The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response.

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.     

Studies on the effect of experimental inflammation on sialyltransferase in the mouse and guinea pig

The effect of inflammation on sialyltransferase was studied in the mouse and guinea pig. There was a three-fold increase in mouse liver sialyltransferase activity reaching a maximum at 72 hr after inflammation; serum levels were increased by five-fold at 72 hr after inflammation. The response of guinea pig sialyltransferase was slower and of lower magnitude compared with the response of the mouse enzyme; liver and serum sialyltransferase increased by about 50% reaching a maximum at 96 hr after inflammation. The specificity of the enzyme that responded to inflammation in the mouse and guinea pig was found to be Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase, the same enzyme activity that was shown to be an acute phase reactant in earlier studies in the rat (Kaplan et al., 1983).     

Studies on the effect of lysosomotropic agents on the release of Gal beta 1-4GlcNAc alpha-2,6-sialytransferase from rat liver slices during the acute-phase response.

The mechanism of release of Gal beta 1-4GlcNAc alpha-2,6-sialyltransferase (CMP-N-acetylneuraminate: beta-galactoside alpha-2,6-sialytransferase, EC 2.4.99.1) from rat liver during the acute-phase response is due to the action of a cathepsin D-like proteinase that cleaves the trans-Golgi membrane-bound enzyme from a membrane anchor; this allows a major portion of the enzyme containing the catalytic site to escape into the extracellular space [Lammers & Jamieson (1988) Biochem. J. 256, 623-631]. The release of sialytransferase was most effective at pH 5.6, suggesting that release of sialyltransferase from the Golgi in whole cells is dependent on maintaining an acidic environment in the trans-Golgi compartment of the hepatocyte. Golgi membranes contain a proton pump that maintains the acidic pH in these compartments [Glickman, Croen, Kelly & Al-Awquati (1983) J. Cell Biol. 97, 1303-1308; Yamashiro, Tycko & Maxfield (1984) Cell (Cambridge, Mass.) 37, 789-800; Zhang & Schneider (1983) Biochem. Biophys. Res. Commun. 114, 620-625; Anderson & Pathak (1985) Cell (Cambridge, Mass.) 40, 635-643]. Lysosomotropic agents, such as NH4Cl, chloroquine and methylamine can penetrate acidic compartments of the cell, such as the Golgi complex, raise the pH, and thus affect proteolytic cleavage events. The present paper describes the effect of lysosomotropic agents on the release of sialyltransferase from the hepatocyte using liver slices as a whole-cell system. Slices were prepared from control rats and rats suffering from the acute-phase response, where release of sialyltransferase is increased substantially [Lammers & Jamieson (1988) Biochem. J. 256, 623-631; Kaplan, Woloski, Hellman & Jamieson (1983) J. Biol. Chem. 258, 11505-11509]. Release of sialyltransferase was almost abolished in presence of 50 mM-NH4Cl, 50 mM-methylamine or 1 mM-chloroquine. Inhibition of release of sialyltransferase was reversed when the lysosomotropic agents were removed from the medium, showing that these agents are not cytotoxic to the cells under the conditions used. The secretion of rat alpha 1-acid glycoprotein, which is not subject to proteolytic processing in the Golgi complex, was not found to be substantially affected by the presence of lysosomotropic agents. The results suggest that proteolytic cleavage of the catalytic site of sialyltransferase is a process that is significantly affected by the intra-Golgi pH.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.     

Membrane-associated carbonic anhydrase from rat lung. Purification, characterization, tissue distribution, and comparison with carbonic anhydrase IVs of other mammals.

Carbonic anhydrase (CA) IV was purified to homogeneity from rat lung microsomal and plasma membranes. The single N-terminal amino acid sequence showed 55% similarity to that reported for human CA IV. A monospecific antibody to the 39-kDa rat enzyme that cross-reacts on Western blots with CA IVs from other mammalian species was produced in rabbits. Digestion of rat lung enzyme with endoglycosidase (peptide-N-glycosidase F) reduced the Mr to 36,000, suggesting that rat CA contains one N-linked oligosaccharide chain. All of eight additional mammalian CA IVs that were examined also contained oligosaccharide chains, as evidenced by reduction in Mr from 52,000 (cow, sheep, and rabbit), 42,000 (pig, guinea pig, and dog), and 39,000 (mouse and hamster) to 36,000 after treatment of the respective lung microsomal membranes with peptide-N-glycosidase F. The 36-kDa human enzyme showed no change in molecular mass with this treatment. Thus, the human CA IV is the exceptional one in lacking carbohydrate. Rat lung CA IV was found to be relatively resistant to sodium dodecyl sulfate and to be anchored to membranes by a phosphatidylinositol-glycan linkage; both properties were found to be shared by other mammalian CA IVs. Western blot analysis indicated distribution of CA IV in rat tissues other than kidney and lung where it was previously known to be present. CA IV was particularly abundant in rat brain, muscle, heart, and liver, all locations where the CA IV enzyme was not known to be present previously. None was detected in rat skin or spleen.     

An effect of colchicine on galactosyl- and sialyltransferases of rat liver Golgi membranes

Colchicine inhibited the activity of the galactosyl- and sialyltransferases of rat liver Golgi membranes. The sialyltransferase was more sensitive to the drug than galactosyltransferase since it was inhibited to a greater extent and at lower concentrations of colchicine than the galactosyltransferase. Two soluble enzymes, i.e. that from rat serum and that isolated from bovine milk, were not inhibited by colchicine. Even with very high concentrations of colchicine a marked stimulation of activity was observed. The data suggest that the inhibition observed in the Golgi membranes is in some way related to the arrangement of the enzymes in the lipid bilayer. In support of this hypothesis, the milk galactosyltransferase became very sensitive to colchicine after incorporation of the enzyme into lipid vesicles. The incorporation of colchicine into Golgi membranes was shown to decrease the order parameter as determined by electron spin resonance which reflects an increased fluidity of the Golgi membranes. A change in fluidity may be responsible for the inhibition of enzyme activity at least in part.     

Further observations on the properties of serum and tissue guanase from man and some animal species. Optimum pH and activation energy in the presence of 8-azaguanine.

The activation energy and the optimum pH of guanine deaminase in man, the rat, guinea pig and mouse were studied using 8-azaguanine as a substrate. The serum guanase in man and in all the animal species studied differs in activation energy from the guanase of the liver. In man, moreover, the serum guanase is also different from the brain and kidney enzyme. In the rat and guinea pig the brain enzyme has thermic activation energy different from the liver and kidney enzyme. The guanase of the serum and tissues of the guinea pig differs from the enzyme of the serum and tissues of man, rat and mouse for optimum pH.     

Topography of glycosyltransferases involved in the initial glycosylations of gangliosides.

We attempted to establish within which organelle UDP-Glc:ceramide beta 1----1'glucosyltransferase (GlcT) is located and moreover to obtain information about its orientation on intracellular membranes as well as that of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) and CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase (SAT-1). An extremely purified Golgi apparatus fraction was the only liver fraction where a ceramide-dependent formation of glucosylceramide could be demonstrated. This Golgi fraction, mainly constituted by stacks of intact cisternae which retained the same topographical orientation as in vivo, was then incubated with liposomal dispersions of glycosphingolipid-glycosyltransferase acceptors in reaction mixtures containing all the requirements for enzyme activity but no detergent. Under such conditions, SAT-1 and other late acting glycosyltransferases were over 90% latent, while both GlcT and GalT-2 were just as active as in the detergent-containing assay; they were still inhibited by EDTA. Sepharose-immobilized ceramide and Sepharose-immobilized glucosylceramide were found to be suitable acceptors for GlcT and GalT-2, respectively, still using intact Golgi cisternae as the enzyme source. Moreover, a part of GlcT and GalT-2 activity was released from intact Golgi cisternae upon cathepsin D treatment. These results provide strong evidence that GlcT and GalT-2 face the cytoplasmic side of the Golgi apparatus, whereas SAT-1 and the other late acting enzymes face the luminal side.     

Cathepsins J and K: high molecular weight cysteine proteinases from human tissues

Human tissue extracts contained two high Mr proteinases active in hydrolyzing the fluorogenic substrate Cbz-phe-arg-aminomethylcoumarin. By gel filtration chromatography, cathepsins J and K had apparent molecular weights of 230,000 and 650,000, respectively. Both enzymes were cysteine proteinases with optimum activity at pH 6.2-6.8; neither had aminopeptidase activity. Human kidney, lung and spleen were rich sources of these enzymes, while liver contained moderate amounts. Cathepsins J and K were partially characterized and appeared to differ from the mammalian high Mr cysteine proteinases described in the literature. In rat liver and kidney and in mouse liver, cathepsin J was localized in the particulate fraction, whereas cathepsin K was not detected in these tissues.     

Light and electron microscopic studies of the paracortical post-capillary high-endothelial venules

Summary The light and electron microscopic structure of the high-endothelial postcapillary venules of lymph nodes were studied in the mouse, rat, guinea pig, and rabbit. The venules were most frequently found in the mouse and rat. In all species, they reached their highest degree of differentiation in the paracortical area. The morphology in the light microscope was identical in all four species. The venules in the rat and mouse were surrounded by a cuff of concentrically-arranged lymphocytes, which was rarely seen in the guinea pig and rabbit.The ultrastructure of the high-endothelial cells in all four species was very complex; a prominent Golgi apparatus was present which often occupied large parts of the cytoplasm. Coated and uncoated vesicles originating in the Golgi apparatus often permeated the cytoplasm. These vesicles emptied their contents into the extracellular space after fusion with the plasma membranes.Numerous lymphocytes traversed vessel walls. During their passage, they were always located between, not inside the high-endothelial cells.     

Comparative studies of the protein composition of red blood cell membranes from eight mammalian species

The polypeptide pattern of red blood cell (RBC) membranes from cow, sheep, horse, rabbit, guinea pig, rat, mouse, analyzed by polyacrylamide gel electrophoresis, was compared to human RBC counterpart. Some qualitative and quantitative differences were noted. Among the high molecular weight components the bands 2.1- 2.3 appeared slightly decreased in rabbit and rat and increased in sheep RBC membranes. Band 3 appeared to have a higher molecular weight in the cow, guinea pig and mouse RBCs, and a lower molecular weight in the sheep RBCs. Band 4.1 from the RBC membranes of cow, sheep, rabbit and guinea pig was splitted into two sub-bands, while band 4.2 overlapped with band 4.1 in horse and guinea pig RBC membranes. There are marked differences in the number and position of bands in the 4.5 region, while band 4.9 is present in higher amounts in horse, rabbit and guinea pig RBC membranes. Band 6 (glyceraldehyde 3-phosphate dehydrogenase) was undetectable in horse, rat and mouse RBC membranes and was decreased in sheep, rabbit and guinea pig. There are also major differences in the region of band 7 and below ("post-7"). Band 8 was undetectable in horse, cow and guinea pig, and was in higher amounts in rat. A band corresponding to a molecular weight of about 22 kD in the "post-8" region was present only in guinea pig RBC membranes.     

Characterization of vasoactive intestinal peptide (VIP) receptors in mammalian lung

125I-VIP bound specifically to sites on human, rat, guinea pig, and rabbit lung membranes with a dissociation constant (KD) of 60-200 pM and binding site maxima of 200-800 fmol/mg of protein. The presence of a second lower affinity site was detected but not investigated further. High affinity 125I-VIP binding was reversible and displaced by structurally related peptides with an order of potency: VIP greater than rGRF greater than PHI greater than hGRF greater than secretin = Ac Tyr1 D Phe2 GRF. 125I-VIP has been covalently incorporated into lung membranes using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrilamide gel electrophoresis of labeled human, rat, and rabbit lung membranes revealed major 125I-VIP-receptor complexes of: Mr = 65,000, 56,000, and 64,000 daltons, respectively. Guinea pig lung membranes exhibited two 125I-VIP-receptor complexes of Mr = 66,000 and 60,000 daltons. This labeling pattern probably reflects the presence of differentially glycosylated forms of the same receptor since treatment with neuroaminidase resulted in a single homogeneous band (Mr = 57,000 daltons). Soluble covalently labeled VIP receptors from guinea pig and human lung bound to and were specifically eluted from agarose-linked wheat germ agglutinin columns. Our studies indicate that mammalian lung VIP receptors are glycoproteins containing terminal sialic acid residues.     

Variations in induction of drug-metabolizing enzymes by trans-Stilbene oxide in rodent species

trans-Stilbene oxide (400 mg/kg) produced a 500% increase in the microsomal in the microsomal epoxide hydratase activity in rat and mouse with little change in the soluble enzyme activity. However, in guinea pig, the soluble epoxide hydratase activity increased by about 33% with only a small increase (47.6%) in the microsomal enzyme activity. The soluble glutathione S-transferase activities were also induced in both rat and mouse, with little change in that of the guinea pig. Increasing dosage of trans-stilbene oxide from 400 mg/kg to 1000 mg/kg had little effect on the above enzyme activities. That the guinea pig was not relatively refractory to all inducing agents was shown by the fact phenobarbital (100 mg/kg) and 3-methylcholanthrene (25 mg/kg) produced relatively similar increases in the activities of aniline hydroxylase and P-aminopyrineP-demethylase in rat, mouse and guinea pig. However, these inducers produced only a 15–20% stimulation in the soluble glutathione S-transferase and microsomal epoxide hydratase activities in guinea pig, when compared to a 50–80% increase in rat and mouse, suggesting a general resistance to induction by the phase II enzymes in guinea liver. In all animal models, the inducer markedly increased th emicrosomal total phospholipid content, although the sphingomyelin content itself was decreased. In both rat and mouse, the microsomal cholesterol content was significantly decreased while that in guinea pig was unaffected. Possible factors responsible for the observed species differences are discussed.     

Characteristic distribution of cathepsin E which immunologically cross-reacts with the 86-kDa acid proteinase from rat gastric mucosa

The antiserum raised against the high-molecular-weight acid proteinase from rat gastric mucosa, termed 86-kDa acid proteinase, has been shown to recognize rat cathepsin E, but not cathepsin D (Muto, N. et al. (1987) J. Biochem. 101, 1069-1075). Using this specific antiserum, characteristic distribution of cathepsin E in rats was demonstrated. The enzyme was detected in a limited number of tissues, such as stomach, thymus, spleen, bladder, and erythrocyte membranes. Among them, the highest activity was observed in the stomach. In contrast, cathepsin D immunoreactive with the antiserum specific to rat gastric cathepsin D was demonstrated in all the tissues examined. Cathepsin E-type enzymes partially purified from these five tissues were precipitated in the same manner by the specific antiserum, and they had the same molecular weight, electrophoretic mobility, and resistance against denaturation by 4 M urea. These results indicate that they could be exactly classified as cathepsin E. This type of enzyme was also detectable in mice and guinea pigs, but they showed relatively weak immunoreactivities with the antiserum. Thus, it is concluded that the distribution of cathepsin E is intrinsically different from ordinary cathepsin D, suggesting that it has a different physiological role from cathepsin D.     

Post-Golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody

During studies on the Golgi apparatus immunolocalization of beta-galactoside alpha 2,6-sialyltransferase in intestinal cells, immunostaining of a number of post-Golgi apparatus structures including mucus droplets and plasma membrane were observed. In order to determine if this labeling was in fact due to sialyltransferase and not carbohydrate-specific antibodies in the polyclonal antiserum preparation, fusion protein to sialyltransferase was used to epitope purify polypeptide-specific antibodies. The affinity purification was performed on a column containing a beta-galactosidase-sialyltransferase fusion protein expressed in Escherichia coli. Using such antibodies we present evidence that in intestinal cells sialyltransferase is not only present in the Golgi apparatus cisternal stack but also its transtubular network and various post-Golgi apparatus structures. In absorptive enterocytes, post-Golgi apparatus vesicles, the brush border and basolateral plasma membrane, multivesicular bodies, and lysosome-like structures were labeled. In goblet cells the limiting membrane and lumen of forming and mature mucus droplets as well as the plasma membrane exhibited label for sialyltransferase. The results provide evidence for "ecto-sialyltransferase" in the plasma membranes of these cells, and suggest that most of the sialyltransferase is released from the Golgi membranes and becomes secreted with the goblet cell mucus. In addition, the polypeptide epitope-purified antibody was also used to examine regional expression of sialyltransferase in the rat intestinal epithelium. Immunolabel was restricted to the large intestine and not found in duodenum, jejunum, and ileum. Direct measurement of the enzyme activity was found to correlate with the immunoelectron microscopic data. This observation suggests that there is regional specific expression of the beta-galactoside alpha 2,6-sialyltransferase.     

Purification and characterization of pig lung carbonyl reductase.

A pyrazole-sensitive carbonyl reductase from pig lung was purified to homogeneity by electrophoretic criteria. Chemical cross-linking study suggested that the native enzyme is a tetramer with a Mr of 103,000, consisting of apparent identical subunits of Mr 24,000. The enzyme reduced aliphatic and aromatic carbonyl compounds with NADPH as a preferable cofactor to NADH and catalyzed the oxidation of secondary alcohols and the aldehyde dismutation in the presence of NAD(P)+. Immunohistochemical study with the antibodies against the enzyme revealed that the enzyme was localized in the ciliated cells, nonciliated bronchiolar cells, Type II alveolar pneumocytes, and the epithelial cells of the ducts of the bronchial glands in the pig lung. In addition to the properties and distribution, the pig lung enzyme was immunochemically similar to the pulmonary enzymes in the guinea pig and mouse. However, the pig enzyme showed the following unusual features. (1) The enzyme exhibited an equatorial specificity in the reduction of 3-ketosteroids; the 4-pro-S hydrogen of NADPH was transferred to the carbonyl carbon atom of 5 alpha- and 5 beta-androstanes, and the respective reduced products were identified as 3 beta- and 3 alpha-hydroxysteroids. (2) Although the NADPH-linked reduction of carbonyl compounds apparently obeyed the Michaelis-Menten kinetics at pH 6.0, the double-reciprocal plots of the velocity vs concentrations of the carbonyl substrates were convex at pH higher than 6.5. The Hill coefficients and [S]0.5 values for the substrates decreased as the pH for reaction increased. The results suggest that the pig enzyme exhibits negative cooperativity with respect to the carbonyl substrates and that the hydrogen ion acts as an allosteric effector abolishing the negative interaction.     

Monospecific antiserum to rat spermidine synthase and its application to rat tissues and several mammals

Monospecific antiserum to rat spermidine synthase was prepared by immunization of rabbits with purified enzyme protein from rat prostate, and its usefulness for analysis of spermidine synthase protein in not only rat tissues but also several other mammals was demonstrated by Western blotting and immunotitration of the enzyme activity. Application of the antiserum for elucidating the relationship between the enzyme activity and protein in normal rat tissues strongly suggested that marked difference in spermidine synthase activity among rat tissues depends solely on the difference in the amount of enzyme protein. Also, application of the antiserum for analyzing spermidine synthase from liver of mouse, rat, guinea pig, pig, and human, showed that the enzymes had a similar subunit molecular weight of 35,000 and a cross-reactivity with the antiserum, exhibiting almost the same immunoreactivity to mouse enzyme as to rat enzyme. Thus, it was suggested that the antiserum would be useful for further studies of mammalian spermidine synthase from the viewpoints of enzymology and molecular biology.     

Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.     

Putative convertase involved in the proteolytic conversion of rat proalbumin to serum albumin

We have found a proteolytic activity in Golgi membranes which efficiently converts [35S]methionine-labeled proalbumin, isolated from pulse-labeled rat hepatocytes in culture, to serum albumin in an in vitro assay system. The proalbumin-converting activity was dependent on Ca2+ and the maximum activity was observed at pH 5.5-6.0. Since the enzyme activity was found to be resistant not only to both leupeptin and E-64 but also to thiol-blocking reagents, it is unlikely that cathepsin B is involved in the proteolytic conversion of proalbumin occurring in the Golgi complex.     

Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D.

Procathepsin D is the intracellular aspartyl protease precursor of cathepsin D, a major lysosomal enzyme. Procathepsin D is rapidly processed inside the cell, and, thus, examination of its proteolyic activation and structure has been difficult. To study this proenzyme, a nonglycosylated form of the human fibroblast procathepsin D was expressed in Escherichia coli, refold in vitro, and purified by affinity chromatography on pepstatinyl agarose. Sequence analysis of the refolded, autoactivated enzyme allowed determination of the autoproteolytic cleavage site. The sequence surrounding this cleavage site between residues LeuP26 and IleP27 (in the "pro" region) resembled the first cleavage site found during activation of other aspartyl proteases. Thus, the autoactivated procathepsin D is analogous to the pepsin activation intermediate, which has been termed pseudopepsin. The enzymatic activity, thermal and pH stability, and fluorescence spectra of pseudocathepsin D were compared to mature, predominantly two-chain, cathepsin D isolated from human placenta. The results indicated that pseudocathepsin D and mature enzyme have a similar Km toward a peptide substrate and cleave a protein substrate at identical sites. Temperature stability of the recombinant enzyme was similar to that of the tissue-derived enzyme. However, the recombinant enzyme had increased stability at low pH when compared to the glycosylated tissue-derived two-chain cathepsin D. Fluorescence spectra of the recombinant and tissue-derived enzymes were identical. Thus, the absence of asparagine-linked oligosaccharides and the presence of the remaining segment of propeptide did not significantly alter the structural and enzymatic properties of the enzyme.