C. Ohrloff

Glutathione peroxidase, glutathione reductase and superoxide dismutase in the aging lens

Glutathione reductase (GR) and glutathione peroxidase (GPx) show in bovine lenses a decrease in specific activity; furthermore, the heat lability of both enzymes increases with age monitoring structural changes of the molecules. GR activity was correlated with type of cataract in human lenses. Its decrease is significantly connected with cortical opacities. Superoxide dismutase activity declines in aging and cataractous lenses. These results support the assumption that in old lens tissue the capacity of the antioxidative scavanger system is diminished.

Separation of lens proteins in rats with tryptophan deficiency cataracts

Abstract Rats on tryptophan-free diet develop posterior subcapsular (saucershaped) cataracts and discrete anterior subcapsular haze. Trp-deficient rats have a lower lens dry weight than normal lenses of the same age. There is a slight significant increase of lens water in the diet group. The water insoluble lens protein showed no changes by the trp-free diet, whereas β-crystallins were severely affected. After 20 days of trp deficiency the amount of β-crystallin was only 72% of the value found in controls. α- and γ-crystallins were less affected and showed only a slight (but significant for α-crystallin) decrease.

Experimental cataracts in rats due to tryptophan-free diet

A Tryptophan-free diet induces posterior subcapsular cataracts and reversible corneal opacities in young Wistar rats. Compared to the controls there is a significant decrease of body weight, lens fresh weight, and water-soluble lens protein. Protein separation by isoelectric focusing shows diminished α-, β-, and γ-crystallin fractions. If tryptophan is restored to the diet after 24 days, new clear lens fibers are laid down again, indicating that the mechanism of protein synthesis has not been permanently damaged by tryptophan-deficiency. Eine tryptophanfreie Diät führte bei jungen Wistarratten zur Ausbildung von reversiblen Hornhauttrübungen und einer hinteren subkapsulären Katarakt. Im Vergleich zu den Kontrolltieren sind das Körpergewicht, das Linsenfrischgewicht und der Anteil an wasserlöslichen Linsenproteinen vermindert. Eine Trennung mit Isoelektrofokussierung zeigt eine Abnahme der α-, β- und γ-Kristalline. Da es nach Absetzen der Diät und Gabe von tryptophanhaltigem Futter wieder zur Entwicklung klarer Linsenfasern kommt, wird der Proteinsyntheseapparat vom Tryptophanmangel nicht bleibend geschädigt.

Immunologic detection of inactive enzyme molecules in the aging lens

The specific activity of nearly all enzymes of the carbohydrate metabolism declines during aging. In rat lenses, this decrease is accompanied by a loss in the heat-unstable portions of the aldolase isoenzymes A and C. By means of the quantitative immunologic titration and the use of enzymespecific antibodies, inactive aldolase molecules, the so-called cross-reacting material, could be determined in 2-year-old rat lenses. The cross-reacting material could also be determined for phosphofructokinase in the nucleus of 6-year-old rabbit lenses.

Investigations on phosphofructokinase (PFK, EC 2.7.1.11) in bovine lenses in dependence on age, topographic distribution and water soluble protein fractions

The specific activity of phosphofructokinase and its affinity to the substrate fructose-6-phosphate was determined with reference to the age of lens. The allosteric behaviour of the enzyme was eliminated by choosing the appropriate assay conditions. Age-dependent changes could be observed in both parameters. The specific activity decreased by 50% during the first 4 years, thereafter remaining almost constant. It was highest in the lens equator followed by anterior and posterior cortical region and lens nucleus. The substrate saturation curve for the calf lens shows a typical hyperbolic course, (Michaelis-Menten) and the slope of Lineweaver-Burk plot gives a Km-value of 5 × 10−5 m for F-6-P. For the nucleus of calf and bovine lenses the Lineweaver-Burk diagram shows convex curves indicating heterogeneity of the kinetic properties due to aging. After separation of the water soluble proteins the activity of phosphofructokinase could be localized in the α-crystallin as well as in the β-crystallin fraction.

Ketohexokinase Activity in Whole Bovine Lenses and Single Lens Parts in Dependence on Age

The specific activity of ketohexokinase (EC 2.7.1.3), initiating the fructose utilization of lenses, depends upon age. In whole bovine lenses, this activity was found to be at a maximum at an age of about 9 months and, after subsequent decrease, the activity shows no further changes after an age of about 3 years. The highest activity at all ages investigated was found in the lens quator, with regional activities decreasing therefrom in the order: anterior cortex, posterior cortex and nucleus. The age dependency of ketohexokinase activity in the different lens parts differs from that of the whole lens. During ageing, there is an increase in specific activity in the equator; anterior and posterior cortex show almost constant values and the activity of the lens nucleus steadily decreases. The importance of these findings is discussed with respect to the age-induced changes in the lens carbohydrate metabolism.

Ageing of Lens Metabolism

The significance of age-related changes of enzyme activities does not only lie in the decrease of the specific activities but also in the changes of their kinetic and physico-chemical properties, whic

Presence of Ketohexokinase (EC 2.7.1.3) in Human Lenses

Ketohexokinase (EC 2.7.1.3) is present in bovine lenses and facilitates fructose degradation to fructose-1-phosphate. The importance of this finding to lens carbohydrate metabolism is discussed.

[Studies on the kinetics of lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (E.C. 1.1.1.37), and fructosediphosphate aldolase (EC 4.1.2.13) in calf and bovine lenses].

The specific activities of LDH, MDH and ALD in whole lenses of bovines as well as in the equatorial zone of bovine and calf lenses were investigated and the individual K M -values determined. Additional investigations were concerned with fractionating the watersoluble protein of the bovine lens equator into α- and β-fractions, and the specific activities of LDH, MDH and ALD and their K M -values in these fractions were determined. In bovine lenses we found less specific activity of the enzymes under investigation than in calf lenses which could be qualified through their K M -values (Table 1). Of special interest is the behaviour of the MDH isoenzyme of the α-fraction, which was inhibited at an oxalacetate concentration of 0.1 mM and more. The isoenzyme of the β-fraction was not influenced by substrate concentration. ALD, however, was only present in the α-fraction. This means that the age induced changes in the α-fraction have less influence on the LDH and MDH activity, because isoenzymes of the β-fraction are present, which may compensate the loss of the enzymatically active protein of the α-fraction. Es wurden die spezifischen Aktivitäten der LDH, MDH und ALD in der ganzen Rinderlinse sowie in der Äquatorzone der Rinder- und Kälberlinse untersucht und außerdem ihre K M -Werte bestimmt. Zusätzlich erfolgte über Sephadex G-200 eine Fraktionierung des wasserlöslichen Proteins des Rinderlinsenäquators in eine α- und β-Fraktion, in denen ebenfalls die Messungen durchgeführt wurden. Im Äquator der Rinderlinse fanden sich stets niedrigere spezifische Aktivitäten als in der Kälberlinse. Für die LDH und MDH wurden Isoenzyme in den beiden Kristallfraktionen festgestellt, die durch jeweils unterschiedliche K M -Werte qualifiziert werden konnten (Tab. 1). Das MDH Isoenzym der α-Fraktion zeigte bei einer Oxalacetatkonzentration von 0,1 mM und darüber eine Aktivitätshemmung. ALD-Aktivität wurde nur in der α-Fraktion nachgewiesen. Die Abnahme von Enzymaktivitäten während des Alterungsprozesses der Linse sind möglicherweise eng mit den Veränderungen im Gehalt an wasserlöslichen Proteinen verbunden. Das Vorkommen von Isoenzymen ist daher von besonderer Bedeutung. Die bekannten Altersveränderungen des α-Kristallins werden für den Linsenstoffwechsel weniger Wirksamkeit besitzen, wenn der Verlust des enzymatisch aktiven Proteins durch Isoenzyme kompensiert werden kann.

Post-Synthetic Alterations of Bovine Lens Enzymes Demonstrated by Heat Lability Measurements

The heat lability of certain enzymes of carbohydrate metabolism was determined in the nucleus and equator of bovine lenses of different age (1, 4–6 and 15 years old).