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K Kasamo

Publications and source records attributed to K Kasamo.

41 records · Page 3Linked to original sources
PubMed1986

Characterization of an essential arginine residue in the plasma membrane H+-ATPase of Neurospora crassa.

Treatment of the plasma membrane H+-ATPase of Neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees C, pH 7.0, leads to a marked inhibition of ATPase activity. MgATP, the physiological substrate of the enzyme, protects against inactivation. MgADP, a competitive inhibitor of ATPase activity with a measured Ki of 0.11 mM, also protects, yielding calculated KD values of 0.125 and 0.115 mM in the presence of phenylglyoxal and 2,3-butanedione, respectively. The excellent agreement between Ki and KD values makes it likely that MgADP exerts its protective effect by binding to the catalytic site of the enzyme. Loss of activity follows pseudo-first order kinetics with respect to phenylglyoxal and 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration yield slopes of 0.999 (phenylglyoxal) and 0.885 (2,3-butanedione), suggesting that the modification of one reactive site/mol of H+-ATPase is sufficient for inactivation. This stoichiometry has been confirmed by direct measurements of the incorporation of [14C]phenylglyoxal. Taken together, the results support the notion that one arginine residue, either located at the catalytic site or shielded by a conformational change upon nucleotide binding, plays an essential role in Neurospora H+-ATPase activity.

Arginine↗
PubMed1986

Purification and Properties of the Plasma Membrane H-Translocating Adenosine Triphosphatase of Phaseolus mungo L. Roots.

The plasma membrane ATPase of mung bean (Phaseolus mungo L.) roots has been solubilized with a two-step procedure using the anionic detergent, deoxycholate (DOC) and the zwitterionic detergent, zwittergent 3-14 as follows: (a) loosely bound membrane proteins are removed by treatment with 0.1% DOC; (b) The ATPase is solubilized with 0.1% zwittergent in the presence of 1% DOC; (c) the solubilized material is further purified by centrifugation through a glycerol gradient (45-70%). Typically, about 10% of the ATPase activity is recovered, and the specific activity increases about 11-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the peak fraction from the glycerol gradient contains three major polypeptides of M(r) = 105,000, 67,000, and 57,000 daltons. The properties of the purified ATPase are essentially the same as those of membrane-bound ATPase, with respect to pH optimum, substrate specificity, inhibitor sensitivity, and ion stimulation.

Journal Article↗
PubMed1984

Electrogenic H+ translocation by the plasma membrane ATPase of Neurospora. Studies on plasma membrane vesicles and reconstituted enzyme.

Fluorescent probes have been used to measure electrogenic proton pumping by the plasma membrane ATPase of Neurospora. In isolated plasma membrane vesicles, greater than 85% of which are inverted, ATP hydrolysis is accompanied by the formation of an inside acid pH gradient (delta pH) which can be detected by acridine orange fluorescence quenching and an inside positive membrane potential (delta psi) which can be detected by oxonol V fluorescence quenching. Maximal values of delta pH were generated in the presence of a permeant anion (SCN-, NO-3, or Cl-) and maximal delta psi, in the absence of such anions. Cation effects were much less pronounced and can probably be accounted for by non-specific salt effects on the rate of ATP hydrolysis. In addition, a rapid method is described for the reconstitution of the [H+]-ATPase, starting from isolated plasma membranes. When the membranes are solubilized with deoxycholate in the presence of asolectin and detergent is removed by passage through a Bio-Gel P-10 column, vesicles are reformed in which the Mr = 104,000 polypeptide of the ATPase constitutes 35% of the protein. Freeze-fracture electron microscopy of the vesicles has revealed intramembrane particles with a diameter of 116 A, equally distributed between the two fracture faces. Measurements with acridine orange and oxonol V indicate that the reconstituted ATPase retains its transport activity, generating both delta pH and delta psi during the hydrolysis of MgATP.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1978

Response of Membrane-bound Mg-activated ATPase of Tobacco Leaves to Tobacco Mosaic Virus.

Infectious material was formed at an early stage, and migrated into the mesophyll from the epidermis of tobacco leaves (Nicotiana tabacum cv. Samsun NN) during the period of 1 to 3 hours after inoculation with tobacco mosaic virus (TMV). The activity of membrane-bound Mg(2+)-activated ATPase from the mesophyll was stimulated two to four times within 30 minutes after inoculation with 1.0 microgram per milliliter of TMV. Maximum TMV stimulation of membrane-bound Mg(2+)-activated ATPase activity in epidermis and mesophyll was observed at 0.5 and 3.0 hours after inoculation, respectively. This stimulation was also observed with ultraviolet irradiated TMV (only RNA was destroyed), whereas, the stimulation was not observed with heat-irradiated TMV (both coat and RNA were destroyed). Stimulation equal to that of TMV was observed by inoculation with cucumber green mottle mosaic virus and to a lesser extent with cucumber mosaic virus.These results illustrate that the stimulus resulting from inoculation with TMV transfers to underlying cells faster than the migration of TMV particles. This stimulus might be closely correlated to the structure of virus, but not to the infectivity of virus.

Journal Article↗