Identification of two distinct protein carboxyl methyltransferases in eucaryotic cells

Two distinct protein carboxyl methyltransferases (PCM) were identified in the electric organ of Torpedo ocellata. They were separated from each other in the active form by means of nondenaturing gel electrophoresis and by p-(chloromercuri)benzoate-agarose chromatography, and were individually identified by specific polyclonal antibodies. The existence of at least two distinct PCMs in eucaryotic cells raises the possibility that these enzymes are involved in distinct transmethylation reactions.     

Low background scintillation counting

Counting radioactive samples with Beckman Instrument's Ready Caps, using a restricted energy window, LL-UL = 400-1000, resulted in machine backgrounds of under 2 cpm and efficiencies of counting relative to liquid scintillation cocktails (LSC) of 51%, 65%, 57%, 62%, and 1% for 32P, 125I, 14C, 35S and 3H, respectively. Signal-to-noise ratios from a quantitative molecular hybridization technique were increased 8-10 fold. There may be a general application for this product in experiments yielding low amounts of radioactivity in liquid samples.     

Evaluation of the use of S1 nuclease to detect small length variations in genomic DNA

A method which utilises S1 nuclease to detect small length variations in cloned and genomic DNA has been evaluated. The methodology of this technique is simple and robust, permitting the rapid analysis of 10(4) base pairs. By employing defined sequence variants, this method is shown to have a sensitivity which should enable the detection of length variations of only a few base pairs in heterozygous individuals.     

alpha-Fetoprotein production and erythropoiesis in cell cultures of human fetal liver.

alpha-Fetoprotein and the synthesis of heme associated with hemoglobin were measured simultaneously in short-term cultures of human fetal liver cells to correlate the relationship of alpha-fetoprotein to erythroid cell function. Both synthetic processes decreased exponentially during the first 5 days of culture. The use of media supplemented with different batches of fetal calf serum and porcine portal vein serum indicated that the optimal conditions for the production of alpha-fetoprotein were different from those required for the synthesis of heme associated with hemoglobin. Moreover, the alpha-fetoprotein-producing cells could be separated from erythroid cells after velocity sedimentation in Ficoll gradients. Although it is well known that erythropoiesis and alpha-fetoprotein production occur simultaneously during ontogenesis, alpha-fetoprotein itself (0.01-100 micron g/ml) did not stimulate heme synthesis in liver erythroid cells. Erythropoietin did not stimulate alpha-fetoprotein production. It is concluded that there is no cause-effect relationship between alpha-fetoprotein production and erythroid cell fuction in human fetal liver cells and that the two processes occur independently in different cell types.     

Density-functional investigation on the mechanism of H-atom abstraction by lipoxygenase

Using experimentally calibrated density functional calculations on models of the active site of soybean lipoxygenase 1 (SLO-1), insight has been obtained into the coordination flexibility of the iron active site and its molecular mechanism of catalysis. The ferrous form of SLO-1 shows a variation in coordination number in solution that is related to a weakly coordinating Asn694 ligand. From the calculations it is determined that the weak Fe-O(694) bond associated with this coordination flexibility is due to a sideways tilted geometry of Asn694 that is imposed on the site by the protein. Release of this constraint (by altering the hydrogen bonding network) leads to a pure six-coordinate site. In contrast, the ferric form of the enzyme stays five-coordinate. In this case, deprotonation of a coordinated water gives a strong hydroxo donor in the cis position to Asn694, weakening the Fe-O(694) bond. Hence, Asn694 is a stronger ligand to the reduced relative to the oxidized site. Using these experimentally calibrated models, the reaction energy for H-atom transfer in SLO-1 has been calculated to be about -18 kcal/mol. The observed change in coordination number going from five-coordinate in ferric to six-coordinate in ferrous SLO-1 increases the reduction potential of the iron active site. Hence, the protein adjusts the active site for optimal reactivity. Analysis of the electronic structure along the reaction coordinate shows that the H-atom transfer in SLO-1 actually corresponds to a proton-coupled electron transfer (PCET). The transferred electron does not localize on the proton, but tunnels directly from the substrate to the ferric active site in a concerted proton tunneling-electron tunneling (PTET) process. The covalently linked Fe-O-H-C bridge in the transition state lowers the energy barrier and provides an efficient superexchange pathway for this tunneling. The thermal barrier for the PTET process is estimated from the calculations to be about +15 kcal/mol including zero-point energy corrections. This corresponds to a thermal reaction rate of k(therm) approximately 1 s(-1). In comparison, the rate of proton tunneling can be as high as 2 x 10(9) s(-1) under these conditions.