hours for the appearance of the second peak may be explained by the overall depression of metabolic reactions. A decrease in respiration of isolated kidney tissues following toxic doses of both inorganic and organic mercurials has been reported. Close