Howard M. Jernigan

Purification and properties of rat lens methionine adenosyltransferase

Methionine adenosyltransferase (MAT) has been partially purified from rat lenses using a combination of ammonium sulfate fractionation and hydrophobic chromatography on phenyl Sepharose columns. The partially purified enzyme resembles purified Type II MAT from non-hepatic tissues. The Km for methionine is 3.0 microM, and the Km for ATP is 80 microM. The enzyme is activated by potassium ions (25-50 mM), and inhibited by higher concentrations of potassium. A divalent cation (magnesium or manganese) is essential for activity. The Vmax with magnesium is about five times higher than with manganese, but the optimal manganese concentration is around 2.0 mM, compared with 10-20 mM for magnesium. The enzyme is active over a broad pH range, with optimal activity between pH 7.0 and 8.0. The enzyme is inhibited by all three of its products, phosphate, pyrophosphate, and S-adenosylmethionine. Individually phosphate and pyrophosphate are weak inhibitors, but in combination they show a marked synergistic inhibitory effect. Tripolyphosphate is also an effective inhibitor. The inhibition of the enzyme by the cataractogenic agent, dimethylsulfoxide, further confirmed the similarity to Type II MAT.

Methionine adenosyltransferase and S-adenosylmethionine in the developing rat lens.

Methionine adenosyltransferase (MAT) activity, and the concentration of its reaction product, S-adenosylmethionine (AdoMet), were measured in the lenses of rats of different ages; ranging from 1 day of age to over 1 yr. The highest specific activity of MAT was found in the lenses of the one day old rats (sp. ac. 0.327 units/mg-1 protein). After 1 week the specific activity had dropped to 0.067, and by 6 weeks had declined to adult levels (0.02 units/mg-1 protein). AdoMet concentrations were measured by HPLC in perchloric acid extracts. The highest concentration of AdoMet was found in the lenses of day-old rats (48.2 microM), and gradually declined with increasing age, reaching 5.5 microM in the oldest rats. In addition, the specific activity of MAT was found to be higher in the lens epithelium than in the cortex plus nucleus. The specific activity of MAT is almost an order of magnitude higher in the lens epithelial fraction (0.099 units mg-1 protein) than in the combined cortex plus nucleus fraction (0.011 units mg-1 protein).

The effect of an aldose reductase inhibitor on lens phosphorylcholine under hyperglycemic conditions: biochemical and NMR studies.

Phosphorylcholine (P-choline) is a precursor of the phospholipids in the lens membrane. A human lens normally contains approx. 1 mM P-choline but this is significantly lowered in some cataractous lenses. A normal rat lens contains a very high concentration (11 mM). We found that rat lens P-choline was depleted drastically when the lenses were exposed to hyperglycemic conditions either in culture, with galactose or xylose, or in vivo by streptozotocin-induced diabetes. The lens P-choline level was measured by fractionating the organic phosphates in the lens homogenate using an ion exchange column, or by quantitating the P-choline 31P NMR intensity in intact lenses. The results of both the chemical method and the noninvasive method agreed remarkably well. Besides the change in P-choline, the choline influx was also drastically reduced both in lenses from diabetic rats and in lenses incubated with 30 mM xylose. In addition, the ATP concentration was greatly diminished under similar conditions. The changes in P-choline, choline, and ATP could all be prevented in the presence of an aldose reductase inhibitor (ARI). It is thus concluded that these changes in phospholipid precursors may result from lenticular membrane defects caused by hyperglycemic stress. The effect of the lowered precursors on lipid biosynthesis was observed, and surprisingly showed a more rapid phospholipid-biosynthesis in the 2-week diabetic rat lens than in the 3-day diabetic rat lens.

Effects of Cataractogenesis on the CDP-Choline Pathway: Changes in ATP Concentration and Phosphocholine Synthesis during and after Exposure of Rat Lenses to Sugars in vitro and in vivo

We measured choline influx and phosphorylation, ATP concentration ([ATP]), choline kinase activity and lens swelling during formation and partial reversal of sugar cataracts in rat lenses incubated with xylose or galactose and in lenses of galactosemic rats. [ATP] and phosphocholine (P-Cho) synthesis decreased about 60 and 40% after 4 h in normal rat lenses incubated up to 24 h in medium containing 30 mM xylose and partially recovered when the lenses were then removed from the xylose. Incubation with the somewhat less cataractogenic sugar galactose decreased P-Cho synthesis but had little effect on [ATP]. P-Cho synthesis decreased rapidly in the lenses of rats fed a 50% galactose diet, but began recovery by the third day on this diet. [ATP] decreased for at least 10 days during the galactose diet and did not recover, even with resumption of the control diet (50% starch) after 4 or 7 days. The results of in vitro and in vivo sugar cataractogenesis differed from each other in several respects, including effects on choline influx and the degree to which the changes were reversible. The in vitro and in vivo sugar cataracts, however, could both produce swelling and opacification of the lens and decreased P-Cho synthesis and [ATP]. Neither model caused a substantial change in the choline kinase activity (as measured in cell-free assays). The data did not generally support the hypothesis that decreased [ATP] causes decreased P-Cho synthesis.

Metabolism of glutamine and glutamate in monkey lens

In rat and bovine lenses, the primary source of intracellular glutamate has been shown to be glutamine transported from the surrounding fluids, whereas extracellular glutamate is less readily utilized. For comparison, glutamine and glutamate metabolism were studied in a primate. Fresh, intact lenses from Rhesus monkeys (Macaca mulatta) were incubated in balanced salt medium containing [15N]glutamate (Group A) or amino-labeled [15N]glutamine (Group B). In contrast to other species, the monkey lenses metabolized the glutamate more rapidly than the glutamine, although glutamine entered the lenses more rapidly than glutamate. Formation of labeled aspartate, alanine, proline, and serine was more rapid in Group A than in Group B, but labeling of the lenticular glutamate + glutamine pool was more rapid in Group B. This indicated that, in monkey lenses, deamidation of glutamine is sufficiently slow to limit the entry of glutamine into pathways requiring or preferring glutamate. The difference in rates of deamidation of glutamine in intact monkey lenses and rat lenses was confirmed by measuring the rate of ammonia release by lenses incubated with glutamine.

Rat lens glutaminase: Separation and characterization of soluble and particulate fractions

The ability of rat lenses to import glutamate from the aqueous humor is limited by the activity of the transporter for dicarboxylic acids in this tissue. The principal source of glutamate for rat lens metabolism appears to be glutamine, which enters the lens much more readily than glutamate and is then deamidated by glutaminase. Both soluble and particulate glutaminase activities were obtained from rat lenses by extraction and differential centrifugation. The lens fractions were compared with previously reported isoenzymes of glutaminase from rat kidney and liver. The apparent Km for glutamine of the soluble preparation from lens was in the range from 17- to 24 mM, while that of the particulate lens fraction was 5 mM. Gel filtration on Sepharose 4B demonstrated that the lens soluble glutaminase in Tris buffer is similar in size to the kidney enzyme and, like the glutaminase from kidney, reversibly formed active aggregates in borate buffer. The liver enzyme did not form aggregates under identical conditions. The glutaminase preparations were all dependent on phosphate for complete activation, but the lens particulate fraction is more active than the lens or kidney soluble fractions at low concentrations of inorganic phosphate. Unlike the soluble enzyme, the particulate glutaminase was partially activated by phosphorylcholine. Rat lenses contain 11 mM phosphorylcholine, and this unusually high concentration of phosphorylcholine may be sufficient to partially activate the enzyme. These properties of the particulate fraction suggest that the membrane-bound glutaminase may be physiologically important in the lens in vivo.

Serine hydroxymethyltransferase : evidence for its presence in human, monkey and rat lenses

Serine hydroxymethyltransferase (SHMT) is present in cultured rat, monkey and human lenses as shown by 15N-serine or 15N-glycine labeling studies. Following incubation with 15N-serine, the percent enrichment of 15N in glycine increases with time, and vice versa, demonstrating the presence of the enzyme, and the reversibility of the reaction in intact cultured lenses. Similar patterns of 15N enrichment were found in all three species, but lenses from young rats showed a higher percent enrichment than lenses obtained from older animals. Label from 15N-serine or 15N-glycine was also incorporated into a number of other amino acids, including aspartate, alanine, glutamate and proline. Conclusive evidence for the presence of SHMT in rat lens homogenates has been obtained by direct enzyme assay. The specific activity of rat lens SHMT was age dependent; approximately 2.4 units per mg protein in day old rats, declining to about 0.15 units per mg in adult animals. The higher specific activity observed in younger animals is consistent with the 15N labeling results obtained with cultured lenses. Lens SHMT has been partially characterized. In the presence of excess tetrahydrofolate the assay was essentially linear with increasing time. With serine as the substrate, the enzyme requires tetrahydrofolate for activity, the pH optimum is between pH 7.5 and 8.3, the Km for serine is about 0.25 mM, and the enzyme is inhibited by cycloserine. In conclusion, this study demonstrates the existence of SHMT in rat, monkey and human lenses. Rat lens specific activity has been shown to decrease with increasing age, and the enzyme has been partially characterized.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of glutamine and glutamate in human lenses

Glutamate is important to lenses as a central intermediate in amino acid metabolism, as well as for synthesis of proteins and glutathione. In rat and calf lenses, the principal source of lenticular glutamate is glutamine, which enters the lens and is deamidated to form glutamate. In contrast, monkey lenses use external glutamate more readily than glutamine. Amino acid metabolism was studied in human lenses by incubating them with amino-labeled [15N]glutamine or [15N]glutamate. The lenticular free amino acids were then isolated and analysed by gas chromatography-mass spectrometry to determine 15N-labeled products. The results were compared with those of similar experiments with lenses from other species. Label was measured in aspartate, alanine, serine and proline, as well as lenticular glutamine and glutamate. Glutamine enters lenses more readily than glutamate in all the species examined. Nevertheless, aspartate, alanine and serine were more rapidly labeled by incubating human lenses in [15N]glutamate than in [15N]glutamine. This observation is similar to reports of experiments with monkey lenses, which unlike rat lenses, preferentially utilize glutamate rather than glutamine. In contrast, human lens proline was more rapidly labeled by incubating lenses with [15N]glutamine than with [15N]glutamate. In human lenses, the relatively slow utilization of glutamine for the transamination reactions which form aspartate, alanine and serine appears to result from slow deamidation. The relative preference for glutamine over glutamate as a precursor for proline synthesis in human lenses may be related to the mitochondrial location of the enzymes involved.