Raymond H. Lindsay

Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent.

Studies of protein-bound (PB-SH) and nonprotein bound sulfhydryl group (NP-SH) concentrations in tissues under various conditions is a prerequisite to understanding the role of sulfhydryls in living organisms. Many methods have been developed for the measurement of sulfhydryl groups, but these have been concerned chiefly only with the estimation of NP-SH in biological fluids or total SH groups (T-SH) in purified protein or other substances. The reviews on SH determinations by Chinard and Hellerman (1) and by Benesh and Benesh (2) give excellent and comprehensive discussions of the general problems involved in sulfhydryl determinations. However, few investigators (3-5) have discussed the concomitant estimation of PB-SH, NP-SH, and T-SH in biological materials and none of the methods used is convenient for the routine estimation of sulfhydryls in these fractions. In addition, Jocelyn’s method (4) has been unsatisfactory in our hands because of poor reproducibility and recoveries. The purpose of this communication is to report a simple spectrophotometric method for the routine concomitant determination of sulfhydryl groups in PB-SH, NP-SH, and T-SH fractions in various tissues. These spectrophotometric procedures are based on the method of Ellman (6, 7), who reported that 5,5’-dithiobis(2,-nitrobenzoic acid) is reduced by SH groups to form 1 mole of 2-nitro-5-mercaptobenzoic acid per mole of SH. The reaction is:

An isotopic assay for uridine phosphorylase

Abstract An isotopic assay for measurements of uridine phosphorylase activity has been presented. Uracil-2- 14 C and uridine-2- 14 C, which would serve either as substrate or product for this reversible reaction, were complexed with borate and separated on an ion-exchange column, making possible their quantitation with the radioactivity in each fraction. Separation of these compounds with 0.7 × 2.5 cm columns was achieved with recoveries of 96 to 101%. The uridine nucleotides UMP, UDP, and UTP were retained on the column and subsequently removed individually or collectively with the appropriate eluent. The method recommended for the assay of crude enzyme is based on utilization of uracil-2- 14 C as a substrate. Incorporation of radioactivity into uridine and uridine nucleotides would then be measured separately or collectively.

Site of glucuronide conjugation to the antithyroid drug 6-n-propyl-2-thiouracil

Abstract Chromatographyically pure, essentially salt-free radioactive 6- n -propyl-2-thiouracil (PTU) glucuronides were isolated from rat bile and urine and synthesized with guinea pig liver microsomes to determine if more than one PTU glucuronide was formed and to determine which group or groups in the PTU molecule was glucuronidated. Analyses on Bio-Gel P-2 and DEAE-Sephadex A-25 columns and on TLC sheets in five solvent systems demonstrated that the three glucuronide preparations were chromatographically identical. Furthermore, the reactivities of the three glucuronides with 1 N HCl, methyl iodide, sodium azide-iodine reagent, 2,6-dichloroquinone-chloroimide and H 2 O 2 were also identical strongly indicating that a single PTU glucuronide was formed. The PTU glucuronide was partially hydrolyzed by 1 N HCl to 6- n -propyl-uracil (PU), a reaction typical of S -conjugated PTU; demonstrated greatly reduced reactivity with methyl iodide whereas the S of PTU was readily methylated; exhibited a negative reaction in the azide-iodine test which was a certain indication that the C—SH or Cue5fbS group was not present; failed to react with 2,6-dichloroquinone-chloroimide which reacts with the Cue5fb=S of PTU and provides the basis for a colorimetric assay for PTU; and was not oxidized by H 2 O 2 to form sulfate as are all PTU derivatives except S conjugates of PTU. Furthermore PU, which possesses identical potential conjugation sites with the exception of the S , was not glucuronidated under conditions in which PTU was readily conjugated. The results obtained strongly indicate that the glucuronide is conjugated to the S of PTU.

Relative Antithyroid Effects of 2-Thiouracil, 2-Thiouridine and 2-Thio-UMP

Summary The presence of uridine phosphorylase and thymidine phosphorylase, enzymes capable of converting thiouracil to thiouridine, was demonstrated in thyroid tissue from several species. Uridine kinase which catalyzes further metabolism of thiouridine to thio-UMP was also present. The activity of UMP pyrophosphorylase which converts thiouracil to thio-UMP in a one-step reaction was insignificant. Comparisons of antithyroidal activity in intact rats and with partially purified porcine thyroid peroxidase demonstrate that thiouridine was approximately 10% as potent as thiouracil in vivo but less than 1% as active as thiouracil as an inhibitor of thyroid peroxidase. Thio-UMP was about 5% as active as thiouracil on thyroid peroxidase. These results indicate that it is highly unlikely that thiouracil conversion to a nucleoside or nucleotide is involved in its antithyroidal action.

Purification of Bovine Thyroid-Stimulating Hormone by Solubility Fractionation with Ammonium Sulfate∗:

Summary Solubility fractionation of relatively crude TSH with ammonium sulfate was investigated as a means of purifying the hormone. The results obtained with three variations in solubility fractionation techniques using decreasing concentrations of ammonium sulfate demonstrated that each method yielded a 5- to 7-fold purification of the 0.49 to 0.84 U/mg starting material. Total recovery of TSH was 90-100%. Recoveries in the high potency fractions were 60-80% when TSH was extracted with ammonium sulfate or recovered by fractional precipitation and 25-35% when batch eluted from diethylaminoethyl (DEAE) cellulose. Solubility fractionation appears to be a simple and rapid method for the purification of TSH to 4-6 U/mg which may be adequate for most purposes. Highly purified TSH may then be obtained by standard column chromatography with DEAE-cellulose.

Metabolism of 2-Thiouracil in Pyrimidine Pathways Leading to Nucleotide Synthesis

Summary The present results demonstrate that the antithyroid drug 2-thiouracil can be metabolized in animal tissues to the corresponding nucleotide by at least two pathways. In one pathway, uridine phosphorylase catalyzes the reversible conversion of thiouracil to thiouridine and further metabolism of 2-thiouridine to thio-UMP is catalyzed by uridine kinase. In the other pathway, thiouracil is converted to thiouridine by thymidine phosphorylase, then to thio-UMP by uridine kinase. Although thymidine phosphorylase can also form 2-thiodeoxyuridine, further metabolism of this compound by thymidine kinase to a deoxyribonucleotide does not appear to occur.

Enzyme secretion invitro in the absence of alterations in oxidation

Abstract The effects of various concentrations of epinephrine and dibutyryl cyclic 3′, 5′-AMP (dcAMP) on enzyme secretion and glucose oxidation by rat parotid in vitro were determined. Epinephrine at 0.33 μg/ml increased α-amylase secretion 43% without significantly altering glucose oxidation. Higher concentrations of epinephrine increased both secretion and glucose oxidation. The cyclic 3′,5′ -AMP (cAMP) derivative dcAMP stimulated amylase secretion 157% at 200 μg/ml without producing any accompanying increase in glucose oxidation. These results indicate that changes in oxidation are not essential for the cAMP induced phase of enzyme secretion and that the increase in glucose oxidation produced by epinephrine is not mediated by cAMP.

Accumulation of I131 by channel catfish (Ictalurus punctatus) ovaries in vivo and in vitro

Ovaries of channel catfish (Ictalurus punctatus) were found to concentrate I131 with accumulation reaching a peak 20–30 hours after injection. A major portion of the injected I131 was recovered in the ovaries which accounted for 45.0% of the injected dose after 24 hours while the thyroid accounted for only 2.43%. Accumulation of I131 in the ovaries occurred against a concentration gradient with ovary to blood ratios after 24 hours of 32:1. Accumulation of I131 was also demonstrated by ovarian tissue in vitro with tissue to medium ratios as high as 96:1. Concentration of I131 in vitro was inhibited by 2,4-dinitrophenol, sodium perchlorate, and sodium thiocyanate, but was not affected by methimazole or thiouracil. The I131 in fish ovaries accumulated in vitro appeared to be predominately in an inorganic form. However, seasonal variations were noted in which I131 uptake by ovaries obtained in June, as opposed to those obtained in January, appeared to be more firmly bound to the tissue. Traces of I131 were present in the protein band after electrophoresis of tissue homogenates and 2–5% of the I131 was associated with protein after paper chromatography.

Metabolism of the Nucleotide (2-Thio-UMP) of the Antithyroid Drug 2-Thiouracil:

Summary The results presented demonstrated that a mixture of rat liver mono- and diphosphokinases metabolized 2-thio-UMP to two substances. One was identified as thio-UTP. The other had some properties similar to thio-UDP but was chromatographically different. The rate of UMP conversion to UDP and UTP by this enzyme system greatly exceeded that of thio-UMP to nucleoside triphosphate. Thio-UMP conversion to thio-UTP demonstrates that pathways are present in animal tissues for the metabolism of 2-thiouracil to 2-thio-UTP. The enzymes involved appear to be identical or very similar to those converting uracil to UTP.

The Relationship of Epinephrine Oxidation to Rat Parotid Metabolism and Secretion in Vitro

Summary Epinephrine conversion to ad-renochrome by rat parotid in vitro was verified by the appearance of a pink color in the medium and an increase in UV absorbance. Preincubation with epinephrine to increase adrenochrome concentration during incubation produced no alteration in the magnitude of catecholamine stimulation of glucose-6-14C oxidation. Inhibition of epinephrine conversion to adrenochrome or of degradation by monamine oxidase also had no effect on the response to this catecholamine. Although epinephrine produced pronounced stimulation of α-amylase secretion, glucose-6-14C oxidation, leucine-U-14C oxidation, and inhibition of leucine-U-14C incorporation into rat parotid protein, no measurable effects were produced by adrenochrome. The present results strongly indicate that epinephrine rather than adrenochrome or other oxidation products is primarily responsible for the alterations in α-amylase secretion and metabolism observed with rat parotid in vitro.