Lysosomal hydrolase secretion by Tetrahymena: A comparison of several intralysosomal enzymes with the isoenzymes released into the medium

Abstract

Tetrahymena pyriformis were grown in proteose-peptone medium and then washed and incubated in a dilute salt solution for one hour. The cells were then discarded and the lysosomal hydrolases that had been secreted were subjected to DEAE cellulose column chromatography. At least three isoenzymes of acid phosphatase, three of acid protease, and two of beta-N-acetylhexoseaminidase were found, as well as single peaks of alpha-mannosidase, beta-galactosidase, and beta-fucosidase. The latter two activities were not resolved by the DEAE column and could not be separated in a second chromatographic step on CM-cellulose. Cells were also grown under identical conditions and homogenized in 0.25 M sucrose in order to allow comparison of some of the intracellular lysosomal hydrolases with their secreted counterparts. Two lysosomal populations were resolved by sucrose density gradient sedimentation, a heavy lysosomal fraction, contered at a density of about 1.25 gm/cm3, and a light lysosomal fraction, centered at a density of about 1.16 gm/cm3. These two populations differed in that the light lysosomes did not appear to contain significant amounts of beta-fucosidase, beta-galactosidase, or acid protease, whereas all six of the hydrolase activities studied were present in the heavy lysosomes. The light lysosomal peak occurred in cells grown to transition phase, but was markedly reduced in cells from cultures grown to stationary phase. In addition to these two fractions a third very light particle, containing only alpha-mannosidase activity, was detected just inside the gradient. Measurements were made of the effect of heat (10 minutes at 66 degrees) and of a change in pH from 4.5 (standard assay condition) to 6.0 on the three acid phosphatases and two beta-N-acetylhexoseaminidase isoenzymes resolved by DEAE column chromatography of the secreted hydrolases and on these hydrolyases in the heavy and light lysosomal fractions on the sucrose gradient. Use of the thermostability and pH criteria permitted computation of the expected properties of the intralysosomal acid phosphatase and hexoseaminidase activities if these consisted of the respective isoenzymes in the proportions secreted. It was found that neither the intralysosomal acid phosphatase nor the intralysosomal hexoseaminidase had the properties expected if they consisted of the secreted mixture of the respective isoenzymes, indicating that modification of some of these isoenzymes may have occurred during the 1-hour starvation period or after secretion.

Purification and Characterization of a Secreted Protease from Tetrahymena pyriformis

“…Dickie and Liener [4] have isolated two different extracellular proteases with neutral pH optimum and one intracellular acid protease having the optimum at pH 5.5 for denatured hemoglobin. Miiller [2] noted the release of an acid protease having pH optimum at 3.3 for denatured hemoglobin and Blum [32] suggested that two or more acid proteases hydrolyzing denatured hemoglobin at pH 3.3 were secreted into the medium. Furthermore, extensive studies have been performed by Levy and coworkers [ll], who isolated several isoenzyme forms of intracellular protease possessing a neutral optimum pH for synthetic substrate and azocasein and an optimum of pH 3.3 for denatured hemoglobin.…”

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

“…Recently, interest has been shown in the role of cysteine proteases in the pathology of parasitic infections. These proteases can be released from the parasites (Blum, 1976;Keene, 1986; Lockwood et al, 1988; Garber and Lemchuk-Favel, 1989;North et al, 1989; Boutignon et al, 1990) andmay beinvolved in the pathology of the diseases and the survival of the parasites within the host (Bremmer et al …”

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

“…Because the content and the composition of the lysosomal enzymes secreted by T. thermophila are strongly media-dependent, 62,63 the portfolio of the different N-linked glycans in T. thermophila might be influenced by the prevailing cultivation conditions. It remains to be determined if the observed range of N-glycan structures is the result of incomplete processing during intracellular transport, whether it is dependent on the resident time in the extracellular medium, or if it is the result of a normal variation.…”