Elucidation of a minimal immunoreactive site of vertebrate calmodulin

The heptapeptide AsnTyrGluGluPheValGlnNH₂ corresponding to residues 137–143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem.256, 4205–4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127–144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only sevenresidue segment in the 135–145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

Structural changes caused by thyrotropin in thyroid cells and in liposomes containing reconstituted thyrotropin receptor

Thyrotropin causes a rapid and significant increase in the fluorescence polarization of DPH when this hydrophobic probe is incorporated into a strain of functioning rat thyroid cells (FRTL5). This increase is ligand-specific and is not related to cAMP production. The phenomenon seems to reflect the interaction of thyrotropin with the glycoprotein component of its membrane receptor, as suggested by experiments in which thyrotropin causes increases in DPH fluorescence polarization in liposomes embedded with this receptor component but not with gangliosides. A strain of nonfunctioning rat thyroid cells (FRT), exhibiting no reactivity with monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, requires two orders of magnitude higher concentrations of thyrotropin to exhibit a comparable phenomenon.     

Sensitivity to values of the rate constants in a neurochemical metabolic model

Equations were derived for the instantaneous relative sensitivities of reaction rates (controllability indices) and metabolite concentrations (response indices) to perturbations in the values of rate constants and were used to analyze the behavior of a model of in vivo glutamate metabolism in rat brain. Controllabilities of reversible reactions were found to increase as the values of the corresponding rate constants (i.e., the rate of approach to equilibrium) increased. Response indices generally declined with the metabolic distance between the metabolite and the rate constant, but they were unexpectedly high for reversible reactions with high controllabilities. The transient response of a given metabolite is most sensitive to reactions involving metabolites which are changing most rapidly relative to their respective pool sizes. Rapidly reversible reactions are most important for communication between metabolite pools.     

Temperature-Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase-Insensitive Toxin-Receptor Complex

Abstract: Energy-dependent internalization of ¹²⁵I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside G_D1b (1–5 μg/ml) also lowers the amount of cell-associated toxin. The fraction of ¹²⁵I-labeled toxin retained by the cells after exposure to high-salt medium at 0–4°C or after addition of G_D1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.     

A graph-theoretical analysis of metabolic regulation

A graph theoretical method is proposed for modeling metabolic networks including enzymic cascades and synergistic binding of ligands to enzymes. Formal operations on the graph of a given network leads to the identification of feedback metabolites and the enzymes which regulate the feedback. These systemic properties are thus isolated from the purely local regulation of individual enzymes. The method was applied to a model of glycogen metabolism. At low cyclic AMP and insulin levels feedback control of the system is predicted to be largely with the glycogen branching and debranching enzymes, which set the amount of glycogen in the metabolically available outer branches.     

Nonsubstrate induction of a soluble bacterial cytochrome P-450 monooxygenase by phenobarbital and its analogs

A soluble, cytochrome P-450-dependent fatty acid hydroxylase--epoxidase complex from Bacillus megaterium ATCC 14581 can be induced more than 100-fold by the addition of phenobarbital or one of its analogs (hexobarbital) to the growth medium. These barbiturate inducers are apparently not substrates for the enzyme nor do they activate the monooxygenase in the cell-free system. The induction efficiency of both phenobarbital and hexobarbital can be significantly increased with respect to monooxygenase activity by autoclaving the inducer in the growth medium rather than by adding it to the medium after autoclaving. Turnover numbers of about 3 000 nmoles of substrate oxygenated per min per nmole of P-450 were obtained in crude cell-free preparations obtained from maximally induced cultures. Our data indicate that products formed by heating phenobarbital or hexobarbital in the growth medium are significantly better inducers of monooxygenase activity than are the unaltered drugs.     

ERYTHROID CELL DIFFERENTIATION AND THE INHIBITION OF CYTOKINESIS BY CYTOCHALASIN

Cytochalasin B produces multinucleated erythroid cells in tissue cultures of very young chick blastoderms. There is no apparent qualitative interference with differentiation and maturation of erythroid cells, but the amounts produced are reduced 4- and 10-fold. These effects of cytochalasin are readily reversible.     

Transduction and Plasmid Deoxyribonucleic Acid Analysis in a Multiply Antibiotic-Resistant Strain of Staphylococcus epidermidis

A genetic analysis of a multiply antibiotic-resistant strain of Staphylococcus epidermidis was performed. Experiments designed to show reversion of organisms to antibiotic susceptibility, as well as studies of the influence of ultraviolet irradiation of phage on the transduction frequencies of the resistance markers, indicated that determinants of chloramphenicol (cml), tetracycline (tet), and neomycin (neo) resistance are present on separate plasmids, but the streptomycin marker is chromosomal. In 2 to 6% of tetracycline-resistant transductants, co-transduction of cml was also observed. By using CsCl-dye density gradients followed by neutral sucrose gradients, the plasmids carrying cml, tet, and neo could be isolated and their molecular weights could be determined. The tetracycline plasmid is shown to be incompatible with one of the cryptic plasmids of a recipient strain.