Species variations amongst proteinases in liver lysosomes

Cathepsin B, H, L and D activities in liver lysosomes were compared between species. Although cathepsin B and D were detected in bovine, pig, chicken and rat liver, striking species differences were evident for cathepsin H and L. Cathepsin L activity was particularly high in chicken lysosomal extracts, but could not be detected in bovine and pig extracts. Whereas there was no significant cathepsin H activity in bovine extracts, rat liver lysosomal extracts contained large amounts of cathepsin H activity.     

A lysosomal factor that interconverts multiple forms of rat liver tyrosine aminotransferase.

A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase $\text{in}$$\text{vitro}$ with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated $\text{in}$$\text{vitro}$ by 2.5 mM MgCl₂ and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to $\text{in}$$\text{vitro}$ interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of $\text{in}$$\text{vitro}$ manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.     

Studies on the conversion of multiple forms of tyrosine aminotransferase in rat liver.

Studies from several laboratories have demonstrated the existence of at least three separable forms of the hepatic enzyme, tyrosine aminotransferase. The significance of these separable forms of the enzyme isolated in vitro for the nature and regulation of the enzyme in vivo has been the subject of some controversy. The studies reported in this paper demonstrate the existence of a heat-labile, pH- and temperature-dependent, nondialyzable component associated predominantly with the lysosomal and mitochondrial fraction of rat liver which catalyzes the conversion of form II to forms III and IV of the enzyme. The activity of this conversion factor is not significantly affected by F^?, molybdate ions, or two inhibitors of proteases. On the other hand, the cyanate ion completely inhibits the conversion of form II to forms III and IV of tyrosine aminotransferase, as do iodoacetate and oxidized glutathione. p-Chloromercuribenzoate also markedly inhibits the conversion. Kinetic studies suggest that the shift from one form to another follows the pathway: II to III to IV. Titration of the available sulfhydryl groups of the three forms of the enzyme demonstrates that form II possesses between 16 and 17 titratable SH groups per mole, while forms III and IV possess 15 and 13 or 14, respectively. The possible catalytic mechanism by which the conversion of the multiple forms of tyrosine aminotransferase is accomplished is discussed.     

Purification of the native form of tyrosine aminotransferase from rat liver

A procedure that provides a homogeneous, native form of tyrosine aminotransferase (l-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) from rat liver in exceptionally high yield is described. This goal is accomplished by rapidly inactivating the lysosomal converting factor that generates two additional, lower-molecular-weight forms of tyrosine aminotransferase, and by separating the native enzyme from the altered forms by chromatography on carboxymethyl-Sephadex C-50 and an optional hydroxylapatite step. Homogeneity appears to be achieved after the carboxymethyl-Sephadex C-50 step, and the final yield of the enzyme exceeds 30%.     

Cathepsin D of mouse leukemia L1210 cells. Unusual intracellular localization and biochemical properties.

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopycnic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18. After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45,000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95,000. Both forms are 4-5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss in activity after treatment with beta-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse leukemia L1210 cells are similar to the situation found for rat thoracic duct lymphocytes.     

Cathepsin D of mouse leukemia L12010 cells unusual intracellular localization and biochemical properties

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopynic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18.After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45 000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95 000. Both forms are 4–5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss activity after treatment with β-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse luekemia L1210 cells are similar to the situation found for rat thoratic duct lymphocytes.     

A liver factor that interconverts multiple forms of tyrosine aminotransferase.

Three activity peaks of rat liver soluble tyrosine aminotransferase have been resolved using hydroxyl-apatite chromatography. These peaks interconvert during storage of the soluble enzyme preparation in ice for 20 h. A component of a particulate fraction of liver which will interconvert the forms of tyrosine aminotransferase in vitro with no alteration of total enzyme activity has been detected. This factor is present in a 31, 000 gh pellet of liver and is solubilized by sonication. When the factor is subjected to dialysis or incubation at 25°C for 30 min. its effect on tyrosine aminotransferase is greatly diminished.     

Physical properties, limited proteolysis, and acetylation of tyrosine aminotransferase from rat liver

The native and one of the modified forms of tyrosine aminotransferase were purified from rat liver and characterized. Several hydrodynamic properties of the native enzyme are: Stokes radius, 46 A; subunit isoelectric point, 5.6; sedimentation coefficient, 5.6 S, frictional ratio, 1.44; diffusion coefficient, 4.65 X 10(-7) cm2 s-1; extinction coefficient of a 1% solution (w:v) at 280 nm, 10.5 cm-1. The molecular weight of the dimeric protein is 110,500 as calculated from the Stokes radius and sedimentation coefficient. The subunit of the modified form is of lower molecular weight than the subunit of the native enzyme and has a pI of about 5.9. During isoelectric focusing, both forms of the enzyme separate into two components. The more acidic component that is resolved from the native enzyme is phosphorylated, but the other component is not. The amino acid composition of native tyrosine aminotransferase differs from values reported for mixtures of the three forms of this enzyme. Neither the native nor the modified forms of the enzyme possess a free alpha-amino group as judged by dansylation, nor can they be digested with leucine aminopeptidase, implying that the NH2-terminus is blocked. The possibility that tyrosine aminotransferase is acetylated was examined by translating poly(A)+RNA from hepatoma cells in a cell-free translational system in the presence and absence of inhibitors of protein acetylation. [35S]Tyrosine aminotransferase synthesized in the presence of the inhibitors has a more basic isoelectric point than the native enzyme as determined by isoelectric focusing, suggesting that the enzyme is acetylated either at the NH2-terminal or the epsilon-amino group of an internal lysine. When digested by either of two lysosomal proteases, tyrosine aminotransferase is cleaved to a smaller size. These data show that tyrosine aminotransferase is susceptible to several post-translational modifications.     

Species distribution and properties of hepatic phenylalanine (histidine):pyruvate aminotransferase.

Hepatic phenylalanine(histidine):pyruvate aminotransferase activity is much higher in the mouse and rat than in other animal species (human, guinea-pig, rabbit, pig, dog and chicken). The activity is elevated in the mouse and rat by the injection of glucagon but not in other species (guinea-pig, rabbit and chicken). The enzyme was purified from the mitochondrial fraction of mouse liver to homogeneity as judged by polyacrylamide disc gel electrophoresis in the presence of dodecylsulphate. With histidine as amino donor, the enzyme was active with pyruvate, oxaloacetate and hydroxypyruvate as amino acceptors but not with 2-oxoglutarate. Effective amino donors were histidine, phenylalanine and tyrosine with pyruvate, and methionine, serine and glutamine with phenylpyruvate. The apparent Km for histidine was about 6.9 mM with pyruvate and that for pyruvate was 21 mM with histidine. The enzyme is probably composed of two identical subunits with a molecular weight of approximately 40000. The pH optimum was near 9.0. Isoelectric focusing of the purified enzyme resulted in the detection of four forms with pI 6.0, 6.2, 6.5 and 6.7, respectively, all of which were responsive to glucagon. These four forms were nearly identical with the purified enzyme before the focusing with respect to physical and enzymic properties. A possible mechanism of this multiplicity is discussed.     

Behaviour of tyrosine amino transferase and convertase during the first hours after hepatectomy in rats

The activity of rat liver tyrosine amino transferase (TAT) increases after hepatectomy with a first prominent peak at 8 h and a second peak at 18 h. This change in activity is probably due to de novo enzyme synthesis since it is prevented by actinomycin-D (AMD). In the same period an increase of the lysosomal converting enzyme (convertase) which catalyses the in vitro transition of TAT from form I to form III, has been observed; this is not accompanied by changes of other lysosomal enzymes, such as acid phosphatase and cathepsin L. The activity of convertase is equal to that of the controls (sham operated animals) 2 h after hepatectomy, increases three times at 5 h, maintains the same value at 8 h and then decreases slowly to control level after 24 h. The correlation between the activity changes of the two enzymes strongly suggests a physiological role of convertase in TAT turnover.     

Age-related changes of liver tyrosine aminotransferase in senescent rats

Tyrosine aminotransferase was induced in adult and senescent rat liver and its properties studied. We show the appearance of a 'cross-reacting material' for induced tyrosine aminotransferase of old rats compared to basal enzyme; this cross-reacting material can be provoked in adult rats after injection of cycloheximide, and suppressed in adult and old rats after injection of a serine protease inhibitor (tosylphenylalanine chloromethylketone). Other properties of induced tyrosine aminotransferase (thermostability, Km for tyrosine, isoelectrofocusing) are identical except for the proportion of the three forms and their sensitivity to trypsin in the absence of pyridoxal phosphate, which is increased in senescent animals. The suppression of cross-reacting material clearly indicates that it is not due to errors on old rat liver DNA but rather to post-translational modifications. This demonstrates also the role of serine proteases in tyrosine aminotransferase degradation. We suggest that induced enzyme of senescent rats would undergo a conformational change, possibly due to a release of pyridoxal phosphate from the enzymic molecules, which would thus become more susceptible to proteolytic attack than those of adult rats.     

Characteristics of hepatic alanine-glyoxylate aminotransferase in different mammalian species.

Mitochondrial extracts of dog, cat, rat and mouse liver contain two forms of alanine-glyoxylate aminotransferase (EC 2.6.1.44): one, designated isoenzyme 1, has mol.wt. approx. 80 000 and predominates in dog and cat liver; the other, designated isoenzyme 2, has mol.wt. approx. 175 000 and predominates in rat and mouse liver. In rat and mouse liver, isoenzyme 1 activity was increased by the injection in vivo of glucagon, but not isoenzyme 2 activity. Isoenzyme 1 was purified and characterized from liver mitochondrial extracts of the four species. Both rat and mouse enzyme preparations catalysed transamination between a number of L-amino acids and glyoxylate, and with L-alanine as amino donor the effective amino acceptors were glyoxylate, phenylpyruvate and hydroxypyruvate. In contrast, both dog and cat enzyme preparations were specific for L-alanine and L-serine with glyoxylate, and used glyoxylate and hydroxypyruvate as effective amino acceptors with L-alanine. Evidence that isoenzyme 1 is identical with serine-pyruvate aminotransferase (EC 2.6.1.51) was obtained. Isoenzyme 2 was partially purified from mitochondrial extracts of rat and mouse liver. Both enzyme preparations were specific for L-alanine and glyoxylate. On the basis of physical properties and substrate specificity, it was concluded that isoenzyme 2 is a separate enzyme. Some other properties of isoenzymes 1 and 2 are described.     

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells

Corticosteroi-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 × g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I → II → III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cells subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.     

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells.

Corticosteroid-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 X g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I leads to II leads to III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cell subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Isolation of two different molecular weight polypeptides copurifying with rat liver tyrosine aminotransferase.

An affinity chromotography resin highly specific for rat liver tyrosine aminotransferase (EC 2.6.1.5) has been synthesized and used in the purification of this enzyme. The structure of the resin, N-(5′-phosphopyridoxyl)-l-tyrosyl-aminoocytl-Sepharose 4B, was designed to resemble the tyrosine-pyridoxal phosphate Schiff's base intermediate in the reaction pathway catalyzed by this enzyme. Use of this resin in combination with octyl-agarose chromatography on partially purified enzyme resulted in a tyrosine aminotransferase preparation with a specific activity of about 450 units/mg protein. When analyzed on one-dimensional polyacrylamide-sodium dodecyl sulfate slab gels, the highly purified enzyme was composed of two polypeptides with molecular weights of about 56,000 and 53,000. Radioiodinated tryptic peptides from each of these polypeptides were essentially identical following two-dimensional analysis. Although the two polypeptides could not be separated from each other in an active form, it was found that (i) both polypeptides have pyridoxal phosphate-binding sites, (ii) the coenzyme is probably bound to both polypeptides as a Schiff's base, (iii) both polypeptides have binding sites for l-tyrosine and l-glutamic acid, the two specific substrates for the enzyme, and (iv) both polypeptides can catalyze the formation of the initial amino acid-pyridoxal phosphate Schiff's base adduct in the overall reaction pathway. Since the ratios of these polypeptides differed from preparation to preparation of purified enzyme, the 53,000 M_r species probably arises by proteolysis of tyrosine aminotransferase in crude liver extracts. These results imply that if tyrosine aminotransferase isozymes exist, they are not the result of translation products produced by different structural genes.     

Stabilization of rat liver tyrosine aminotransferase by tetracycline.

Rat liver tyrosine aminotransferase was purified 200-fold and an antiserum raised against it in rabbits. 2. Hepatic tyrosine aminotransferase activity was increased fourfold by tyrosine, twofold by tetracycline, 2.5-fold by cortisone 21-acetate and ninefold by a combination of tyrosine and cortisol administered intraperitoneally to rats. 3. Radioimmunoassay with 14C-labelled tyrosine aminotransferase, in conjunction with rabbit antiserum against the enzyme, revealed that cortisol stimulates the synthesis of the enzyme de novo, but that tetracycline has no such effect. 4. Incubation of rat liver homogenates with purified tyrosine aminotransferase in vitro leads to a rapid inactivation of the enzyme, which tetracycline partially inhibits. 5. The inactivation is brought about by intact lysosomes, and the addition of 10mM-cysteine increases the rate of enzyme inactivation, which is further markedly increased by 10mM-Mg2+ and 10mM-ATP. Here again tetracycline partially inhibits the decay rate, leading to the inference that the increase of tyrosine aminotransferase activity in vivo by tetracycline is brought about by the latter inhibiting the lysosomal catheptic action.     

Cathepsin L. A new proteinase from rat-liver lysosomes.

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex. 2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8-6.1 ascertained by analytical isoelectric focusing. 3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6-7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30-40% at pH 7.0. 5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor. 6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.     

Subcellular distribution of a factor inactivating tyrosine aminotransferase. Study of its mechanism and relationship to different forms of the enzyme.

The subcellular distribution of a tyrosine aminotransferase inactivating factor in rat liver has been investigated. Most of its activity is associated with plasma membranes, with minor amounts in mitochondria and endoplasmatic reticulum. The factor is also found in kidney and inactivates the enzyme reversibly in presence of cysteine, most likely by modification of -SH groups. ATP counteracts this inactivation only, when crude enzyme extracts are inactivated by purified subcellular fractions or when the purified enzyme is inactivated in presence of liver or kidney cortex homogenates. The relationship of this inactivation to reported different forms of the enzyme has been investigated. Form I of three different forms, that can be obtained by hydroxyl-apatite chromatography, is readily inactivated, form III can be partly converted to form I by incubation in presence of purified plasma membranes. The relationship of these findings to a possible multistep mechanism in the turnover of the enzyme discussed.     

The influence of starvation and tryptophan administration on the metabolism of phenylalanine, tyrosine and tryptophan in isolated rat liver cells.

Liver cells from fed Sprague-Dawley rats metabolized phenylalanine, tyrosine and tryptophan at rates consistent with the known kinetic properties of the first enzymes of each pathway. Starvation of rats for 48 h did not increase the maximal activities of phenylalanine hydroxylase, tryptophan 2,3-dioxygenase and tyrosine aminotransferase in liver cell extracts, when results were expressed in terms of cellular DNA. Catabolic flux through the first two enzymes was unchanged; that through the aminotransferase was elevated relatively to enzyme activity. This is interpreted in terms of changes in the concentrations of 2-oxoglutarate and glutamate. Cells from tryptophan-treated animals exhibited significant increases in the catabolism of tyrosine and tryptophan, but not of phenylalanine. The activities of tyrosine aminotransferase and tryptophan 2,3-dioxygenase were also increased, although the changes in flux and enzyme activity did not correspond exactly. These results are discussed with reference to the control of aromatic amino acid catabolism in liver; the role of substrate concentration is emphasized.