Correlated biochemical and radioautographic studies of protein turnover in developing rat incisor enamel following pulse‐chase labeling with L‐[³⁵S]‐ and L‐[methyl‐³H]‐methionine

Abstract

The movement of proteins into and out of enamel was followed over time using a highly sensitive microprecipitation technique to quantify the amount of TCA-insoluble radioactivity present within small pieces of freeze-dried enamel and cells (enamel organ) dissected from the mandibular incisors of rats injected with L-[35S]-methionine. Conventional image processing techniques were also used to estimate the number of silver grains over enamel and cells in radioautographs of mandibular incisors from rats similarly injected with L-[methyl-3H]-methionine. Data from both techniques indicated that the average half-life for labeled proteins secreted into enamel was about 8.9 days. Typically, radioactive proteins accumulated in increasing amounts for 8 hours after which they were lost slowly up to 4 days and more rapidly thereafter when enamel formed during the secretory stage underwent maturation. The half-life for radioactive proteins in cells was only about 20.7 hours. No significant accumulation of radioactivity could be detected in the TCA-soluble or TCA-insoluble fractions of cells as enamel development proceeded. Results from this study suggest that radioautographs provide an accurate estimate of changes occurring to proteins in enamel and cells except at early time intervals (less than 1 hour) when a high percentage of total radioactivity is present within the TCA-soluble fraction of cells.

Injectable intratumoral depot of thermally responsive polypeptide–radionuclide conjugates delays tumor progression in a mouse model

“…Samples were collected from this mixture at 0, 2, 4, 8, 24, 48, 72, and 168 hr and immediately stored at −20 °C. Radiolabel stability in the conjugate was quantified by precipitating large ELP fragments (> 10 kDa) with 100 μL 16.5% cold trichloroacetic acid [38]. Low MW degradation products (< 10 kDa) in the supernatant and larger ELP species in the pellet were measured on a γ-counter and the loss of label was obtained by dividing the supernatant radioactivity by the total radioactivity in the supernatant and pellet at each time point.…”

Cellular and Chemical Events During Enamel Maturation

“…(3) during the first half of the maturation stage, modulating ameloblasts secrete proteins some of which appear to be amelogenins (Nanci et al, 1987a(Nanci et al, ,b, 1989aMcKee et al, 1988;Inage et al, 1989Inage et al, , 1996Smith andNanci, 1989, 1996;DenBesten and Li, 1992;Smith et al, 1992;Fong et al, 1996a;Wurtz et al, 1996), and therefore protein secretion is not an activity excluded from "resorptive" ameloblasts;…”

Biological Calcification

“…Studies on the components of the enamel matrix have encountered a series of difficulties chiefly arising from the heterogeneous composition of the matrix, which is partly due to the secretion of a variety of proteins by the ameloblasts, and partly to the post-secretory sequential degradation of these proteins Smith et al 1992). Probably because it is secreted by ameloblasts, that is, by ectodermal cells, and also because of its high sulfur content, it has been suggested that the organic matrix of immature enamel could consist of keratin-related proteins (Eastoe 1963).…”