Association of hydrophobic substances with hemin. Characterization of the reverse type I binding spectrum and its relationship to cytochrome P-450.

When hydrophobic compounds were added to a solution of protoferriheme, a a reverse type I spectral change was produced when observed by difference spectroscopy. The spectrum had a peak at 422 nm and a trough at 387 nm, and the characteristics were dependent on the pH of the sample. An association constant for the complex could be determined and was also found to be pH sensitive, with the association constant dropping to zero at values below pH 7.0 and above pH 8.5. The determination of the delta Absmax for the ethylbenzene-hemin complex at various hemin concentrations indicates monomeric heme to be the species responsible for binding the hydrocarbon with the concomitant generation of the reverse type I spectral change.

Rat pancreatic duct epithelium cultured on a porous support coated with extracellular matrix.

A method was developed for the isolation and culture of rat pancreatic duct epithelium of predominantly interlobular duct origin. Purified duct epithelial fragments were cultured on a porous support (HATF filters, Millipore) at 37 degrees C in a 1:1 mixture of Dulbecco's Modified Eagle's and Ham's F-12 media supplemented with insulin, cholera toxin, epidermal growth factor, bovine pituitary extract (BPE), and Nu-Serum (Collaborative Research) in a humidified atmosphere of 95% air and 5% CO2. The filters were coated with an extracellular matrix of either rat tail collagen or Matrigel (Collaborative Research), both of which significantly enhanced growth of the duct epithelium in comparison with untreated filters. The cells grew from the tissue fragments as epithelial islands, which merged to form a confluent sheet of epithelium covering at least 80% of the filter within 10 days in culture. The mitotic index of the spreading epithelium increased with time, reaching a maximum of 0.6% on days 3 and 5 and then declining. The epithelial monolayer consisted of tightly packed cells, with a few large cells and a few cells undergoing abnormal mitoses. Fibroblast contamination was negligible. The cells retained carbonic anhydrase activity, consistent with their pancreatic ductal origin and with the maintenance of differentiation in culture. The epithelium could be subcultured but with a low efficiency. A defined, serum-free medium was established with the addition of ethanolamine, bovine serum albumin, and transferrin and the deletion of serum and BPE. The epithelial cells grew nearly as well in this medium as in the serum-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS)