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.     

An isoelectric focusing study of acid phosphatase heterogeneity in rat liver

The different forms of acid phosphatase (EC 3.1.3.2) in rat liver homogenates, lysosomal, mitochondrial, microsomal fractions and cytosol were studied with isoelectric focusing. Evidence is presented that isoelectric focusing of acid phosphatase in subcellular fractions shows individual changes and time related patterns. Mild autolysis shifted all enzyme activity peaks of isoelectric focusing patterns to the one at pH 7.04.     

An Inhibition and Kinetic Study of Acid Phosphatase in Paramecium caudatum and Paramecium tetraurelia1

ABSTRACT. Inhibition, inactivation, pH, and kinetic studies using both homogenates and purified lysosomal fractions of Paramecium caudalum and of P. tetraurelia were carried out to examine the lysosomal acid phosphatase (AcPase) and its relationship to p-nitrophenylphosphatase (pNPPase), glucose-6-phosphatase (G6Pase), and 5′-nucleotidase (AMPase). The results generally support the idea that Paramecium cells contain a distinct lysosomal AcPase with a broad substrate specificity. The hydrolysis of glucose-6-phosphate (G6P) and adenosine 5′-monophosphate (AMP) was shown to be due to this enzyme, suggesting that true G6Pase and AMPase may be lacking in these two species; however, some hydrolysis of AMP at pH 7.5 catalyzed by an unknown soluble enzyme distinct from alkaline phosphatase and Na⁺-K⁺-ATPase was observed. Since the hydrolysis of p-nitrophenylphosphate (pNPP) at acid pH was also shown to be due to AcPase alone, pNPPase could be used as a rapid assay for Paramecium AcPase. At an alkaline pH, however, this activity was catalyzed by an alkaline phosphatase located in the cytosol fraction. P. caudatum AcPase was shown to have kinetic properties similar to those of purified rat liver and human prostatic AcPase and to have relative substrate affinities in the order of G6P < β-glycerophosphate < pNPP < AMP. These different substrate affinities might account for the observed differences in the inhibition of the four lysosomal activities by NaF, L(+)-tartrate, and molybdate, all of which inhibited the hydrolysis of G6P, β-glycerophosphate, and pNPP competitively, but which exhibited a noncompetitive inhibition of a mixed type with the hydrolysis of AMP.     

The use of phospholipid vesicles for in vitro studies on cholesteryl ester hydrolysis.

Radiolabeled cholesteryl oleate was incorporated into vesicles prepared from egg yolk lecithin and utilized as a substrate for studies of sterol ester hydrolases present in rat liver homogenates. The cholesteryl oleate was shown to be associated with vesicles (unilamellar liposomes) using Sepharose 4B chromatography. With this substrate, two different cholesteryl ester hydrolytic enzymes were demonstrated in subcellular fractions from the liver homogenates. In the lysosome-rich fraction an acid hydrolase was present, while in the cytosol fraction (150,000 g supernatant), hydrolytic activity was shown to occur with an optimum pH between 8 and 8.5. The substrate was characterized by Sepharose chromatography both before and after incubation with the liver fraction and was not dramatically altered even by rigorous incubation conditions. The lysosomal enzyme preparation was capable of hydrolyzing almost all the cholesteryl oleate in the vesicles. Hydrolysis of the phospholipid was proportionately much less than that of the cholesteryl oleate. Comparisons were performed between the vesicle preparation and an alternate substrate preparation involving the direct addition of cholesteryl oleate in acetone solution. The vesicles appeared to be a better substrate for the lysosomal enzyme whereas the activity in the cytosol fraction did not distinguish between the two substrate preparations. Unsonicated suspensions of cholesteryl oleate and lecithin did not serve as suitable substrates for the enzymes. These studies demonstrate the applicability of cholesteryl ester-containing vesicles as a useful substrate for studying cholesteryl ester hydrolysis in vitro.     

The subcellular distribution of rat liver l-alanine–glyoxylate aminotransferase in relation to a pathway for glucose formation involving glyoxylate

1. The distribution of l-alanine-glyoxylate aminotransferase activity between subcellular fractions prepared from rat liver homogenates was investigated. The greater part of the homogenate activity (about 80%) was recovered in the ;total-particles' fraction sedimented by high-speed centrifugation and the remainder in the cytosol fraction. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients revealed a specific association between the aminotransferase and the mitochondrial enzymes glutamate dehydrogenase and rhodanese. 3. The aminotransferase activities in the cytosol and the mitochondria are due to isoenzymes. The solubilized mitochondrial enzyme has a pH optimum of 8.6, an apparent K(m) of 0.24mm with respect to glyoxylate and is inhibited by glyoxylate at concentrations above 5mm. The cytosol aminotransferase shows no distinct pH optimum (over the range 7.0-9.0) and has an apparent K(m) of 1.11mm with respect to glyoxylate; there is no evidence of inhibition by glyoxylate. 4. The mitochondrial location of the bulk of the rat liver l-alanine-glyoxylate aminotransferase activity is discussed in relation to a pathway for gluconeogenesis involving glyoxylate.     

Insulin and glucagon degradation by the kidney II. Characterization of the mechanisms at neutral pH

Insulin and glucagon degradation by rat kidney homogenates and subcellular fractions was examined under a variety of conditions including high and low substrate concentrations, at pH 4 and pH 7, with and without glutathione. At high insulin concentration (4.1 · 10⁻⁵ M) insulin degradation by the homogenate was greatest at pH 4 but at low insulin concentration (1 · 10⁻¹⁰ M) insulin degradation was greatest at pH 7. At either high or low glucagon concentration glucagon degradation by the homogenate was greatest at pH 7. Glutathione at pH 7 stimulated insulin degradation at high insulin concentrations and inhibited insulin degradation at low concentrations. Glucagon degradation at pH 7 was inhibited at both high and low concentrations of glucagon by glutathione.Separation of kidney into cortex and medulla prior to homogenation produced a pattern of insulin and glucagon degradation identical to the whole homogenate but glucagon degradation by the medulla was greater than by the cortex.Examination of degradation by subcellular fractions revealed that at high concentration at neutral pH most insulin was degraded by the 100 000 × g pellet but at low insulin concentrations over 90% of the activity was in the 100 000 × g supernatant. At pH 7, at both high and low concentrations, most glucagon-degrading activity was in the 100 000 × g pellet, although the cytosol also had activity. At pH 4 most degradation occurred in the lysosomal fractions.Separation into cortex and medulla again showed similar distribution of activity as the whole gland with the medulla having more glucagon-degrading activity than the cortex. With low insulin concentrations the cortex 100 000 × g supernatant had higher relative specific activities than the medulla supernatant.Examination of recoveries of enzyme activity revealed that the subcellular fractions consistently had markedly less insulin-degrading activity than the original homogenate. This loss of activity was only discernible when insulin degradation was performed at pH 7 at low substrate concentrations. Comparable losses of glucagon-degrading activity were not seen.     

Arylsulphatase activity of corn (Zea mays L.) seedling roots

The crude lysosomal fraction of corn seedling root tips contains an arylsulphatase (E.C. 3.1.6.1) which hydrolysed p-nitrophenyl sulphate at pH 8.0 but had no activity towards p-nitrocatechol sulphate. The Km value for p-nitrophenyl sulphate was 1.24 mM. The hydrolysis of p-nitrophenyl sulphate was linear up to 2 h and the rate was proportional to the amount of enzyme added. The enzyme was strongly inhibited by cyanide, fluoride and phosphate ions and did not resemble the arylsulphatases of bacterial and animal origin.     

The effect of TEPC-183 plasmacytoma growth on beta-glucuronidase activity in serum and tissues of tumor-bearing mice

beta-Glucuronidase activity increased in the serum of BALB/c mice during the growth of the IgM-secreting plasmacytoma, TEPC-183. The increase appeared to correlate with tumor burden. The beta-glucuronidase activity in tissue homogenates of spleen, liver, and kidney from tumor-bearing mice also increased significantly compared to the levels found in corresponding tissues from normal control mice. Assays of lysosomal and microsomal fractions from livers of TEPC-bearing mice indicated that approximately 70% of the enzyme activity was associated with the lysosomal fraction and the remainder with the microsomal fraction. A similar distribution was found in homogenates prepared from the plasmacytoma itself. In contrast to this the beta-glucuronidase activity in livers from normal BALB/c mice is nearly equally distributed between lysosomal and microsomal fractions.     

STRUCTURE-LINKED ACTIVITY OF LYSOSOMAL ENZYMES IN THE DEVELOPING MOUSE BRAIN

—A longitudinal study of the maturation of mouse cerebral lysosomal enzymes has been completed. Activity of the enzymes, acid phosphatase (I.U.B. 3.1.3.2), β-glucuronidase (I.U.B. 3.2.1.31) and β-acetylglucosaminidase (I.U.B. 3.2.1.30) was assayed spectrofluorimetrically on portions of supernatant from 0.25 M sucrose homogenates spun at 6 x 10³ -min. Activities were obtained in native (free) and Triton X-100 activated samples (total). The neonatal period was characterized by relatively low free and high total acid phosphatase activities. An abrupt rise in free activity occurred during the period 10–20 days. Discontinuous anion exchange DEAE cellulose chromatography (0.01 m -tris–maleate, pH 6.3) with elution by ascending molarities of NaCl of the Triton X-100 activated supernatant revealed three major peaks in the adult. A fourth peak, designated as fraction II (‘maturation fraction’) occurred only during the neonatal period, a time also characterized by increased specific activity of fraction I, with no change in fraction IV. The chromatographic fractions were further characterized by optimal pH, ascorbate, fluoride, Cu²⁺ and Fe²⁺ ions. The maturation profiles of total, β-glucuronidase and total, β-acetylglucosaminidase differed from each other, and from that of total acid phosphatase. Comparable differences existed in the profiles of the free activities, and the ratio of free:total activity differed for each enzyme at any selected time especially during the neonatal period. These findings are are discussed with reference to the maturation of isoenzyme fractions with age, and suggest that the changes in structure-linked organization of individual lysosomal hydrolases are functions of heterogeneity in enzyme complement of individual lysosomes.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.     

Properties of mouse α-galactosidase

α-Galactosidase has been examined in various murine tissues using the substrate 4-methylumbelliferyl-α-galactoside. Mouse liver appears to contain a single major form of the enzyme, as judged by chromatography and electrophoresis. The enzyme was purified 467-fold with a yield of about 40% by a method involving chromatography on Concanavalin A-Sepharose. It has maximal activity at pH 4.2, a K_m value of 1.4 mM, an energy of activation of 16 400 cal/mol, and a molecular weight of 150 000 at pH 5.2. It is inhibited at high concentrations of myoinositol and appears to contain N-acetylneuraminic acid. In these characteristics it resembles human α-galactosidase A.The enzyme from various tissues differs in electrophoretic mobility. After treatment with neuraminidase, however, the enzyme from all tissues comigrates as a single band of activity. By this criterion the α-galactosidase of liver is most heavily sialylated and that from kidney the least. As estimated by gel filtration, the enzyme from liver and kidney exists as species of molecular weight 320 000, 150 000 and 70 000, depending upon pH and ionic strength. This appears to be the result of aggregation of the enzyme, since the forms are interconvertible and under some conditions a single molecular weight species is observed. The liver enzyme is primarily lysosomal, while the kidney enzyme is distributed approximately equally between lysosomal and microsomal fractions.     

Hepatic alkaline phosphatase isoenzymes: isolation, characterization and differential alteration.

Although it is generally believed that hepatic alkaline phosphatase is localized to liver plasma membranes, 63% is present in the cytosol fraction after ultracentrifugation of rat liver homogenates. Divalent cation requirements, heat inactivation, pH optima, Km and chemical inhibition characteristics of partially purified alkaline phosphatase enzymes prepared from membrane and cytosol fractions suggested different structural forms. Furthermore, bile duct obstruction and ethinyl estradiol administration preferentially increased membrane-bound alkaline phosphatase activity, while cytosol activity was unaltered. In contrast, phenobarbital treatment decreased membrane-bound alkaline phosphatase and increased cytosol activity. These studies support the presence of two forms of hepatic alkaline phosphatase in rat liver which are regulated by different control mechanisms.     

Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells

1. 1. The specific activity of the lysosomal hydrolases in cultured skin fibroblasts varies according to the phase of growth in culture.

2. 2. Diagnosis of heterozygous genotypes for lysosomal enzyme deficiency diseases is unreliable with cultured fibroblasts, at least partly because of the growth curve-associated variations in specific activity.

3. 3. Fluctuations in specific activity during the beginning of the growth curve in vitro can be avoided by initiating cultures with cells which are in the early log phase of growth.

4. 4. Primary amniotic fluid cell cultures show no relationship between length of time in culture and lysosomal enzyme specific activity.

5. 5. Secondary amniotic fluid cell cultures exhibit growth curve-related variations in lysosomal enzyme specific activity as they assume fibroblast-like growth kinetics.

6. 6. Prenatal and postnatal diagnosis on cultured amniotic fluid cells and fibroblasts requires the use of appropriate controls which are matched for stage of growth and length of time after the last trypsinization.

     

Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum.

Lysosome formation was induced in cells of the renal medulla by feeding rats on a K+-deficient diet. The role of the endoplasmic reticulum in the production of acid phosphatase, a typical lysosomal enzyme, was examined. Lysosomal and microsomal fractions were prepared for study by differential centrifugation of homogenates of renal papilla and inner stripe of red medulla. Acid phosphatase activity in the microsomal fraction was distinguished from the activity in the lysosomal fraction in normal tissue by differences in pH optima, tartrate inhibition, distribution of multiple forms after polyacrylamide-gel electrophoresis and detergent-sensitivity. During progressive K+ depletion, acid phosphatase activity in both microsomal and lysosomal fractions of the tissue increased 3-fold. In the lysosomes, K+ depletion was associated with the appearance of a new band of acid phosphatase. The neuraminidase-sensitivity of this band on polyacrylamide-gel electrophoresis indicated that the enzyme protein had been modified by the addition of sialic acid residues. K+ depletion also altered the lysosomal enzyme so that thiol compounds were able to stimulate its activity.     

Transformation of Pseudomonas putida with chromosomal DNA

The $\text{in vitro}$ deiodination of [¹²⁵I]-4-iodobiphenyl, [¹²⁵I]-4-iodonitrobenzene and [¹²⁵I]-4-iodoaniline was investigated. No deiodination was detected in rat thyroid homogenates. However, at least three biodeiodination mechanisms were indicated for substrates in rat liver subcellar fractions. Microsomal dehalogenation occurred to a minor extent with increased dehalogenation taking place in the cytosol fraction. The cytosol deiodination was extensive for 4-iodonitrobenzene and was mediated by glutathione. A second cytosol deiodination mechanism, not mediated by glutathione, was evident when 4-iodobiphenyl was the substrate. This soluble enzyme system could be enhanced by Arochlor 1254 or 4-iodobiphenyl pretreatment.     

Multiple forms of synthase D phosphatase and phosphorylase a phosphatase in liver and regulatory effects of metabolites on their activities

The smooth endoplasmic reticulum (ER) and cytosol fractions of liver homogenates exhibit phosphoprotein phosphatase activity towards glycogen synthase D and phosphorylase a. The following observations suggest that liver contains multiple forms of these phosphatases. Synthase phosphatase activity in either fraction was more readily inactivated by heating than phosphorylase phosphatase activity. Both synthase phosphatase and phosphorylase phosphatase activities in smooth ER were non-competitively inhibited by Mg2+, but were activated by this ion in the cytosol. Synthase phosphatase activities in cytosol and smooth ER were stimulated by a number of sugar phosphates, particularly glucose-1-phosphate, galactose-6-phosphate and fructose-6-phosphate. Erythrose-4-phosphate stimulated synthase phosphatase activity in the cytosol, but inhibited the microsomal enzyme. Phosphorylase phosphatase activities in either fraction were inhibited by most sugar phosphates. Adenosine mono-, di- and tri-phosphates inhibited phosphatase activities in both fractions. Low concentrations of AMP and ADP inhibited phosphorylase phosphatase activities to a greater extent than synthase phosphatase activities. Chromatography of the smooth ER fraction on DEAE-cellulose resulted in the separation of synthase phosphatase from phosphorylase phosphatase, as soluble proteins. The elution profile for the microsomal phosphatase was different from that for the cytosol enzymes. It is concluded that: both synthase phosphatase and phosphorylase phosphatase in liver have at least two isoenzyme forms; synthase phosphatase and phosphorylase phosphatase are separate enzymes; the different behaviour of microsomal and cytosol phosphatases towards divalent cations and sugar phosphates provides a potential mechanism for the differential regulation of these activities in liver.     

The occurrence and intracellular localization of sucrase activity in rat liver

The presence of an enzyme in rat liver which hydroluzes sucrose is demonstrated in this report. The hydrolysis of sucrose was studied in vivo after injecting [¹⁴C]sucrose into rats, and a method was developed for the extraction and analysis of the radioactive sugars stored within subcellular particles. The results show that, besides sucrose, glucose and fructose are also found in the lysosomal fraction of the liver homogenate. In vitro studies reveal the presence of a sucrase, although the activity of the enzyme is very low. Intracellular distribution studies indicate that sucrase is present in the lysosomes as well as in the microsomes, the microsomal enzyme having a pH optimum different from that of the lysosomal enzyme.     

The relative stability of liver cytosol enzymes incubated in vitro

1. Relative rates of enzyme inactivation were measured in liver slices, homogenates and cytosol fractions as well as in the presence of trypsin and at acid pH. The enzymes chosen are all present in the cytosol fraction of rat liver, and have widely different degradation rate constants in vivo. 2. The inactivation rates of lactate dehydrogenase, fructose bisphosphate aldolase, glucose 6-phosphate dehydrogenase, glucokinase, phosphoenolpyruvate carboxykinase (GTP), l-serine dehydratase and thymidine kinase in liver preparations at neutral pH are in a similar order to the rate constants of degradation of these enzymes in the intact animal. 3. The two exceptions of this general correlation were tyrosine aminotransferase, which was stable in vitro but not in vivo, and glyceraldehyde phosphate dehydrogenase, which shows the reverse pattern. 4. These findings generally support the concept that the same factors are responsible for enzyme inactivation in vitro as occur in the intact tissue.