J.M.W. Bouma

Role of individual cathepsins in lysosomal protein digestion as tested by specific inhibitors

Abstract We have investigated the effect of some inhibitors on the breakdown of proteins at 38 °C and pH 5 by highly purified Triton WR-1339-filled lysosomes obtained from rat liver. When cathepsin D (EC 3.4.23.5) was completely inhibited by pepstatin, the rate of degradation of native serum albumin was not and that of carboxymethylated serum albumin only slightly influenced. Leupeptin or antipain, used at a concentration which resulted in more than 95% inhibition of cathepsin B1 (EC 3.4.22.1) activity, strongly decreased the rate of degradation of native albumin, but affected the degradation of carboxymethylated albumin or hemoglobin only to a slight degree. Leupeptin or antipain in combination with pepstatin inhibited proteolysis of the three proteins strongly, but not completely. Omission of Cl − , essential for cathepsin C (EC 3.4.14.1) activity, from incubation mixtures containing pepstatin, leupeptin and antipain had only a slight additional inhibitory effect. Addition of both pepstatin and monoiodoacetate fully suppressed the degradation of these protein substrates. Some of the experiments have also been done using Triton WR-1339-filled lysosomes obtained from purified hepatocytes. Our results indicate that (1) cathepsin D is not essential for degradation of proteins by liver lysosomal enzymes: (2) cathepsin B1 (or possibly another leupeptin-inhibited thiol protease) is the most important enzyme in the degradation of native albumin; (3) thiol enzymes, other than cathepsin B1, C or D, are involved in lysosomal protein degradation; (4) hepatocyte lysosomes contain a set of proteases sufficient for rapid and extensive degradation of albumin.

The plasma volume of the Wistar rat in relation to the body weight.

The plasma volume of 43 male Wistar rats, weighing between 140 and 350 g, was determined. A close linear relationship between plasma volume and body weight was found: plasma volume (ml)=0.0291×body weight (g)+2.54.

Intracellular distribution of cathepsin B and cathepsin C in rat liver.

Summary 1. The intracellular distribution of cathepsin B and cathepsin C (EC 3,4,4.9) in rat liver has been investigated by means of differential and density-equilibrium centrifugation, activation and solubilization experiments. 2. The enzymes appear to be localized in the lysosomes. No evidence of the presence of an inhibitor of cathepsin B in the supernatant fraction was obtained. 3. The dehydration of mitochondria by exposure to very concentrated sucrose solutions is probably slowly reversible.

Involvement of thiol enzymes in the lysosomal breakdown of native and denatured proteins

Abstract We have investigated the effect of thiols on the breakdown of some proteins by extracts from highly purified Triton WR-1339-filled rat liver lysosomes at pH 5 and 38°C. The rate as well as the final degree of hydrolysis of serum albumin, a protein which remains presumably native at pH 5, was stimulated by thiols like dithiothreitol and strongly inhibited by monoiodoacetic acid. These effects were also found with native and performic acid-oxidized ribonuclease, with cytochrome c and with horse radish peroxidase. The digestion of hemoglobin was not stimulated by dithiothreitol, but partly inhibited by monoiodoacetic acid; yeast invertase was not hydrolysed at all. Our results indicate (a) that thiol-cathepsins are essential for the breakdown of many proteins by lysosomes; (b) that apparently native proteins like albumin and ribonuclease can be degraded extensively by lysosomal cathepsins; (c) that this degradation is, to a large extent, an all-or-none reaction.

Endocytes and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo: II. Effect of charge

Experiments presented in this paper suggest that sinusoidal rat liver cells recognize basic groups on proteins and that this recognition results in endocytosis of the proteins. Evidence for involvement of basic groups was obtained in two ways. Firstly, we changed the positively charged amino groups of the cross-linked ribonuclease molecules to neutral or negative by acetylation or succinylation, respectively. The modified proteins did not contain easily reducible disulfide bonds and they were not very sensitive to endoproteases, suggesting that they were not denatured by the acetylation procedures. Acetylation and succinylation reduced uptake of the injected cross-linked ribonuclease derivatives by liver and spleen and abolished their rapid clearance from plasma. In nephrectomized rats about 75% of the polymer, 36% of the acetylated polymer and 32% of the succinylated polymer were endocytosed by liver after 6 h. For the dimer fractions these values were 59%, 23% and 27%, respectively. Autoradiography and subcellular fractionation of liver 30 min post-injection localized the acetylated polymer in the lysosomal/microsomal fraction of sinusoidal liver cells, probably endothelial cells. Secondly, a positive correlation was found between binding of a number of ribonuclease derivatives to the cation exchanger SP-Sephadex G-25 and the rate of endocytosis by sinusoidal liver cells.

The digestion of the B chain of oxidised insulin by extracts of rat liver lysosomes

Abstract Optimal conditions for the hydrolysis of the B chain of oxidised insulin by extracts of 60-fold purified rat liver lysosomes have been studied. The digestion at pH 5 was activated by dithiothreitol and partly inhibited by iodoacetamide. Hydrolysis of 65% of the peptide bonds was obtained after exhaustive digestion at optimal conditions. The breakdown products were identified after gel filtration and separation of amino acids and peptides as their copper complexes on DEAE-cellulose. The results show that the lysosomal enzymes, acting concertedly, can split the polypeptide to amino acids, dipeptides and three cystiec acid-containing tetrapeptides. Free cysteic acid was not found, indicating that peptide bonds formed by this unnatural amino acid are especially resistant to the lysosomal enzymes. Estimation of the molecular size of the digestion products after short periods of incubation suggests the involvement of exo- and endopeptidases.

Effect of size and charge on endocytosis of lysozyme derivatives by sinusoidal rat liver cells in vivo

Hen egg-white lysozyme has been modified by intermolecular cross-linking with dimethyl suberimidate or by acylation with acetic or succinic anhydride. Retention of the native conformation of the modified enzyme was checked by measuring enzyme activity, resistance of disulfide bridges to reduction by thiols, and susceptibility to proteases. Unmodified lysozyme and its derivatives (labelled with 125I) were intravenously injected into nephrectomized rats, and plasma clearance and uptake by liver cells were determined. Under these conditions, about 6% of the unmodified lysozyme was taken up by liver 15 min after injection. Cross-linking led to a greatly increased uptake (up to 89% of the dose in 15 min), whereas acylation reduced the uptake to 3-4%. Cell isolations showed that the unmodified enzyme and the cross-linked derivatives were taken up by sinusoidal cells. Differential fractionation of liver homogenates indicated tht the unmodified enzyme was taken up in lysosomes. The cross-linked derivatives were concentrated in the nuclear and microsomal fractions as well as in the lysosomal fraction, suggesting adsorption on plasma membranes besides uptake in lysosomes. The experiments described in this paper, together with previous results on ribonuclease and lactate dehydrogenase, indicate that endocytosis of some proteins by sinusoidal liver cells is positively correlated with size and positive charge of the molecules.

Proteolysis at neutral pH in a lysosomal-cytosol system can not be attributed to the uptake of proteins into lysosomes

Although intracellular protein degradation is a major factor in determining the cellular concentration of a protein, there is little known about the mechanism of this process [ 1,2] . It has not been possible to imitate this process in vitro; the energy requirement, noticed in liver slices and isolated cells is lost after cell disruption [3]. Auricchio et al. [4], however, suggested from experiments on the inactivation of tyrosine aminotransferase (TAT) in a liver homogenate at neutral pH, that this enzyme is taken up into and degraded within intact lysosomes. The formerly noticed [5] absence of TAT inactivation and protein degradation in general at neutral pH should, according to Auricchio [4] , be attributed to a homogenization procedure, which ruptured the lysosomes. We have tested the validity of this hypothesis for a labelled protein fraction from rat liver cytosol. We did not find any evidence for the uptake of shortor longlived cytosol proteins into intact lysosomes.

LOCALIZATION AND SOME PROPERTIES OF LYSOSOMAL DIPEPTIDASES IN RAT-LIVER

1. The rates of hydrolysis of 26 synthetic dipeptides by extracts from highly purified lysosomal fractions from rat liver at pH 5.0 and by whole liver homogenates at pH 7.4 have been determined. Extracts from the lysosomal fractions hydrolysed most peptides at a lower rate per mg protein than the homogenates, and some peptides not at all. 2. Properties of two dipeptidases present in the extracts from the lysosomal fractions, splitting Ile-Glu and Leu-Gly, respectively, were studied in greater detail. The enzyme that hydrolysed Ile-Glu was strongly activated by dithiothreitol, showed optimal activity at pH 4.5 and had a molecular weight of about 120 000. Leu-Gly dipeptidase did apparently not contain an essential thiol group and had a molecular weight of approx. 90 000. It showed maximal activity at pH 6.5. 3. After differential centrifugation of liver homogenates, Ile-Glu and Leu-Gly-splitting activities were determined in the fractions, under the optimal conditions mentioned above. The Ile-Glu-hydrolysing enzyme activity showed about the same distribution as the lysosomal marker enzyme acid phosphatase. Leu-Gly-splitting activity, however, was largely present in the cytosol fraction, with only a small peak in the lysosomal fraction. We obtained evidence that the activities present in the lysosomal fraction and in the cytosol fraction were due to different enzymes, and that one of these enzymes was localized exclusively in lysosomes. 4. It is concluded that some dipeptides originating from intralysosomal proteolysis might be split by lysosomal dipeptidases, whereas others are probably hydrolysed only in the extra-lysosomal compartment of the cell.

INFLUENCE OF THIOLS, ATP AND COA ON PROTEIN BREAKDOWN BY SUBCELLULAR-FRACTIONS FROM RAT-LIVER

Abstract The effects of thiols, ATP and CoA on the breakdown of 14 C-labelled rat serum albumin by mitochondrial and lysosomal fractions from rat liver have been studied. The breakdown of serum albumin at neutral pH was negligible in comparison with the digestion at pH 5. At acid pH the hydrolysis was strongly activated by thiols like dithiothreitol, cysteine and glutathione and also to some extent by CoASH, but not by ATP. The digestion was enhaced by those treatments which rupture the lysosomal membrane. In contrast with reports in the literature, our results do not point to the presence in subcellular fractions of a proteolytic system that could be responsible for the energy dependence of intracellular protein turnover.