Enzyme inactivation by a cellular neutral protease: Enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation

Abstract

A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.

Effects of cycloheximide and actinomycin D on the amino acid transport system of tetrahymena

“…A neutral protease whose synthesis is inhibited by cycloheximide and by actinomycin D is present in Tetrahymena (Suprynowicz and Allewell, 1979) and its activity drops by a factor of two to five within 3 hours after transfer to a nonnutrient medium. Although this protease is sensitive in vitro to inhibition by leupeptin and chymostatin (Levy et al, 1976;Levy and McConkey, 1977), it is not known whether these protease inhibitors are effective intracellularly in this organism. Regardless of the possible involvement of a protease in the loss of transport capacity, the present results therefore seem to be consistent with Schaeffer's proposal that the transport system has a short lifetime and that it is coded for by a short-lived message.…”

Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH‐14: Comparison of Extracellular with Intracellular Proteases

“…These values are close to ID,, toward azocasein of T . pyriformis proteases described by Levy et al (16) but show a slight difference from that of the secreted acid protease (8). Leupeptin inhibition for extracellular proteases was similar to that reported for papain (32) but was stronger than for cathepsin B (5).…”

“…pyriformis W cells. Extensive studies have been performed by Levy and co-workers (16,17), who identified several intracellular neutral proteases in strain E which were thioldependent and also demonstrated an increase in a neutral protease activity following shaking of statically grown cultures. Furthermore, they provided evidence that Tetrahymena has a protease capable of degrading cellular enzymes at physiological pH, which would play a key role in regulation of cellular enzyme levels.…”

Stability Properties of Activated Tryptophan Hydroxylase from Rat Midbrain

“…Concentrations of 0.8 and 1.0 mM-CaC1, simply increased the initial activity and facilitated the subsequent loss of activity. At a concentration of 50 PM, leupeptin (Sigma), which has a relatively wide range of specificities with regard to proteases and their substrates (Aoyagi and Umezawa, 1975; Levy and McConkey, 1977) and is an especially effective inhibitor of calcium-activated proteases (Libby and Goldberg, 1978; Banik et al, 1979; Kameyama and Etlinger, 1979), prevented the entire sequence of effects induced by 0.5 mwcalcium and had no influence on control preparations (Fig. 2).…”

“…We are encouraged to think speculatively about these results because it has been demonstrated that many labile enzymes manifest the same stability properties in v i m as in vivo (Hopgood andBallard, 1974: Bond, 1975;Ballard, 1980). tend to be regulatory, and are often stabilized by either substrate or cofactor (Litwack andRosentield, 1973: Levy andMcConkey, 1977;Vitto and Gaertner, 1978). By comparison, we have found two associated enzymes.…”