A kinetic analysis of the effects of inhibitor-1 and inhibitor-2 on the activity of protein phosphatase-1

The steady-state interaction between protein phosphatase-1 and its two inhibitor proteins was studied in vitro at low enzyme concentrations where the assumptions of the Michaelis-Menten equation appeared to be valid. Under these conditions, and in the absence of divalent cations, inhibitor-1 behaved as a mixed inhibitor using phosphorylase alpha as a substrate, whereas inhibitor-2 was a competitive inhibitor. The results demonstrate that inhibitor-1 and inhibitor-2 do not interact with protein phosphatase-1 in an identical manner. Inhibitor-1 was only a substrate for protein phosphatase-1 in the presence of Mn2+, and its dephosphorylation was inhibited competitively by inhibitor-2 (Kis = 8 nM). Inhibitor-1 did not inhibit its own dephosphorylation in the presence of Mn2+. Its Km as a substrate (190 nM) was very much higher than its Ki as an inhibitor (1.5-7.5 nM). The results are consistent with a model in which a single binding site for inhibitor-1 is present on protein phosphatase-1, distinct from the binding site for phosphorylase alpha. It is envisaged that the binding of inhibitor-1 to this site not only inhibits the dephosphorylation of other substrates but permits access of its phosphothreonine to the same catalytic group(s) responsible for the dephosphorylation of other substrates. G-substrate, a protein phosphorylated exclusively on threonine residues, did not inhibit the dephosphorylation of phosphorylase alpha and its dephosphorylation was potently inhibited by inhibitor-1 or inhibitor-2. The role of the phosphothreonine residue in inhibitor-1 is discussed in the light of these results.     

Studies of activating and nonactivating metal ion binding to yeast enolase

Measurements of binding of certain divalent cations to yeast apoenolase were made using a pH-meter, chromatography, a divalent cation electrode, and ultrafiltration. The binding of the activating metal ions Mg2+ and Co2+ and the nonactivator Ca2+ were studied as functions of the presence or absence of substrate/product, phosphate, and fluoride or level of Tb3+. The data suggest phosphate and fluoride increase Mg2+ binding but not Ca2+ binding. Substrate/product appears to increase Ca2+ binding as well as that of Mg2+ and Co2+. In the presence of substrate, Co2+ binding was 5-6 mol/mol dimer. In the absence of substrate/product, Tb3+ reduced Co2+ binding from 4 mol/mol to 2. These data are interpreted in terms of binding to "conformational," "catalytic" (substrate/product dependent), and "inhibitory" sites. Measurements of Tb3+ fluorescence quenching by Co2+ suggested that the distance between "conformational" sites on the two subunits was large, while the distance between "conformational" and "inhibitory" sites was ca. 17 +/- 4 A. Potentiometric titrations of apoenzyme with Ca2+ and Mg2+ showed that the metal ions produced the same proton release in the presence or absence of substrate/product. If phosphate and fluoride were present, then more protons were released if Ca2+ was the titrant rather than Mg2+, suggesting a difference in ionization state in the complex with the activating metal. Electron paramagnetic resonance studies of Co2+ binding to the various sites in the enzyme are presented. The Co2+ bound to all three sites appears to be high spin, consistent with a preponderance of oxyligands in an octahedral environment. Substrate, citrate, and a strongly binding substrate analogue strongly enhance the hyperfine structure of conformational Co2+. This is interpreted as the result of a change in interaction of an axial ligand to conformational Co2+ produced by carbon-3 of substrate or analogue.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Plant lectins specific for N-acetyl-β-D-galactosamine

N-Acetyl-D-galactosamine in β-linkage being ubiquitous in cell surface glycoproteins, their interaction with lectins specific for this sugar moiety may be a significant event in cell adhesion phenomena. This article discusses the common β-N-acetyl galactosamine-specific lectins, with particular stress on the lectin from winged beans (Psophocarpus tetragonolobus).     

LARGE-SCALE ISOLATION AND FRACTIONATION OF ORGANS OF DROSOPHILA MELANOGASTER LARVAE

Methods for the mass isolation of diverse organs from small animals are described. They involve novel devices: a mechanical dissecting system, a centrifugal agitator for the separation of fibrillar from globular particles, and a settling chamber for the fractionation at unit gravity of particles with sedimentation velocities above the useful range for centrifugation. The application of these methods to the isolation of polytene and nonpolytene nuclei from Drosophila melanogaster larvae is described.     

Demonstration of an “Excitation-Contraction Recoupling” Mechanism in Mammalian Ventricular Myocardium

A PROCESS called “excitation-contraction coupling” has been generally accepted to take place only in the direction of excitation to contraction. Through this mechanism a propagated action potential initiates an active state in skeletal or cardiac muscle and the muscle contracts. We propose that, in the mammalian ventricular myocardium at least, the process is not unidirectional and an important reverse coupling between the contractile system and the excitable plasma membrane has been overlooked. Through this feedback interaction the mode of contraction (that is, isotonic or isometric) not only determines the instantaneous electrical state of the plasma membrane, but also influences the mechanical events of the subsequent beats. Thus when Kaufmann et al.¹ recorded intracellular action potentials from cat papillary muscle, the time course of the repolarization was altered depending on the mode of contraction. Some kind of contraction-excitation feedback has also been suggested by Stauch² and Lab^3,4. They showed a difference in the shape of the monophasic action potential, as recorded by a suction electrode, when comparing isotonic and isovolumic contraction of the intact ventricle. But their experimental conditions did not allow satisfactory analysis of the phenomenon.     

Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sokawa et al. suggest that rel^- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited.     

“Pleiotypic Response”

A hypothesis has been developed to relate stringent control in bacteria to a set of interactions involved in the regulation of growth of transformed and untransformed mammalian cells.     

Inhibition of DNA Synthesis but not of Poly-dAT Synthesis by the Arabinose Analogue of Cytidine <Emphasis Type="Italic">in vitro</Emphasis>

Kimball and Wilson¹ reported that the arabinose analogue of cytidine (ara-C) inhibited DNA polymerase in a crude extract prepared from Ehrlich ascites cells. Furth and Cohen² observed cytosine arabinoside triphosphate (ara-CTP) inhibited DNA polymerase in extracts from either calf thymus or bovine lymphosarcoma tissue, although these investigators³ had already found no effect of ara-CTP on DNA polymerase from Escherichia coli. The inhibition in both of these cases could be substantially reversed by dCTP; but incorporation of the arabinose nucleotide (ara-CMP) into DNA could not be unequivocally demonstrated. Graham and Whitmore⁴ reported the incorporation of ara-C into DNA in vivo and the inhibition of a DNA polymerase from L cells by ara-CTP. They found that ara-CMP was initially incorporated into small DNA strands but subsequently appeared in long strands. Momparler⁵ has presented evidence that, in vitro, ara-C incorporation was limited to the 3′-hydroxyl end of DNA chains. Such incorporation might be expected to block further chain elongation but this expectation was not supported by the evidence presented by Graham and Whitmore.