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K R Fleshman

Publications and source records attributed to K R Fleshman.

3 recordsLinked to original sources

Age changes in bovine lens endopeptidase activity.

Lens endopeptidase activity and thermal stability have been determined as a function of cell development, cell age, and animal age. Lenses from animals aged 3 months to 15 years (lens weights 1.15-2.80 g) were divided into epithelial (outermost), cortical (peripheral), and nuclear (central) regions. Changes accompanying cell development were determined by measuring specific activity in epithelial (undifferentiated), outer cortical (differentiating), inner cortical (mature) and nuclear (aged) regions of individual lenses. Thermal stability of the enzyme activity obtained from the outer cortical and nuclear regions of the same lenses was also determined. Specific activity and thermal stability were found to decrease as a function of lens cell development. Changes with cell development represent the effects of both differentiation and increasing cell age. To determine the effects of cell age alone, activity was determined in the same population of aged, fully differentiated cells in lenses of different ages. Specific activity decreased as a function of cell age alone. Changes with animal age were determined by comparing cells of the same developmental stage from animals of different ages (e.g., differentiating cells of the cortex in animals 3 months to 15 years old). Specific activity for the cortical region increased with animal age while specific activity in the nuclear region appeared to remain constant or decrease slightly with increasing animal age. Thermal stability of the enzyme activity from the cortex was different in young and adult lenses. The change in stability occurred early in the lifespan and was therefore more closely related to animal development than to aging.

Aging↗

Changes during aging in rat lens endopeptidase activity.

Protein degradation has been implicated in lens aging and cataract development. We propose that proteolytic enzymes are important in these processes. In this study, lens neutral proteinase activity and thermal stability have been measured as a function of cell age and animal age in rat lens. Epithelial, cortical, and nuclear lens regions from animals between 1 and 25 months were analyzed. Specific activity and thermal stability were found to decrease with lens cell age, that is, from epithelium to nucleus. Specific activity in the cortex increased with animal age, while specific activity in the nuclear region remained constant with animal age. In the epithelium, specific activity showed no correlation with animal age. In 15 day old rats, thermal stability curves for the cortical enzyme were linear, indicating only one form of activity hydrolyzing synthetic substrate was present. At 1.25 months of age, thermal stability curves for the cortical enzyme were non-linear indicating that multiple forms of the activity were present.

Aging↗

A synthetic endopeptidase substrate hydrolyzed by the bovine lens neutral proteinase preparation.

Lens neutral proteinase is thought to exhibit primarily endopeptidase activity. We have identified a synthetic endopeptidase substrate which is hydrolyzed by the bovine lens neutral proteinase preparation. Among 11 fluoro- and chromogenic endopeptidase substrates, only carbobenzoxy-glycylglycyl-L-leucyl-p-nitroanilide is effectively hydrolyzed. The activity hydrolyzing this substrate co-elutes with neutral proteinase activity upon gel filtration and specifically attacks the leucyl-p-nitroaniline bond. Optimal hydrolysis of the synthetic substrate is at neutral pH and high temperature (53 degrees C), analogous to the alpha-crystallin protein substrate obtained from lens. The rate of hydrolysis of the synthetic substrate increased proportionally with temperature between 20 and 60 degrees C, in contrast to alpha-crystallin. The rate of hydrolysis was linear for at least 1 h at 37 degrees C and there was no evidence of enzyme activation at high temperature.

Animals↗