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PubMed · 6813855

Viroids and prions.

Abstract

Viroids are small "naked" infectious RNA molecules that are pathogens of higher plants. The potato spindle tuber viroid (PSTV) is composed of a covalently closed circular RNA molecule containing 359 ribonucleotides. The properties of PSTV were compared with those of the scrapie agent, which causes a degenerative neurological disease in animals. PSTV was inactivated by ribonuclease digestion, psoralen photoadduct formation, Zn2+ -catalyzed hydrolysis, and chemical modification with NH2OH. The scrapie agent resisted inactivation by these procedures, which modify nucleic acids. The scrapie agent was inactivated by proteinase K and trypsin digestion, chemical modification with diethylpyrocarbonate, and by exposure to phenol, NaDodSO4, KSCN, or urea. PSTV resisted inactivation by these procedures, which modify proteins. Earlier evidence suggested that the scrapie agent is smaller than PSTV. Its small size seems to preclude the presence of a genome coding for the protein(s) of a putative capsid. The properties of the scrapie agent distinguish it from both viroids and viruses and have prompted the introduction of the term "prion" to denote a small proteinaceous infectious particle that resists inactivation by procedures that modify nucleic acids.

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BibTeXRIS

T O Diener, M P McKinley, S B Prusiner. 1982. Viroids and prions.. https://doi.org/10.1073/pnas.79.17.5220

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Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.

Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pKa of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes Vmax but not Km values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.

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