Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.     

Purification and characterization of chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica)

1. A chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica) was purified by acid extraction, salt fractionation and chromatographic separation on CM-cellulose and Sephadex G-100, and gave a single protein band on SDS-PAGE. 2. Quail chymotrypsinogen had a mol. wt of 26,100 calculated from amino acid composition data, an isoelectric point of 7.68, a Km of 3.1 mM and K0 of 40.7 sec-1 for tyrosine ester substrate. 3. The activated chymotrypsinogen of quail had a maximum activity at pH 7.0-8.0 and at 45 degrees C, and was stable at pH 4.0-6.0 below 55 degrees C. 4. Comparison of quail and bovine chymotrypsinogens indicates that the activities of the enzymes from quail and bovine are more constant than their physical characteristics.     

Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium

Summary The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [³H]lathosterol to [³H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesserone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells. This work was supported by National Institute of Health grants CA40294 and CA37589 to G. H. Sato and by a grant from RJR nabisco Inc. Editor's Statement These results help identify the defect in myeloma cells leading to cholesterol auxotrophy. The use of these cells in hybridoma derivation adds practical utility to a detailed appreciation of cholesterol metabolism in these cultures.     

Biosynthesis of various types of collagen by human hepatoma cells in vitro

A cloned human hepatoma cell line (HH2-1) produced and formed collagen fibers in vitro. The relative rate of collagen synthesis by the cells was increased with an enhancement of the cell density. An analysis of the components of the collagen using sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the cells synthesized interstitial collagen, types I and III, and other collagenous proteins. Thus, human hepatoma cells may play an important role in the formation of stromal collagen in the tumor.     

Enhancement of Nitrogen Dioxide-Induced Lipid Peroxidation and Dna Strand Breaking by Cysteine and Glutathione

Nitrogen dioxide less than 100 ppm in air induced lipid peroxidation of liposome composed of l-palmitoyl-2-arachidonylphosphatidylcholine as assessed by thiobarbituric acid reactivity. The nitrogen dioxide-induced lipid peroxidation was enhanced by cysteine, glutathione and bovine serum albumin. While the activity of nitrogen dioxide in air to induce single strand breaks of supercoiled plasmid DNA was low, the breaking was remarkably enhanced by cysteine, glutathione and bovine serum albumin. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that certain strong oxidant(s) were generated by interaction of nitrogen dioxide and cysteine. The spin trapping using 3,5-dibromo-4-nitrosobenzene-sulfonate suggested that sulfur-containing radicals were generated by interaction of nitrogen dioxide and cysteine or glutathione. Hence, certain sulfur-containing radicals generated by the interaction which could effectively induce lipid peroxidation and DNA strand breaks.     

Detection of G protein-activator regions in M4 subtype muscarinic, cholinergic, and alpha 2-adrenergic receptors based upon characteristics in primary structure.

The 14-residue region Arg2410-Lys2423 of the human insulin-like growth factor II receptor possesses the ability to stimulate Gi, the activity being dependent on two structural characteristics: (i) at least two basic residues at the N-terminal side and (ii) the C-terminal motif, B-B-X-B or B-B-X-X-B (where B is a basic residue and X is a non-basic residue). The regions satisfying (i) and (ii) with 10 less than or equal to residue length less than or equal to 26 were located in all of the third inner loops and some of the other intracellular domains of the Gi-coupled M4 sub-type muscarinic cholinergic receptor (M4AChR) and the alpha 2-adrenergic receptor (alpha 2AR). Both the second inner loop 130-147 and the C-terminal portion of the third inner loop 382-400 (MIII) of human M4AChR had the ability to stimulate G proteins with the order Gi approximately Go greater than Gs, but only MIII could activate Gi/Go at nanomolar concentrations. In contrast, the N-terminal portion of the third inner loop 218-228 of human alpha 2AR-C10 activated Gi, Go, and Gs at micromolar concentrations with equal potency, whereas the further C-terminal portion of the third inner loop 301-313 of this receptor lacked the ability to activate any G protein. Among these active regions, only MIII indicated Mg(2+)-dependent Gi-stimulating function. Therefore, the search for the regions satisfying (i) and (ii) was useful to localize the G protein-activating activity of Gi-coupled receptors in limited regions, which were not always in the C-terminal portions of the third intracellular loops and activated G proteins in various modes of actions.     

The cytoplasmic tail of CD4 targets chimeric molecules to a degradative pathway.

Many different cell surface receptors undergo endocytosis via coated pits. Once having entered the cell, the receptors are sorted into diverse pathways. Which path a given receptor will follow is determined by signals inherent in the receptor's structure. The nature of these structural features is not yet known. In this study, we have taken the approach of constructing chimeric molecules to localize the domain of the T-cell surface molecule CD4 which is responsible for targeting it for degradation. Chimeric molecules bearing the cytoplasmic domain of CD4 and the extracellular domain of either the low-density lipoprotein receptor or a major histocompatibility complex (MHC) class I molecule were both internalized in response to phorbol 12-myristate 13-acetate and were subsequently degraded, indicating that the cytoplasmic tail of CD4 contains all the information required for both processes. The ability to modulate the level of MHC class I molecules on the cell surface offers an approach to investigating quantitative aspects of antigen presentation, the initial possibilities of which are explored herein.