Modulation of amyloid beta-protein clearance and Alzheimer's disease susceptibility by the LDL receptor-related protein pathway.

Susceptibility to Alzheimer's disease (AD) is governed by multiple genetic factors. Remarkably, the LDL receptor-related protein (LRP) and its ligands, apoE and alpha2M, are all genetically associated with AD. In this study, we provide evidence for the involvement of the LRP pathway in amyloid deposition through sequestration and removal of soluble amyloid beta-protein (Abeta). We demonstrate in vitro that LRP mediates the clearance of both Abeta40 and Abeta42 through a bona fide receptor-mediated uptake mechanism. In vivo, reduced LRP expression is associated with LRP genotypes and is correlated with enhanced soluble Abeta levels and amyloid deposition. Although LRP has been proposed to be a clearance pathway for Abeta, this work provides the first in vivo evidence that the LRP pathway may modulate Abeta deposition and AD susceptibility by regulating the removal of soluble Abeta.

Mechanisms of Cellular Uptake of Thrombin-Antithrombin II Complexes Role of the Low-Density Lipoprotein Receptor-Related Protein as a Serpin-Enzyme Complex Receptor.

Serine proteinase inhibitors (serpins) such as antithrombin III inhibit target proteinases by forming a stable complexwith the enzyme. Once formed, several serpin-enzyme complexes (SECs) are removed from the circulation by a receptor, termed the SEC receptor, that is present in the liver. Until recently, the identity of this clearance receptor remained unknown; however, data are now available that strongly implicates one member of the low-density lipoprotein (LDL) receptor family as a candidate for the SEC receptor. This receptor, known as the LDL receptor-related protein (LRP), is a prominent liver receptor that is known to bind numerous ligands that include proteinase-inhibitor complexes, matrix proteins, and certain apolipoprotein E- and lipoprotein lipase-enriched lipoproteins. © 1997, Elsevier Science Inc. (Trends Cardiovasc Med 1997;7:9-16).

Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins.

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI-1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell-bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.

Cellular internalization and degradation of antithrombin III-thrombin, heparin cofactor II-thrombin, and alpha 1-antitrypsin-trypsin complexes is mediated by the low density lipoprotein receptor-related protein.

The inhibition of proteinase activity by members of the serine proteinase inhibitor (serpin) family is a critical regulatory mechanism for a variety of biological processes. Once formed, the serpin enzyme complexes (SECs) are removed from the circulation by a hepatic receptor. The present study suggests that this receptor is very likely the low density lipoprotein receptor-related protein (LRP), a prominent liver receptor. In vitro binding studies revealed that antithrombin III (ATIII)-thrombin, heparin cofactor II (HCII)-thrombin, and alpha1-antitrypsin (alpha1AT)-trypsin bound to purified LRP, and their binding was inhibited by the 39-kDa receptor-associated protein (RAP), an antagonist of LRP-ligand binding activity. In contrast, native or modified forms of the inhibitors were unable to bind to LRP. Mouse embryonic fibroblasts, which express LRP, mediate the cellular internalization leading to degradation of these SECs, while mouse fibroblasts genetically deficient in LRP showed no capacity to internalize and degrade these complexes. SECs were also degraded by HepG2 cells, and this process was inhibited by LRP antibodies, RAP, and chloroquine. The cellular-mediated uptake and degradation was specific for SECs; native or modified forms of the inhibitors were not internalized and degraded. Finally, in vivo clearance studies in rats demonstrated that RAP inhibited the clearance of ATIII-125I-thrombin complexes from the circulation. Together, these results indicate that LRP functions as a liver receptor responsible for the plasma clearance of SECs.

LDL receptor-related protein: a multiligand receptor for lipoprotein and proteinase catabolism.

The accumulation of excessive cholesterol-rich lipoproteins within vascular cells, the proliferation of vascular cells, and fibrin deposition are hallmark features of atherosclerosis. Evidence accumulated over the past few years supports the hypothesis that one member of the LDL receptor family, the low density lipoprotein receptor-related protein (LRP), affects the dynamics of each of these processes. LRP is expressed in several vascular cell types, including smooth muscle cells, and in macrophages, and is also expressed in these cells in atherosclerotic lesions. This receptor is a large endocytotic receptor that mediates the catabolism of a number of molecules known to be important in vascular biology, including apolipoprotein E- and lipoprotein lipase-enriched lipoproteins, thrombospondin, and plasminogen activators. The capacity of LRP to mediate lipoprotein catabolism may be a factor in the development of the lesion by contributing to the formation of foam cells. LRP has recently been shown to mediate the catabolism of thrombospondin, a molecule that has potent biological effects on cells of the vasculature. The regulation of its extracellular accumulation by LRP might modulate the dynamic processes of tissue remodeling associated with the response to vascular injury. In addition, LRP regulates the expression of plasmin activity by directly binding and mediating the cellular internalization of urokinase- and tissue-type plasminogen activators. The cellular removal of these two enzymes decreases the local profibrinolytic potential, possibly leading to a thrombotic state at lesion sites.

LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted beta-amyloid precursor protein and mediates its degradation.

The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor-related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimer's disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD.

gp330 on type II pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex.

Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30-50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin.

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.

The cellular internalization and degradation of hepatic lipase is mediated by low density lipoprotein receptor-related protein and requires cell surface proteoglycans.

Hepatic lipase (HL) and lipoprotein lipase (LpL) are structurally related lipolytic enzymes that have distinct functions in lipoprotein catabolism. In addition to its lipolytic activity, LpL binds to very low density lipoproteins and promotes their interaction with the low density lipoprotein receptor-related protein (LRP) (Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., Pladet M. W., Iverius, P. H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175). In vitro binding assays revealed that HL also binds to purified LRP with a KD of 52 nM. Its binding to LRP is inhibited by the 39-kDa receptor-associated protein (RAP), a known LRP antagonist, and by heparin. 125I-Labeled HL is rapidly internalized and degraded by HepG2 cell lines, and approximately 70% of the cellular internalization and degradation is blocked by either exogenously added RAP or anti-LRP IgG. Mouse fibroblasts that lack LRP display a greatly diminished capacity to internalize and degrade HL when compared to control fibroblasts. These data indicate that LRP-mediated cellular uptake of HL accounts for a substantial portion of the internalization of this molecule. Proteoglycans have been shown to participate in the clearance of LpL, and consequently a role for proteoglycans in HL clearance pathway was also investigated. Chinese hamster ovary cell lines that are deficient in proteoglycan biosynthesis were unable to internalize or degrade 125I-HL despite the fact that these cells express LRP. Thus, the initial binding of HL to cell surface proteoglycans is an obligatory step for the delivery of the enzyme to LRP for endocytosis. A small, but significant, amount of 125I-HL was internalized in LRP deficient cells indicating that an LRP-independent pathway for HL internalization does exist. This pathway could involve cell surface proteoglycans, the LDL receptor, or some other unidentified surface protein.

Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates the cellular internalization and degradation of thrombospondin. A process facilitated by cell-surface proteoglycans.

Thrombospondin (TSP) is a cell and matrix glycoprotein that interacts with a variety of molecules. Newly synthesized thrombospondin is either incorporated into the extracellular matrix, or binds to the cell surface where it is rapidly internalized and degraded (McKeown-Longo, P. J., Hanning, R., and Mosher, D. F. (1984) J. Cell Biol. 98, 22-28). In the current investigation we identify the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) as a receptor responsible for mediating the internalization of TSP leading to its degradation. LRP is a large cell surface receptor consisting of a 515-kDa heavy chain and an 85-kDa light chain proteolytically derived from a 600-kDa precursor. A specific and high affinity interaction between purified LRP and TSP was demonstrated by homologous ligand competition experiments, where a KD of 3-20 nM was measured using different preparations of TSP. The binding of TSP to purified LRP was completely inhibited by the 39-kDa receptor-associated protein, a known antagonist of ligand binding by LRP. Cultured fibroblasts rapidly internalize and degrade 125I-labeled TSP via a receptor-mediated process. This process is inhibited by receptor-associated protein and by antibodies against LRP, indicating that LRP is mediating the cellular internalization of TSP. Our studies also confirm that the efficient catabolism of TSP requires the participation of cell surface proteoglycans, since digestion of cells with heparitinase markedly reduces the extent of LRP-mediated TSP degradation. The ability of LRP to directly bind and mediate the cellular internalization and degradation of TSP indicates that this receptor may play an important role in the catabolism of TSP in vivo.

Interaction of surfactant protein A with cellular myosin.

The goal of the current investigation was to characterize, purify, and identify the proteins that bind surfactant protein A (SP-A). Several polypeptides were purified by SP-A affinity chromatography, and the 200 kD major polypeptide that reacted with SP-A on ligand blots was purified further by preparative SDS-PAGE. Protein sequencing of proteolytically derived subfragments of this polypeptide gave sequences that corresponded completely with nonmuscle (cellular) myosin heavy chain. The 200 kD polypeptide was then found to be immunoreactive with antibodies against cellular myosin. A smaller polypeptide of 135 kD also binds SP-A and appears to be a proteolytic fragment of the 200 kD peptide. The ability of SP-A to bind myosin was confirmed in a microtiter well assay and was found to be concentration dependent. We speculated that the physiologic relevance of the interaction of SP-A with myosin might be to facilitate clearance of myosin from the alveolar subphase following its release during lung injury. In support of this hypothesis, we found that there were detectable levels of myosin in lavage fluid and that SP-A could indeed enhance uptake and degradation of myosin by alveolar macrophages.

Immunological localization of glycoprotein 330, low density lipoprotein receptor related protein and 39 kDa receptor associated protein in embryonic mouse tissues.

In this study we have immunologically examined the expression of the structurally and functionally related receptors, LRP and gp330, and their associated 39 kDa protein (RAP) in tissues of the embryonic mouse. One aim was to determine whether these proteins were coordinantly expressed. The data reveals that gp330 is expressed on the apical surfaces of many specialized absorptive epithelia most prominently, choroid plexus, ependyma, metanephric tubules, ear, thyroid, pericardium, and intestine. LRP was detected in all epithelia expressing gp330 with the exception of the epicardium and metanephric tubule epithelium. However, the subcellular pattern of LRP deposition in polarized epithelium was distinct from the apical pattern of gp330, perhaps indicating that LRP was either sequestered intracellularly or distributed basolaterally. The pattern of LRP expression in tissues of the mouse embryo was however much wider than that of gp330. Prominent expression of LRP was observed in skin, myocardium, mesenchyme, liver, pancreas, and marginal regions of the brain. In the developing liver, LRP was not detected in day 10.5 but was detected in megacaryocyte-like cells of 12.5 day and in hepatocytes of 14.5 day embryonic liver. RAP was observed to be coexpressed with either or both of the receptors but its subcellular pattern of distribution coincided with that of LRP. The coexpression of gp330 and LRP in epithelial cells and the observation that gp330 staining was always localized apically while LRP was distributed basolaterally or sequestered intracellularly suggests that these receptors have distinct functions in polarized epithelial cells.

Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates cellular uptake of pro-urokinase.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) is a large cell surface receptor consisting of a 515-kDa heavy chain and an 85-kDa light chain proteolytically derived from a 600-kDa precursor. Previous work has shown that LRP is responsible for mediating the internalization of urinary-type plasminogen activator (uPA) complexed to plasminogen activator inhibitor type I (PAI-1) (Nykjaer et al., 1992; Herz et al., 1992). The current study indicates that pro-urokinase (pro-uPA) and two chain urokinase (tc-uPA) bind directly to purified LRP, and that LRP mediates their internalization and degradation in Hep G2 cells. In vitro binding assays demonstrated that pro-uPA and tc-uPA bind to purified LRP with affinities (Kd = 45 and 60 nM, respectively) that are approximately 15 to 20-fold weaker than the affinity of uPA.PAI-1 complex for LRP (Kd = 3 nM). Competitive binding experiments revealed that pro-uPA and tc-uPA completely inhibit binding of uPA.PAI-1 complexes to purified LRP. The binding of 125I-pro-uPA to LRP is blocked by the 39-kDa receptor-associated protein, but not by an amino-terminal fragment of uPA, which is known to block binding of uPA to the urokinase receptor. 125I-Pro-uPA can be internalized and degraded by Hep G2 cells independent of PAI-1. Both the internalization and degradation are completely blocked by receptor-associated protein or affinity-purified LRP antibodies, indicating that LRP is mediating this process. These processes are also blocked by the amino-terminal fragment, which suggests that the favored pathway for uPA metabolism is initial binding to the urokinase receptor, followed by ligand transfer to LRP, then internalization leading to degradation.

Glycoprotein 330, a member of the low density lipoprotein receptor family, binds lipoprotein lipase in vitro.

Glycoprotein 330 (gp330), a cell-surface protein that is localized in clathrin-coated pits, is structurally related to both the low density lipoprotein receptor (LDLR) and the LDLR-related protein/alpha 2-macroglobulin receptor (LRP). We recently demonstrated that gp330 and LRP may be functionally related as well; both bind the 39-kDa polypeptide referred to as receptor-associated protein (Kounnas, M. Z., Argraves, W. S., and Strickland, D. K. (1992) J. Biol. Chem. 267, 21162-21166). In this report, we tested several other LRP ligands for their ability to interact with human and rat gp330 in vitro. Gp330 did not exhibit detectable binding to the LRP ligands, alpha 2-macroglobulin protease complex or Pseudomonas aeruginosa exotoxin A. However, we found that gp330 (purified from human or rat) bound the lipolytic enzyme lipoprotein lipase (LPL) with high affinity (Kd = 6.1 and 2.7 nM, respectively). The binding was saturable, divalent cation dependent, and inhibited by heparin or receptor-associated protein. Because LRP has also been shown to bind LPL, the present findings further extend the functional similarities between gp330 and LRP. By analogy to the postulated role of the LRP-LPL interaction in facilitating hepatic clearance of LPL-associated lipoproteins from the blood (Beisiegel, U., Weber, W., and Bengtsson-Olivercrona, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8342-8346; Chappell, D. A., Fry, G. L., Waknitz, M. A., Iverius, P. H., Williams, S. E., and Strickland, D. K. (1992) J. Biol. Chem. 267, 25764-25767), we speculate that the gp330-LPL interaction described herein may contribute to the uptake of LPL-associated lipoproteins in tissues expressing gp330. Consistent with this possibility, we found that LPL promoted in vitro binding of 125I-lipoproteins to gp330.

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.

Meprin-A and -B. Cell surface endopeptidases of the mouse kidney.

The proteinase meprin-A is a disulfide-linked tetramer of 90-kDa glycoprotein subunits. It is expressed at high levels in kidney brush border membranes of random bred and certain inbred strains of mice. Some mouse strains (e.g. C3H/He) do not express meprin-A subunits, but do produce a similar but less well characterized metalloendopeptidase, meprin-B. In the present study, meprin-B was purified from C3H/He mouse kidneys to electrophoretic homogeneity, and the relationship between it and meprin-A was investigated. The papain-solubilized form of meprin-B was similar to meprin-A in amino acid composition, molecular mass, secondary, and quaternary structure. However, immunoblots indicated that the enzymes have some common and some distinct epitopes. Lectin blots indicated both enzymes have high mannose and/or complex biantennary oligosaccharides, but there are differences in the complex-type glycosylation. Peptide maps and sequencing of cyanogen-bromide fragments of the enzymes revealed some different amino acid sequences. Thermal inactivation studies indicated that meprin-B was much less stable than meprin-A; the half-life for inactivation at 58 degrees C for meprin-A was 50 min, whereas for meprin-B it was less than 3 min. Both enzymes hydrolyzed azocasein and insulin B chain, but limited proteolysis of the enzymes with trypsin activated meprin-B 5-20-fold, whereas meprin-A was activated 2-fold at most. Analysis of hydrolysis products of the oxidized insulin B chain revealed some common and some distinct sites of cleavage. Bradykinin was a good substrate for meprin-A, while it was not hydrolyzed by meprin-B. A synthetic peptide, YLVC(SO3-)GERG, derived from insulin B chain was hydrolyzed faster by meprin-B than meprin-A, and neither enzyme was activated by trypsin treatment against this substrate. Taken together, the data indicate that the two metalloendopeptidases have many similarities but are distinct enzymes.

Separation and differential activation of rat liver cytosolic cholesteryl ester hydrolase, triglyceride lipase and retinyl palmitate hydrolase by cholestyramine and protein kinases.

Cholesteryl ester hydrolase (CEH), triacylglycerol lipase (TGL) and retinyl palmitate hydrolase (RPH) were measured in 104,000 x g supernatants from rat liver under optimal conditions for measurement of cytosolic CEH. Similar levels of hydrolytic activity were seen with oil droplet dispersions of cholesteryl oleate, trioleoylglycerol and retinyl palmitate. No cytosolic TGL activity was seen with substrate presented in the triton-albumin emulsion used for measurement of lipoprotein lipase-like TGL associated with hepatic plasma membrane. Cytosolic CEH, TGL and RPH were differentially partially purified by both ammonium sulfate precipitation and anion exchange fast protein liquid chromatography (FPLC). Of the tree activities, only CEH was stimulated by cholestyramine feeding and by activators of protein kinases A and C. All three activities were inhibited by alkaline phosphatase treatment, although to different degrees. It is concluded that these activities are catalyzed by at least three differentially regulated enzymes with a high degree of specificity for their respective substrates.