Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes

Hepatic lipase, a glycoprotein synthesized and secreted by the hepatocyte, binds to sinusoidal endothelium where it is involved in metabolism of lipoprotein phospholipid and triglyceride. To better understand the regulation of hepatic lipase, we investigated the synthesis, post-translational processing, and secretion of the enzyme by isolated rat hepatocytes. Metabolically labeled [35S]methionine hepatic lipase protein, produced by the collagenase-dispersed hepatocytes, was immunoisolated from detergent-solubilized cells and incubation medium at designated times, using a polyclonal rabbit anti-rat hepatic lipase antibody raised against hepatic lipase purified to homogeneity from rat liver post-heparin perfusates. Following polyacrylamide gel electrophoresis and fluorography, radiolabeled hepatic lipase was quantitated by densitometry. Newly synthesized hepatic lipase was rapidly secreted and accumulated in the medium as a 59,000-dalton protein in a manner consistent with a constitutive process. An intracellular 53,000-dalton precursor of the mature 59,000-dalton hepatic lipase was identified by immunoprecipitation. The 53,000-dalton form could also be generated by endoglycosidase digestion of the secreted 59,000-dalton protein. In pulse-chase experiments, the 53,000-dalton protein was converted into the 59,000-dalton form. A 47,000-dalton form of hepatic lipase was immunoisolated from cell lysates only after tunicamycin treatment and could be generated from the secreted 59,000-dalton enzyme by prolonged endoglycosidase digestion. These data show that hepatic lipase is synthesized and rapidly secreted by isolated rat hepatocytes. Further, an intracellular 47,000-dalton precursor peptide can be identified after tunicamycin treatment, which may represent the hepatic lipase polypeptide, presumably after removal of its signal sequence; a 53,000-dalton partially glycosylated peptide exists as a major precursor form in the cell; and the mature 59,000-dalton hepatic lipase is present in the hepatocyte, but it is rapidly secreted.     

Proteolytic cleavage of haptoglobin occurs in a subcompartment of the endoplasmic reticulum: evidence from membrane fusion in vitro

The primary translation product of haptoglobin mRNA is a 45-kD polypeptide which is proteolytically cleaved shortly after its synthesis. Previous studies have indicated that the cleavage of this proform of haptoglobin occurs in the ER. In an attempt to characterize the cleaving enzyme, we found that upon incubation of microsomes from rat hepatocytes pulse labeled with [35S]methionine, little cleavage of labeled prohaptoglobin occurred. In contrast, when cells whose cytoplasmic proteins had been released by saponin treatment were incubated, 30-40% of the prohaptoglobin was cleaved. The addition of GTP caused a twofold stimulation, which was abolished by the nonhydrolyzable analog GTP gamma S. With a homogenate of the cells, the addition of GTP resulted in a fourfold stimulation of the degree of cleavage--from 15 to 60%. Differential centrifugation revealed that most of the cleaving activity resided in membranes sedimenting similarly to mitochondria and to a small fraction of the ER. These rapidly sedimenting membranes were therefore prepared from a rat liver homogenate. Upon treatment with high salt, light membranes were released which, when incubated with microsomes of pulse-labeled hepatocytes in the presence of detergent (and in the absence of GTP), induced specific cleavage of prohaptoglobin. The cleaving enzyme had an alkaline pH optimum indicating that it was not of lysosomal origin. These results suggest that cleavage of prohaptoglobin occurs in a subcompartment of the ER. Apparently, the connection between this compartment and the bulk of the ER is broken upon saponin treatment or homogenization but can be reestablished through a process requiring GTP hydrolysis.     

Effects of weakly basic amines on proteolytic processing and terminal glycosylation of secretory proteins in cultured rat hepatocytes.

We examined the effects of weakly basic amines on the secretion and post-translational modifications of secretory proteins in cultured rat hepatocytes. Weakly basic amines such as methylamine, chloroquine and NH4Cl strongly inhibited not only protein secretion, but also the proteolytic conversion of a proform of complement C3, allowing the precursor to be released into the medium. The amines, however, had no effect on the proteolytic conversion of prohaptoglobin into its subunits. Since available evidence indicates that the conversion of pro-C3 occurs at the Golgi complex while that of prohaptoglobin takes place in the endoplasmic reticulum, it is most likely that the weak bases specifically affect the proteolytic event occurring at the Golgi complex. Electron microscopic observations confirmed that the amines caused morphological changes of the Golgi complex, consisting of dilated cisternae and swollen vacuoles. When the glycosylation of alpha 1-protease inhibitor and haptoglobin was examined, it was found that the amines caused a marked accumulation in the cells of both glycoproteins corresponding to the mature secreted forms. Neuraminidase digestion demonstrated that the glycoproteins accumulating in response to the amines had acquired terminal sialic acid. The results indicate that the amines do not significantly affect terminal glycosylation, in contrast with their definite effect on proteolytic processing, despite the fact that both modifications take place in the Golgi complex.     

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.     

Tris inhibits both proteolytic and oligosaccharide processing occurring in the Golgi complex in primary cultured rat hepatocytes

Tris caused the distention of the Golgi cisternae in primary cultured rat hepatocytes and perturbed the functions occurring there. Proteolytic cleavage of precursors of both albumin and complement C3 was inhibited, whereas that of prohaptoglobin was not affected by Tris. These effects on the proteolytic cleavages resemble those of acidotropic amines (Oda, K., and Ikehara, Y. (1985) Eur. J. Biochem. 152, 605-609; Oda, K., Koriyama, Y., Yamada, E., and Ikehara, Y. (1986) Biochem. J. 240, 739-745). However, the effects of Tris significantly differed from acidotropic amines on the basis of its effects on the processing of N-linked oligosaccharides of glycoproteins. Both alpha 1-protease inhibitor and haptoglobin secreted from the Tris-treated cells were found to contain almost equal amounts of endo-beta-N-acetylglucosaminidase H-sensitive and -resistant oligosaccharides, whereas the glycoproteins from both the control and methylamine-treated cells were resistant to the enzyme. The endo-beta-N-acetylglucosaminidase-sensitive oligosaccharides were analyzed to be Man8-5GlcNAc by high resolution gel permeation chromatography, suggesting that trimming of alpha-mannose residues from the precursor Man9GlcNAc2 is incomplete in the Tris-treated cells. On the other hand, Tris did not significantly inhibit incorporation of radioactive monosaccharides (N-acetylglucosamine, galactose, and fucose) into the glycoproteins. However, two-dimensional gel electrophoresis in combination with neuraminidase digestion demonstrated that sialylation was markedly inhibited by Tris. Taken together, our results reveal that Tris inhibits not only the sialic acid addition which takes place in the trans Golgi region, but also the trimming step of high mannose-type oligosaccharides, which is thought to occur before glycoproteins reach the trans Golgi region.     

Lysosomal cysteine protease, cathepsin B, is targeted to lysosomes by the mannose 6-phosphate-independent pathway in rat hepatocytes: site-specific phosphorylation in oligosaccharides of the proregion

Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [(32)P]phosphate, (32)P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of (32)P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated (35)S- or (32)P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular (35)S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the (32)P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH(4)Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized (35)S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.     

Tissue-specific posttranslational modification of rat apoE. Synthesis of sialated apoE forms by neonatal rat aortic smooth muscle cells

We have studied the synthesis, modification, and secretion of rat apoE in primary cultures of neonatal aortic smooth muscle cells and adult rat hepatocytes. The cultures were pulsed with [35S]methionine and the intracellular and secreted apoE were immunoprecipitated and analyzed by two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis and autoradiography. A short pulse (10 min) showed the presence of a major unmodified apoE form in both cultures. This form comigrated on two-dimensional gels with the major rat plasma apoE isoprotein. A longer pulse (15-120 min) resulted in the progressive appearance of intracellularly modified apoE isoproteins in both cultures. The apoE secreted by aortic smooth muscle cells consisted exclusively of sialated apoE isoproteins that were sensitive to neuraminidase treatment. In contrast, the apoE secreted by primary cultures of adult rat hepatocytes, organ cultures of neonatal rat liver, as well as rat plasma apoE, contained several minor modified isoproteins. Nascent apoE secreted by the aortic smooth muscle cell cultures floats in the density range of 1.09 to 1.186 g/ml. We conclude that aortic smooth muscle cells can synthesize and secrete sialated apoE isoproteins associated with nascent lipoproteins floating in the high density lipoprotein region.     

Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein

As a model of ligand-dependent protein secretion the biosynthesis, intracellular transport, and release of the retinol-binding protein (RBP) were studied in primary cultures of rat hepatocytes pulse-labeled with [35S]methionine. After various periods of chase RBP was isolated by immunoprecipitation and identified by SDS PAGE. Both normal and vitamin A-deficient hepatocytes synthesized RBP. The normal cells secreted the pulse-labeled RBP within 2 h. RBP synthesized by deficient cells was not secreted, and intracellular degradation of the protein appeared to be slow. Deficient cells could be induced to secrete RBP on the addition of retinol to the culture medium. This occurred also after protein synthesis had been blocked by cycloheximide. Since retinol induces the secretion of RBP, accumulated in the endoplasmic reticulum (ER), it seems reasonable to conclude that the transport of RBP from the ER to the Golgi complex is regulated by retinol.     

Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin. There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D. (1999) J. Biol. Chem. 274, 14818-14822). By using mammalian cell lines, we have now determined that removal of the heparin-binding region occurs after passage through the Golgi network but before being secreted into the extracellular space. Specific protease inhibitors and overexpression of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines. A mutation in Arg(213) renders EC-SOD resistant to furin processing. These results indicate that furin-dependent processing of EC-SOD is important for determining the tissue distribution and half-life of EC-SOD.     

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].     

Biosynthesis of plasma proteins in serum-free medium by primary monolayer culture of rat hepatocytes

Morphologically intact rat hepatocytes separated by collagenase perfusion were cultured in L-15/fetal calf serum medium to form a monolayer. Thereafter the hepatocytes were grown in serum-free L-15 medium in which they produced and continuously released plasma proteins. The secreted plasma proteins were collected, separated and characterized by crossed immunoelectrophoresis. Most of the newly biosynthesized plasma proteins secreted into the medium during incubation for thirty hours had the same electrophoretic mobility, antigenicity and staining characteristics as their counterparts in rat serum. The addition of tritium labelled amino acid mixture to the culture medium revealed that the release of radioactively labelled plasma proteins into the culture medium was essentially linear during the thirty hour incubation period. However, saturation of the intracellular pool took place after ca. ten hours of incubation. Addition of leukocytic endogenous mediator, LEM, to cultures of rat hepatocytes caused a profound increase in the relative concentration of acute-phase proteins secreted into the culture medium.     

Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier's disease

The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I.     

The AprV5 Subtilase Is Required for the Optimal Processing of All Three Extracellular Serine Proteases from Dichelobacter nodosus

Dichelobacter nodosus is the principal causative agent of ovine footrot and its extracellular proteases are major virulence factors. Virulent isolates of D. nodosus secrete three subtilisin-like serine proteases: AprV2, AprV5 and BprV. These enzymes are each synthesized as precursor molecules that include a signal (pre-) peptide, a pro-peptide and a C-terminal extension, which are processed to produce the mature active forms. The function of the C-terminal regions of these proteases and the mechanism of protease processing and secretion are unknown. AprV5 contributes to most of the protease activity secreted by D. nodosus. To understand the role of the C-terminal extension of AprV5, we constructed a series of C-terminal-deletion mutants in D. nodosus by allelic exchange. The proteases present in the resultant mutants and their complemented derivatives were examined by protease zymogram analysis, western blotting and mass spectrometry. The results showed that the C-terminal region of AprV5 is required for the normal expression of protease activity, deletion of this region led to a delay in the processing of these enzymes. D. nodosus is an unusual bacterium in that it produces three closely related extracellular serine proteases. We have now shown that one of these enzymes, AprV5, is responsible for its own maturation, and for the optimal cleavage of AprV2 and BprV, to their mature active forms. These studies have increased our understanding of how this important pathogen processes these virulence-associated extracellular proteases and secretes them into its external environment.     

Regulation of apo-A-I processing in cultured hepatocytes

Apo-A-I, the major protein component of high density lipoproteins, appears intracellularly as an intermediate precursor (pro-apo-A-I) with a hexapeptide extension (RHFWQQ) at its amino terminus. Proteolytic processing of pro-apo-A-I to apo-A-I has been shown to occur extracellularly in cell and organ cultures from rat and human tissues. Recently, however, intracellular conversion has been detected in chickens. To determine what distinguishes and regulates these two processing methods, the proteolytic processing and secretion of apo-A-I was studied by metabolic labeling in chick hepatocytes and in Hep-G2 cells (derived from a human hepatocellular carcinoma). The proportions of intracellular and secreted pro-apo-A-I and apo-A-I were measured by sequencing NH2-terminal portions of the proteins and determining the location of radio-labeled amino acids. Chick hepatocytes cultured in the absence of hormones or fetal bovine serum secreted primarily processed apo-A-I (83%). In the presence of serum these cells secreted only pro-apo-A-I, whereas incubation with a combination of hormones (insulin, triiodothyronine, dexamethasone) resulted in secretion of a nearly equal mixture of the pro- and processed forms of the protein. In contrast, Hep-G2 cells, maintained in the absence of serum, secreted only pro-apo-A-I; when grown in the presence of serum these cells secreted a mixture of pro- and processed apo-A-I. Under conditions in which chick hepatocytes and Hep-G2 cells secreted both forms of the protein, a mixture of pro- and processed apo-A-I was also found intracellularly; when only the pro-form was secreted, the cells likewise contained only pro-apo-A-I. Under all the above conditions, the secreted apo-A-I exhibited similar isoform patterns in two-dimensional gel electrophoresis. These data show that both chick hepatocytes and human hepatoma cells are capable of intracellularly processing pro-apo-A-I to apo-A-I, and that the extent of intracellular processing is controlled by the cell's hormonal environment.     

Ornithine transcarbamylase in liver mitochondria

Summary Ornithine transcarbamylase (ornithine carbamoyltransferase, EC 2.1.3.3), the second enzyme of urea synthesis, is localized in the matrix of liver mitochondria of ureotelic animals. The enzyme is encoded by a nuclear gene, synthesized outside the mitochondria, and must then be transported into the organelle. The rat liver enzyme is initially synthesized on membrane-free polysomes in the form of a larger precursor with an amino-terminal extension of 3 400–4 000 daltons. In rat liver slices and isolated rat hepatocytes, the pulse-labeled precursor is first released into the cytosol and is then transported with a half life of 1 2 min into the mitochondria where it is proteolytically processed to the mature form of the enzyme. The precursor synthesized in vitro exists in a highly aggregated form and has a conformation different from that of the mature enzyme. The precursor has an isoelectric point (pI = 7.9) higher than that of the mature enzyme (pI = 7.2).The precursor synthesized in vitro can be taken up and processed to the mature enzyme by isolated rat liver mitochondria. The mitochondrial transport and processing system requires membrane potential and a high integrity of the mitochondria. The transport and processing activities are conserved between mammals and birds or amphibians and is presumably common to more than one precursor. Potassium ion, magnesium ion, and probably a cytosolic protein(s), in addition to the transcarbamylase precursor and the mitochondria, are required for the maximal transport and processing of the precursor.A mitochondrial matrix protease which converts the precursor to a product intermediate in size between the precursor and the mature subunit has been highly purified. The protease has an estimated molecular weight of 108 000 and an optimal pH of 7.5–8.0, and appears to be a metal protease. The protease does not cleave several of the protein and peptide substrates tested. The role of this protease in the precursor processing remains to be elucidated.Rats subjected to different levels of protein intake and to fasting show significant changes in the level of enzyme protein and activity of ornithine transcarbamylase. The dietary-dependent changes in the enzyme level are due mainly to an altered level of functional mRNA for the enzyme. In contrast, during fasting, the increase in the enzyme level is associated with a decreased level of translatable mRNA forthe enzyme.Pathological aspects of ornithine transcarbamylase including the enzyme deficiency and reduced activities of the enzyme in Reye's syndrome are also described. A possibility that impaired transport of the enzyme precursor into the mitochondria leads to a reduced enzyme activity, is proposed.Abbreviation pOTC precursor of ornithine transcarbamylase     

Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membranes

Bovine pancreatic trypsin-inhibitor (bPTI) is required for survival of adult rat hepatocytes for more than 2 days in primary cultures in serum-free medium. Of the various protease inhibitors tested, all trypsin inhibitors increased the survival of rat hepatocytes in serum-free medium, their potencies being in the order bPTI greater than alpha 2-plasmin inhibitor greater than leupeptin greater than soybean trypsin inhibitor greater than alpha 1-antitrypsin = alpha 2-macroglobulin. Elastatinal, a specific inhibitor of elastase, was also effective. bPTI did not inhibit the degradation of proteins with short or long lives, suggesting that it did not increase the survival of hepatocytes by inhibiting cellular protein degradation. alpha 2-Plasmin inhibitor immobilized on Sepharose 4B caused dose-dependent increase in survival. Plasma membranes purified from adult rat liver had significant protease activity, about 80% of which was sensitive to bPTI, alpha 2-plasmin inhibitor and leupeptin. From its specificity for substrates and sensitivity to inhibitors, the membrane-bound protease was characterized as a trypsin-like protease. The effects of various inhibitors on the membrane-bound protease correlated well with their abilities to increase survival of rat hepatocytes. Therefore, it seems that bPTI acts on the cell surface and increases hepatocyte survival in serum-free cultures by inhibiting a trypsin-like protease associated with the plasma membranes.     

Rat platelets contain growth factor(s) distinct from PDGF which stimulate DNA synthesis in primary adult rat hepatocyte cultures

Adult rat hepatocytes in primary cultures are stimulated to synthesize DNA in response to rat serum, whereas rat plasma is considerably less active. Biological activity is present in rat platelets and is secreted during aggregation in response to thrombin. The material secreted by rat platelets is heat labile and is sensitive to digestion with trypsin, suggesting that it is a protein. When assayed on 3T3 cells this material also stimulates DNA synthesis; however, the trypsin-sensitive activity is heat stable (100 degrees C, 10 min). These results indicate that rat platelets contain hepatotrophic activities which by virtue of their heat sensitivity are distinct from heat-stable platelet-derived growth factor (PDGF)-like mitogenic activities required by 3T3 cells for growth. It is possible that hepatotrophic platelet factors might be involved in mediating liver regeneration in the rat after partial hepatectomy.     

Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cleavage of membrane secretory component to soluble secretory component occurs on the cell surface of rat hepatocyte monolayers

Rat liver secretory component is synthesized as an integral membrane protein (mSC) and cleaved to an 80-kD soluble form (fSC) sometime during transcellular transport from the sinusoidal to the bile canalicular plasma membrane domain of hepatocytes. We have used 24-h monolayer cultures of rat hepatocytes to characterize the conversion of mSC to fSC. Cleavage of mSC in cultured hepatocytes is inhibited by the thiol protease inhibitors leupeptin, antipain, and E-64, but not by other inhibitors, including disopropylfluorophosphate, pepstatin, N-ethylmalemide, p-chloromercuribenzoic acid, and chloroquine. Leupeptin-mediated inhibition of cleavage is concentration dependent and reversible. In the presence or absence of leupeptin, only 10-20% of mSC is accessible at the cell surface. To characterize the behavior of surface as opposed to intracellular mSC, cell surface mSC was labeled with 125I by lactoperoxidase-catalyzed iodination at 4 degrees C. Cell surface 125I-mSC was converted to extracellular fSC at 4 degrees C in the absence of detectable internalization. Cleavage was inhibited by leupeptin and by anti-secretory component antiserum. Cleavage also occurred at 4 degrees C after cell disruption. In contrast, 125I-mSC that had been internalized from the cell surface was not converted to fSC at 4 degrees C in either intact or disrupted cells. Hepatocytes metabolically labeled with [35S]cys also released small quantities of fSC into the medium at 4 degrees C. The properties of fSC production indicate that cleavage occurs on the surface of cultured rat hepatocytes and not intracellularly. Other features of the cleavage reaction suggest that the mSC-cleaving protease is segregated from the majority of cell surface mSC, possibly within a specialized plasma membrane domain.