William B. Rathbun

Estimation of enzymically produced orthophosphate in the presence of cysteine and adenosine triphosphate.

Abstract A method utilizing phosphomolybdate is described for determination of enzymically formed orthophosphate in the presence of high concentrations of cysteine, ATP, and ADP. This sensitive procedure allows the rapid estimation of phosphate for the large number of samples that are usually encountered in enzyme studies. The variables of the procedure were examined in detail, and the method which evolved minimizes errors in practical enzyme assay procedures.

Age-related cysteine uptake as rate-limiting in glutathione synthesis and glutathione half-life in the cultured human lens

The study included human lenses of ages ranging from newborn to 92 years. Protein-free reduced glutathione decreased 14-fold, whereas protein-free oxidized glutathione increased 2.6-fold with increasing age. L-Cyst(e)ine uptake g-1 lens of very old cultured lenses decreased 70% from that exhibited in newborn lenses, demonstrating a marked decline of L-cyst(e)ine uptake as a function of age. In these same lenses the synthesis of reduced glutathione (mumol g-1 lens) decreased 73% with age. It was concluded that the glutathione decrease observed in the aging human lens was associated with decreased uptake of L-cyst(e)ine, decreased glutathione synthesis and possibly an increase in protein-free oxidized glutathione. The high correlation of L-cyst(e)ine uptake and glutathione synthesis supports the hypothesis that L-cyst(e)ine uptake is a rate-limiting factor of glutathione synthesis in the intact human lens. By the use of buthionine sulfoximine, the half-life of glutathione was estimated to be 90 hr in the cultured human lens.

Activity of glutathione peroxidase and glutathione reductase in the human lens related to age

Lenses from 42 eye bank eyes were assayed for glutathione peroxidase and glutathione reductase activities. The activity of glutathione peroxidase, when considered as a function of age, was lowest in the neonate lens, increasing with age to reach maximal values in young adult lenses, and thereafter progressively decreasing with ages greater than 40 years. Glutathione reductase activity was little affected by age when expressed as activity per lens, per gram lens or per mg soluble protein, indicating that activity of this enzyme did not increase with lens size as would a representative lenticular protein. However, the activity of this enzyme per gram lens was among the highest of any species yet examined.

γ-Glutamyl-cysteine synthetase from bovine lens: I. Purification and properties

Abstract The bovine lens enzyme catalyzing the first step of glutathione synthesis, γ-glutamyl-cysteine synthetase, was purified 260-fold with a recovery of 60% of the original activity. The instability of the enzyme required the rapid use of each solvent near its freezing point. Progress in fractionation was followed by immunoelectrophoresis with rabbit antisera produced in response to two of the purified fractions. Inhibition by ADP required extrapolation to initial velocity for apparent K m valus determinations of glutamate, ATP, cysteine, and α-aminobutyrate. The requirements for the glutamate moiety were a five-carbon dicarboxylic acid with a free amino group in the l -configuration. Allohydroxyglutamate, α-methylglutamate and β-hydroxyglutamate were capable of substituting for l -glutamate. The l -configuratioii was required for the cysteine moiety; α-amino butyrate substituted for l -cysteine. The enzyme from bovine lens and hog liver differ in their pH optima and instability to a variety of conditions. This indicates the enzymes from the two sources may be distinctly different proteins.

Glutathione metabolism in lenses of dogs and rabbits: activities of five enzymes.

Several biochemical parameters were examined in clear dog and rabbit lenses as functions of age, and in posterior subcapsular cataracts in the Alaskan malamute. Tabulated data include soluble protein, reduced sulfhydryl content of soluble protein, reduced glutathione, water, and activity of five enzymes of glutathione metabolism. The enzymes include the glutathione biosynthesis system consisting of gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione peroxidase, glutathione reductase and glutathione-S-transferase. Each enzyme, acting last in a sequential reaction of either two or three reactions, was in excess activity over the preceding enzyme(s) in every case but one. In the exception, the ratio of glutathione reductase to glutathione peroxidase activity was about 1:600 and 1:155 in the dog and rabbit lens, respectively.

Bovine lens γ-glutamyl transpeptidase

Abstract An enzyme, γ-glutamyl transpeptidase, capable of using glutathione as a substrate, was demonstrated in the bovine lens. The capsule-epithelium fraction contained the highest concentration of enzyme activity, but a substantial proportion of the total was contained in the cortical fraction at a low level. The enzyme, purified from the capsule-epithelium fraction with the aid of a spectrophotometric assay based upon γ-glutamyl- p -nitroanilide cleavage, was solubilized by sodium deoxycholate and further purified by butanol extraction and gel exclusion chromatography. The enzymic synthesis of a transpeptidation product, γ-glutamylmethionine, from glutathione or γ-glutamyl- p -nitroanilide and l -methionine was demonstrated. A possible role of the enzyme in amino acid transport is discussed.

Glutathione peroxidase from bovine lens: a selenoenzyme

Abstract Glutathione peroxidase was purified 12 000-fold to homogeneity from the bovine lens. Purification utilized ammonium sulfate, acetic acid and zinc sulfate precipitations, and covalent chromatography on activated Thiolsepharose 4B. The specific activity of the purified enzyme is 1600 and its estimated molecular weight is 140 000. It is a selenoenzyme, containing 4 mol of selenium per mol of enzyme. Its pH maximum is 7·9. The apparent K m s for glutathione, tert-butylhydroperoxide, cumene hydroperoxide and H 2 O 2 are 2·9, 0·54, 0·65 and 0·045 m m , respectively. Iodoacetic acid irreversibly inhibits both the reduced and oxidized forms of the enzyme, whereas p -chloromercuriphenyl sulfonic acid only inhibits the oxidized form and N -ethyl maleimide, the reduced form.

γ-Glutamyl-cysteine synthetase from bovine lens: II. Cysteine analogue studies

Abstract Seventy-one compounds have been tested for their ability to couple with l -glutamic acid to form a γ-glutamyl peptide bond through the action of a purified preparation of γ-glutamyl-cysteine synthetase from bovine lens. The most active amino acids in the system were l -cysteine, l -α-aminobutyrate, and their esters, β-chloro- l -alanine, S -methyl- l -cysteine, l -cycloserine, l -norvaline, allothreonine, allylglycine, β-aminoisobutyrate, and l -homocysteine. The specificity pattern displayed by the enzyme allowed certain conclusions to be drawn concerning its requirements for the cysteine moiety. Several of the isolated enzymic products were hydrolyzed, and formed glutamic acid and the original cysteine analogue. The values for the apparent K m and maximal velocity were determined for many of the reactions.

Inhibition of Na, K-ATPase by sodium selenite and reversal by glutathione

Sodium selenite has been shown to inhibit Na,K-ATPase. Glutathione, at sufficient excess, is able to prevent or reverse the inhibition. Dithiothreitol can also reverse much of the inhibition, but KCN cannot. Selenomethionine does not inhibit Na,K-ATPase. The interactions of sodium selenite with Na,K-ATPase and glutathione may aid in understanding the early events in selenium cataractogenesis.

Species Survey of Glutathione Peroxidase and Glutathione Reductase: Search for an Animal Model of the Human Lens

Lenses from representative species of eight mammalian orders were assayed for glutathione peroxidase and glutathione reductase activities. Wide variation of glutathione peroxidase activity was noted, the lowest activity being that of the prosimian galago while the highest activities were from three Old World monkey species of the genera Macaca and Papio. The hominoids, including the human, all exhibited lower activities of this enzyme. Glutathione reductase activity was lowest in the dog, the rabbit, and all species of cats. The higher Old World monkeys and hominoids, including the human, exhibited enzyme activities many times greater than any other species except the woodchuck. Since glutathione reductase is the rate-controlling enzyme of the glutathione redox cycle, it may be concluded that the most suitable model for the human lenticular glutathione redox cycle will only be found among the higher primates.