Sharon E. Porter

MONOCLONAL ANTIBODY TO CALMODULIN : DEVELOPMENT, CHARACTERIZATION, AND COMPARISON WITH POLYCLONAL ANTI-CALMODULIN ANTIBODIES

Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free calmodulin than for Ca(2+)-calmodulin. Other selected members of the calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism.

Restoration of cell volume and the reversal of carbohydrate transport and growth inhibition of osmotically upshocked Escherichiacoli

Resumption of growth in osmotically upshocked Escherichia coli was effected only by an external stimulus (betaine treatment) in severe upshock, but was spontaneous in less severe upshock. In either case, growth resumption was preceded by a reversal of glucose transport inhibition, and that reversal was preceded by a recovery of cell volume. We hypothesize that deformation of the membrane by osmotic stress results in conversion of a membrane component of the transport system to a less functional conformation, which results in the inhibition of transport and the consequent inhibition of growth. Relief of the deformation would then allow recovery to a more functional conformation, reversal of transport inhibition, and then resumption of growth.

Evidence for the regulation of bacterial glycogen synthesis by cyclic AMP.

Summary Exogenous 3′,5′-cyclic AMP causes a 1.5-fold increase in the rate of glycogen synthesis in E. coli W4597(K) using glucose. This increase is effected without increases in known factors (i.e., the cellular level of the energy charge, fructose-P 2 or glucose-1-P) which increase the velocity of the rate-limiting enzyme of bacterial glycogen synthesis, ADP-glucose synthetase and without an increase in the level of this enzyme. The inhibition by glucose of the rate in cultures using succinate and the antagonism of this inhibition by cyclic AMP cannot be explained by changes in the known factors or the enzyme level. These results and others presented here provide the first evidence that cyclic AMP plays a role in regulating bacterial glycogen synthesis.

The relA gene is not required for glycogen accumulation during NH4+ starvation of Escherichia coli

Abstract When glucose is the carbon source, glycogen accumulates in partial (NH 4 + ) or total (NH 4 + and required amino acids) nitrogen starvation in Escherichia coli strains that are relA + or relA − . But glycogen accumulates only in the relA + strain when required amino acids are depleted or isoleucine starvation is induced by valine addition. However, when glycerol is the carbon source, glycogen accumulates in both relA + and relA − strains after valine addition. We conclude that the relA gene is not required for glycogen accumulation when synthesis of all nitrogen-containing compounds of the cell is limited or abolished. We also conclude that, although the relA gene is needed for glycogen to accumulate in amino acid starvation, this requirement can be replaced by a high cellular concentration of 3′,5′-cyclic AMP.

Regulation of the basal and cyclic AMP-stimulated rates of glycogen synthesis in Escherichiacoli by an intermediate of purine biosynthesis

Abstract In Escherichia coli an abrupt increase in the rate of glycogen synthesis occurs at the onset of total nitrogen starvation. We present here both in vivo and in vitro data indicating that this increase occurs because of the loss of a nitrogen-containing intermediate of purine biosynthesis (apparently 5-aminoimidazole-4-carboxamide ribonucleotide) that inhibits glycogen synthesis. We also show that this inhibitory intermediate antagonizes the stimulation of glycogen synthesis by 3′,5′-cyclic AMP. The uncovering of the regulation of glycogen synthesis by this inhibitor apparently provides the first link in understanding the 23-year-old observation of a reciprocal relationship between growth rate and glycogen accumulation in E. coli .

Identification of GTP as a physiologically relevant inhibitor of Escherichia coli ADP-glucose synthetase

We show that physiological concentrations of GTP can significantly inhibit wild-type Escherichia coli ADP-glucose synthetase (the rate-limiting enzyme of bacterial glycogen synthesis) and that mutant-strain enzymes known to show less inhibition by physiological AMP levels also show less inhibition by physiological levels of GTP. This decreased inhibition by both AMP and GTP can almost totally account for the higher cellular rates of glycogen synthesis observed in the mutant strains. In addition, in metabolic conditions where we have shown that cellular glycogen synthesis increases, cellular GTP levels are known to decrease. Thus, we conclude that GTP inhibition is physiologically relevant.

Logit-log calibration curves for EMIT assays*

The logit-log and four-parameter logistic procedures when appropriate for calculation of enzyme-multiplied immunoassay (EMIT) data have the advantage that they can be applied regardless of the kinetic analyzer or reaction conditions. To use these procedures correctly one must determine the change in absorbance at an infinite drug concentration (delta A infinity). The marked variation of delta A infinity with equipment and reaction conditions and the difficulty in determining this value have hindered broad use of these otherwise universally applicable procedures. We have evaluated two simple methods for determining delta A infinity, both based on its equivalence to delta A in the absence of specific antibody: (1) cross-kit reaction using antibody/substrate and enzyme-drug reagents from kits for different drugs, and (2) substitution of an antibody-free substrate reagent with composition based on direct analysis. The cross-kit procedure was tested with EMIT assays for phenobarbital, primidone, phenytoin, carbamazepine, ethosuximide, and theophylline. In some cases an unexpected type of cross-reaction occurred, giving an erroneously low value for delta A infinity. The antibody-free substrate reagent always permitted accurate determination of delta A infinity.

Evidence that cyclic AMP stimulates bacterial glycogen synthesis by relieving AMP inhibition of and by increasing the cellular level of ADP-glucose synthetase.

Using Escherichia coli mutants that possess an ADP-glucose synthetase (EC 2.7.7.27, the rate-limiting enzyme of bacterial glycogen synthesis) that differs in its inhibition by physiological levels of AMP, evidence was obtained that cyclic AMP stimulates cellular glycogen synthesis during nitrogen starvation by relieving AMP inhibition of this enzyme (without altering the cellular AMP level). Deinhibition for AMP of an enzyme controlled by the adenylate energy charge allows selective release from this control despite the maintenance of a constant cellular energy charge value. It was also shown that an additional increase in rate, not accounted for by AMP deinhibition, was due to an increase in the cellular level of ADP-glucose synthetase.