Edwin A. Popenoe

Interaction of human DNA polymerase β with ions of copper, lead, and cadmium

Under appropriate conditions, divalent copper, lead, and cadmium ions significantly inhibit human DNA polymerase ..beta.. (following accepted convention, the term DNA polymerase ..beta.. refers to the low-molecular-weight, 3 to 4 S DNA polymerase of eukaryotic cells) at concentrations below 10/sup -5/ M. Each metal showed apparent linear noncompetitive inhibition kinetics with respect to the template primer and the deoxynucleoside triphosphate substrate, indicating that complex formation with these components does not account for the inhibition. Apparently, neither lead nor cadmium inhibits by displacing required zinc atoms from the polymerase. The interaction of the metals with the enzyme can be reversed or prevented by EDTA or by thiol compounds, except that inhibition by cadmium ions can be reversed by monothiols but not by dithiols. The metals probably do not inhibit through reaction with thiol groups since the inhibition is not decreased by pretreating the enzyme with N-ethylmaleimide. Although divalent zinc is moderately inhibitory in manganese activated poly(dT) synthesis on a poly(dA) template, it can fill the requirement for a divalent metal ion and, under the conditions tested, is about 60% as effective as Mn/sup 2 +/.

The sulfhydryl nature of collagen proline hydroxylase

Abstract A partially purified preparation of collagen proline hydroxylase from chick embryos is strongly inhibited by sulfhydryl reagents such as N -ethylmaleimide and p -chloromercuribenzoate. The presence of α-ketoglutarate, one of the substrates of the enzyme, affords considerable protection against inhibition by N -ethylmaleimide. The activity of collagen proline hydroxylase is stimulated by the presence of bovine serum albumin in the assay mixture, as Rhoads, Hutton, and Udenfriend have shown. However, the effect consists of two parts. The larger of the two is found to be independent of the sulfhydryl content of the albumin, but a small amount of further stimulation can be abolished by pretreating the albumin with N -ethylmaleimide. Dithiothreitol, which was shown by Rhoads, Hutton, and Udenfriend to stimulate the enzyme when present at concentrations up to 0.3 m m , is found to destroy the enzymic activity at higher concentrations (0.5 or 1.0 m m ). It seems probable that both a sulfhydryl group and one or more disulfide links in the enzyme are necessary for its activity. The sulfhydryl group may be in or near the site for the binding of α-ketoglutaric acid.

Changes in rat lung structure and composition as a result of subchronic exposure to acrolein

Groups of Fischer-344 rats were exposed to either filtered air, 0.4, 1.4, or 4.0 ppm acrolein for 62 days (6 h/day, 5 days/week). Mortality was observed only in the 4.0 ppm chamber, where 32 of 57 male rats died, but none of the 8 exposed females died. The lungs of the 4.0 ppm group were heavier than those of the larger control animals. Relative to controls, there was a 20% increase in total dry lung weight while the percent dry weight decreased 1.5% in the high dose group. This increased dry weight and the absence of significant changes in the DNA and protein content per unit dry weight indicated that the greater lung weight observed in this group was in part due to increased cellularity. Lung connective tissue content was increased as a result of subchronic acrolein exposure. The amount of elastin per unit dry weight was 173% of control values in the animals exposed to 4.0 ppm acrolein. Collagen levels were elevated in both the 1.4 and 4.0 ppm groups, 113 and 137%, respectively, of control values. Histologically, the 4.0 ppm animals demonstrated bronchiolar epithelial necrosis and sloughing, bronchiolar edema with macrophages, and focal pulmonary edema. Exposure related lesions were observed in only 3 of the 31 rats examined from the 1.4 ppm chamber and in none of the animals exposed to 0.4 ppm acrolein.

Partial purification and properties of collagen lysine hydroxylase from chick embryos

Abstract The enzyme collagen lysine hydroxylase, which converts some of the lysine residues in a newly synthesized collagen chain to 5-hydroxylysine residues, has been partially purified from extracts of chick embryo tissues by ammonium sulfate fractionation, calcium phosphate gel treatment, and stepwise elution from DEAE-cellulose. The enzyme so prepared resembles collagen proline hydroxylase in that its activity is increased by bovine serum albumin and it is protected from inactivation by glycine. It differs from the latter enzyme in that it has a higher pH optimum. The most purified preparations are completely devoid of proline hydroxylating activity, which confirms previous assumptions that two separate enzymes are responsible for hydroxylating the two amino acids in collagen.

Labeling of acid mucopolysaccharides with tritium : I. Labeling of heparin with tritium and sulfur-35

Abstract Heparin has been labeled with tritium by exposing it to tritium gas under an electric discharge or to tritiated water in presence of palladium black. The tritium label is stable under a variety of conditions, and, at least in the heparin labeled by catalytic exchange, is equally distributed between hexosamine and hexuronic acid moieties. Tritium-labeled heparin has also been labeled with 35 S by replacing some N -sulfate groups with similar groups labeled with 35 S. The final products retain the original physicochemical and biological properties, and appear to be suitable for metabolic experiments.

Variation of deoxyribonucleic acid polymerase activities during avian erythropoiesis

Amounts of DNA polymerase alpha and beta were determined in extracts of chicken erythroid cells at various stages of development. Concentrations of both polymerase activities are high in erythroblasts which are still dividing, decline after the cells cease dividing and begin maturation, and become almost undetectable in the fully mature erythrocytes. While DNA polymerase alpha activity declines gradually, firmly bound DNA polymerase beta activity in the nuclei drops abruptly after the cells finish DNA synthesis and dividing. The amount of a low molecular weight DNA polymerase extractable with the cytoplasmic fraction, possibly DNA polymerase beta, is low in erythroblasts, increases in the more mature erythroid population and then declines to an undectable level in the fully mature erythrocytes.