Avian hepatic T3 generation by 5'-monodeiodination: characterization of two enzymatic pathways and the effects of goitrogens

The enzymatic nature of 5'-monodeiodination (5'-D) in avian liver homogenates was demonstrated by abolishment of activity by iopanoic acid (IOP). T3 production from T4 was dependent on enzyme and substrate concentrations, incubation time, incubation temperature, and pH. Two pathways of 5'-D activity were present in avian liver and exhibited characteristics similar to those described in mammalian tissues. Type II activity was identified as propylthiouracil (PTU)-insensitive activity. Type I (PTU-sensitive) was determined by difference between Total and Type II. Km values were 1.58 microM T4 for Total activity and 0.90 nM T4 for Type II, corresponding to the characteristics of the mammalian pathways. The effects of goitrogens on avian hepatic 5'-D were equivalent to those reported for the mammalian enzyme.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Insulin release from a cloned hamster B-cell line (HIT-T15). The effects of glucose, amino acids, sulphonylureas and colchicine

Insulin release from statically incubated HIT-T15 cells was maximally stimulated by glucose, L-arginine and L-leucine. L-arginine stimulated insulin release in the absence of glucose. Glucose induced insulin release was potentiated by the addition of L-leucine, L-arginine and the two in combination. Both glibenclamide and chlorpropamide stimulated insulin release from HIT-T15 cells. Glibenclamide was the more potent and equivalent in insulinotrophic action to 7.5 mmol/l glucose. Only chlorpropamide significantly potentiated glucose induced insulin release. Perifused HIT-T15 cells produced a reproducible biphasic insulin response to glucose challenge which was characterised by a pronounced and sustained first phase and a reduced second phase. The stimulation of phase I by glibenclamide alone and the inhibition of phase II of glucose induced insulin release by colchicine suggested the presence of a readily available pool of insulin granules which was not rapidly restored by insulin biosynthesis and granule margination.     

Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA.

This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface. The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface. Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision. Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface. To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate. The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface. Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates. The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer.