Retrovirus-transformed rat cells that actively express transforming growth factor type a (TGF-a) release into the medium a soluble protein of 17-19 kDa that has been identified as a precursor for TGF-a. The identification of this protein as a TGF-a precursor is based on its recognition by specific antibodies and by the ability of elastase to convert this protein into a 6-kDa polypeptide with the properties of mature TGF-a. This TGF-a precursor binds to epidermal growth factor/TGF-a receptors and activates the receptor-associated tyrosine kinase activity in intact cells. The biological potency of this precursor is not markedly increased by conversion into mature TGF-a in vitro. These studies demonstrate the ability of transformed cells to release a TGF-a precursor capable of strong mitogenic action in vivo.
The fate of 125I-labeled transforming growth factor-beta (125I-TGF beta) after binding to its cells surface receptor has been investigated in BALB/c 3T3 mouse fibroblasts. Binding of 125I-TGF beta to cellular receptors at 4 degrees C is pH-sensitive, being markedly decreased at pH less than 6. Most (approximately 90%) of the 125I-TGF beta bound to cells at 4 degrees C can be removed by a brief treatment with acidic medium but is converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Cell-bound 125I-TGF beta is degraded at 37 degrees C and the degradation products are released into the medium. The lysosomotropic bases chloroquine, methylamine, and ammonium and the carboxylic ionophore monensin inhibit the degradation and release of 125I-TGF beta from the cells. Cells allowed to accumulate 125I-TGF beta intracellularly by the action of chloroquine or monensin were treated with the bifunctional agent disuccinimidyl suberate in the presence of detergent Triton X-100; this treatment caused the cross-linking of internalized 125I-TGF beta with the 280-kilodalton TGF beta receptor component. Under conditions in which sustained binding and degradation of saturating 125I-TGF beta concentrations occurs, there is no marked decrease in the binding capacity of the cells even when protein synthesis is blocked with cycloheximide. These results indicate that after TGF beta binding the TGF beta:receptor complex becomes rapidly internalized and that TGF beta is directed towards lysosomes where it is degraded and released. However, the cell surface is replenished with TGF beta receptors recycled after internalization or supplied by a large intracellular pool.
␥-Glutamylcysteine synthetase (␥-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes. Although all eukaryotic ␥-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial ␥-GCS is resistant to BSO. We have confirmed the latter finding with Escherichia coli ␥-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms. We also find that substitution of Mn 2؉for Mg 2؉ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation. Similarly, the specificity of E. coli ␥-GCS for its amino acid substrates is broadened in the presence of Mn 2؉ , and the rate of reaction for some very poor substrates is improved. These results suggest that divalent metal ions have a role in amino acid binding to E. coli ␥-GCS. Electron paramagnetic resonance (EPR) studies carried out with
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Solid-state parameters such as radius ratios, packing efficiencies, and crystal densities may be calculated for various crystal structures from basic Euclidean geometry relating to the Pythagorean theorem of right triangles. Because simpler cases are often discussed in the standard inorganic chemistry texts, this article only presents calculations for closest-packed A-type lattices (one type of particle) and several compound AB lattices (A and B particles) including sodium chloride, cesium chloride, zinc blende (sphalerite), wurtzite, and fluorite. For A-type metallic crystals, the use of recommended values of atomic radii results in calculated densities within 1% of observed values. For AB lattices, assuming ionic crystals, the use of recommended values of ionic radii results in density determinations that are usually but not always close to observed values. When there is covalent character to the bonding, the use of covalent radii results in calculated densities that correlate well with observed values. If interionic or interatomic spacings are used, the calculated densities are always close to the observed values. As indicated by a survey of the standard inorganic texts, these calculations are generally not presented. However, as an illustration of the application of simple mathematical principles to the study of chemistry, discussion of the methods presented in this manuscript may be of value in classroom presentations pertaining to the solid state.
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