Tissue distribution of a major mevalonate pyrophosphate decarboxylase in rats.

The 45- and 35-kDa subunits of mevalonate pyrophosphate decarboxylase (MPD) have been purified from rat liver. In this study, we examined the relationship between 45- and 35-kDa MPD and the tissue distribution of a major MPD in rat liver. When the crude extract of rat liver fed on normal chow was subjected to immunoblot analysis using anti-rat 45-kDa MPD antibody, only the 45-kDa band was detected. In a pulse-chase experiment using anti-rat 45-kDa MPD antibody, there was no precursor-product relationship between the 45- and the 35-kDa MPD. In immunoprecipitation, more than 85% of MPD activity in the rat liver was depleted from the crude extract with an excess of the above antibody. When 45-kDa MPD contents in tissues were analyzed by immunoblotting, a single protein band with an apparent molecular weight of 45 kDa was detected in all tissues. The specific protein content of 45-kDa MPD in liver was markedly higher than in other tissues. The activity/amount ratio varied among brain, liver, and testis, being significantly highest in the liver. From these data, it is suggested that 45-kDa MPD serves as a major enzyme involved in cholesterol biosynthesis in rat liver and that a tissue-specific regulator or isozyme of 45-kDa MPD is present in rat liver.

Mevalonate pyrophosphate decarboxylase is predominantly located in the cytosol of rat hepatocytes.

Mevalonate pyrophosphate decarboxylase (MPD) is found in the 100000 x g supernatant fraction of cells or tissues and is considered to be a cytosolic protein. Recently, other groups reported that MPD is mostly located in the peroxisomes. In this study, we used two different methods to determine whether MPD is predominantly located in the peroxisomes or the cytosol of rat hepatocytes. 1) In permeabilized rat hepatocytes or normal rat kidney cells treated with digitonin, which lack cytosolic enzyme, MPD was mainly present in the medium. 2) Double immunofluorescent labeling of cells with both anti-MPD antibody and anti-hexokinase antibody yielded an immunofluorescent pattern for both enzymes typical of the cytosolic protein. These results indicate that MPD is predominantly located in the cytosol of rat hepatocytes.

Difference in subcellular distribution between 45- and 37-kDa mevalonate pyrophosphate decarboxylase in rat liver.

We previously reported that the CP diet (a diet containing 5% cholestyramine and 0.1% pravastatin)-induced new species of 37-kDa mevalonate pyrophosphate decarboxylase (MPD) was characteristically and immunologically very similar to the well-known 45-kDa MPD. In the present study, we found a difference in subcellular distribution between 45- and 37-kDa MPD by cell fractionation and immunoblot analysis. The cytosol fraction contained 45- and 37-kDa MPD. Peroxisomal fraction contained a small amount of 45-kDa MPD, but not 37-kDa MPD. Also, 45-kDa MPD in peroxisome is localized in the matrix. From these data, the difference in subcellular distribution between 45- and 37-kDa MPD may be due to differences in the physiological role of cholesterol biosynthesis in rat liver.

Mevalonate pyrophosphate decarboxylase in stroke-prone spontaneously hypertensive rat is reduced from the age of two weeks.

We carried out a comparison of tissue distribution of mevalonate pyrophosphate decarboxylase (MPD) between normotensive Wistar Kyoto rat (WKY) and stroke-prone spontaneously hypertensive rat (SHRSP) using Western blotting. However, there was no difference in tissue distribution of MPD between WKY and SHRSP, expect in brain and liver. We then compared the MPD between WKY and SHRSP liver at several weeks of age. We found that MPD in the liver as well as brain of SHRSP was significantly reduced from two weeks of age. This data is useful to identify or understand the mechanism underlying the reduced amount of MPD in SHRSP.

Distribution of a major lysosomal membrane glycoprotein, LGP85/LIMP II, in rat tissues.

We previously identified and characterized a major lysosomal membrane glycoprotein, termed LGP85 (identical to LIMP II), in rat liver lysosomes. This study describes the distribution of the mRNA and protein of LGP85 in rat tissues. LGP85 protein and mRNA were detectable in all tissues when analyzed by Western and Northern blotting. The 4.2- and 2.2-kb transcripts of LGP85 were detected in all tissues. Liver and lung have the highest and lowest levels of LGP85 mRNA, respectively. A single protein band with an apparent molecular weight (Mr) of approximately 85000 was detected in each tissue. The specific protein content of LGP85 in spleen was markedly higher than in other tissues. LGP85 protein is distributed in the tissues independently of LGP85 mRNA. Furthermore, there was a less significant relationship between LGP85 protein and another lysosomal membrane glycoprotein, lamp-1, in the tissue distribution (a regression coefficient of 0.086), which suggests that LGP85 may function in vivo independently of lamp-1.

Distribution of verectin in Aloe vera leaves and verectin contents in clonally regenerated plants and the commercial gel powders by immunochemical screening.

Verectin antiserum raised in white rabbits was immunochemically applied to examine verectin distribution in Aloe vera leaves during growth and flowering seasons, and to quantify verectin in clonally regenerated plants and commercial A. vera gel products.

Two acidic amino acid residues, Asp(470) and Glu(471), contained in the carboxyl cytoplasmic tail of a major lysosomal membrane protein, LGP85/LIMP II, are important for its accumulation in secondary lysosomes.

Lysosomal membrane glycoprotein termed LGP85 or LIMP II has a COOH-terminal cytoplasmic tail whose amino acid sequence is R(459)GQGSMDEGTADERAPLIRT(478). Two acidic amino acid residues, D(470) and E(471), in the cytoplasmic tail of LGP85 are crucial for its binding to adaptor-like complex AP-3. In the present study we investigated their role(s) in intracellular distributions of LGP85 using two alanine substitution mutants at D(470) and E(471) (defined as D470A and E471A, respectively). Immunofluorescence analysis showed that D470A and E471A are localized to endocytic organelles as well as wild-type LGP85. However, the subcellular fractionation study revealed that D470A and E471A are different from wild-type LGP85 in the distribution among early endosomes, late endosomes, and lysosomes. A major portion of wild-type LGP85 existed in the densest lysosomal fraction. In contrast, a significant amount of D470A existed in the early endosomal fraction with a light buoyant density, while less D470A resided in the lysosomal fraction. E471A broadened from the early endosomal fraction to the lysosomal fraction without the high lysosomal peak. These findings indicate that the two acidic residues, D(470) and E(471), play an important role in regulation of LGP85 movement within the endocytic pathway, which finally makes the highest concentration of LGP85 in the dense secondary lysosomes.

Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts.

Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post- and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts.

Purification and characterization of a soluble form of lysosome-associated membrane glycoprotein-2 (lamp-2) from rat liver lysosomal contents.

Lysosomal membrane of rat liver contains a highly glycosylated protein referred to as lamp-2. Lamp-2 occurs to a significant extent in a soluble fraction of rat liver lysosomes. The soluble form of lamp-2 (SF-lamp-2) was purified to electrophoretic homogeneity. An apparent molecular weight M(r) of SF-lamp-2 on sodium dodecy sulfate-polyacrylamide gel electrophoresis was determined to be 91,000 which is 5,000 less than that of the membranous form of lamp-2 (MF-lamp-2). SF- and MF-lamp-2 were very similar to each other in terms of sialic acid content, NH2-terminal amino acid sequence and isoelectric point. Gel filtration data indicated that native SF-lamp-2 has an M(r) = 360,000. Taken together, SF-lamp-2 forms a tetrameric structure consisting of a homogenous polypeptide lacking a membrane-spanning domain and a cytoplasmic tail near the COOH-terminus.

Immunochemical distinction of Aloe vera, A. arborescens, and A. chinensis gels.

Verectin antiserum raised in white rabbits was immunoprecipitated with the Aloe vera nondialysable fraction. Analysis of the immunoprecipitation revealed that verectin accounted for about 1.25% of the total proteins in the nondialysable fraction of Aloe vera gel. The verectin antibody showed differential immunoreactivities against nondialysable fractions of A. arborescens, A. chinensis, and A. vera: 1) an immunopreciptin line was formed against the fraction of A. vera, but not against those of A. arborescens and A. chinensis gel in an Ouchterlony double immunodiffusion test and 2) an immunopositive band was detected in the A. vera and A. chinensis nondialysable fractions but not in that of A. arborescens in immunoblotting. These findings indicate that the verectin antibody can be used to distinguish Aloe materials.

Identification and characterization of a major lysosomal membrane glycoprotein, LGP85/LIMP II in mouse liver.

We previously have purified and characterized a major lysosomal membrane glycoprotein termed LGP85 (LIMP II) in rat liver lysosomes. In this study, LGP85 in mouse liver lysosomes was identified and characterized by biochemical and molecular biological methods. Lysosomal membranes were isolated from murine liver by differential centrifugation. LGP85 was present in the lysosomal membrane fraction from mouse liver in a comparable amount to another lysosomal membrane glycoprotein, lamp-2. Mouse LGP85 (M-LGP85) from liver lysosomal membranes exhibited an Mr of 80,000 on SDS-PAGE, which is smaller by 5,000 than that of rat LGP85 (R-LGP85). M-LGP85 was immunochemically detected in the extracts of brain, heart, lung, liver, and kidney. A cDNA encoding M-LGP85 was cloned from mouse liver cDNA library. The primary protein structure deduced from a nucleotide sequence of M-LGP85 cDNA indicated that M-LGP85 consists of 478 amino acids with Mr of 54,069. M-LGP85 showed 93.3 and 86.0% sequence similarities to its rat and human counterparts in amino acids, respectively. M-LGP85 contains 11 potential N-glycosylation sites which are heavily glycosylated, resulting in the increased Mr of M-LGP85 present in the mouse liver lysosomes. It is likely that M-LGP85 traverses the lysosomal membrane twice, with an NH2-terminal transmembrane domain, and another hydrophobic domain near the COOH-terminus. M-LGP85 has a protruding COOH-terminal cytoplasmic tail consisting of amino acid residues including the leucine-isoleucine sequence shown to be the lysosomal targeting signal of R-LGP85 and human LGP85 (H-LGP85). The high level of expression of M-LGP85 in the lysosomal membrane, the high structural similarities among M-, R-, and H-LGP85, and the occurrence of M-LGP85 in all the mouse tissues examined suggest the essential and constitutive function of LGP85 in lysosomes.

Biosynthetic transport of a major lysosome-associated membrane glycoprotein 2, lamp-2: a significant fraction of newly synthesized lamp-2 is delivered to lysosomes by way of early endosomes.

Lysosomal membranes contain two highly glycosylated proteins, designated as lamp-1 and lamp-2, as major components. Lamp-1 and lamp-2 are similar to each other in the protein structure. Here, we investigated the biosynthetic transport of lamp-2 through the endocytic vacuoles in cultured rat hepatocytes in comparison with that of lamp-1, which has previously been studied [Akasaki et al. (1995) Exp. Cell Res. 220, 464-473]. Newly synthesized lamp-2 (NS-lamp-2) was transported to the trans-Golgi from rough endoplasmic reticulum with a half time (t1/2) of 32 min, more slowly than NS-lamp-1 (t1/2 = 13 min). After leaving the trans-Golgi, NS-lamp-2 is transferred to at least three compartments; the cell surface (t1/2 = 47 min), cell peripheral early endosomes (t1/2 = 38 min) and perinuclear late endosomes (t1/2 = 48 min). NS-lamp-2 transported to any compartment is delivered finally to lysosomes (t1/2 = 90 min). A significant fraction of NS-lamp-2 (45% of the total) was transported from the trans-Golgi to early endosomes, and then delivered to dense lysosomes via perinuclear late endosomes, whereas a major portion of NS-lamp-1 follows an intracellular route to late endosomes without passing through the cell periphery. NS-lamp-2 leaves the cell peripheral region more rapidly than NS-lamp-1. The kinetic and quantitative data for biosynthetic transport of NS-lamp-2 to early endosomes and the cell surface indicate that NS-lamp-2 may be transported first to early endosomes, from which a small portion of it (approximately 3.5% of the total) moves to the plasma membrane via a recycling system. In contrast, a small fraction of NS-lamp-1 is transported to the plasma membrane directly from the trans-Golgi, since NS-lamp-1 is delivered to the plasma membrane and early endosomes with almost the same half times.

Biosynthetic transport of a major lysosomal membrane glycoprotein, lamp-1: convergence of biosynthetic and endocytic pathways occurs at three distinctive points.

We studied the kinetics of the biosynthetic transport of lysosome-associated membrane glycoprotein-1 (lamp-1) to the endocytic compartments in cultured rat hepatocytes. Newly synthesized lamp-1 (NS-lamp-1) was transported to the trans-Golgi from rough endoplasmic reticulum with a half time (t1/2) of 13 min. From the trans-Golgi, at least 25% of NS-lamp-1 was delivered to the cell periphery: to the cell surface and early endosomes with t1/2 s of 32 and 33 min, respectively. A comparison of the kinetics of the biosynthetic transport of lamp-1 to both compartments demonstrated that NS-lamp-1 takes two peripheral routes from the Golgi apparatus; it is delivered to early endosomes directly and after reaching the cell surface. A major portion of NS-lamp-1 follows a direct intracellular pathway to late endosomes (t1/2 = 45 min) and subsequently to lysosomes (t1/2 = 85 min). The kinetic data of the biosynthetic transport to these endocytic vacuoles suggested that a significant fraction of NS-lamp-1 returns to the late endosomes immediately after its arrival at lysosomes and that there is a unique retrograde delivery of NS-lamp-1 from late to early endosomes prior to its transport to lysosomes. Thus, in cultured rat hepatocytes, the lamp-1 biosynthetic and the endocytic pathways converge at the three distinctive points. Late endosomes are centrally situated in the complex biosynthetic route of lamp-1.

Purification and characterization of a major kyotorphin-hydrolyzing peptidase of rat brain.

We purified a major kyotorphin (L-Tyr-L-Arg)-hydrolyzing peptidase (KTPase) from the rat brain, to electrophoretic homogeneity using conventional chromatographic techniques. KTPase was purified 1,660-fold with a specific activity of 161 mumol/min/mg protein and 6.8% recovery. The purified enzyme was composed of a single polypeptide with a molecular mass of 67 kDa and an isoelectric point (pI) of 5.5. KTPase has the ability to hydrolyze a variety of natural dipeptides. It also liberated NH2-terminal tyrosine from Tyr-Gly-Gly and Tyr-Tyr-Leu. Bestatin and arphamenine B were potent inhibitors of this enzyme, while amastatin and puromycin had little effect. An excess of anti-KTPase antibody raised in a white rabbit precipitated approximately 80% of the kyotorphin-hydrolyzing activity in the cytosol of rat brain. These data suggested that 67 kDa KTPase has a role in the degradation of kyotorphin within neuronal cells of the rat brain.

Cycling of an 85-kDa lysosomal membrane glycoprotein between the cell surface and lysosomes in cultured rat hepatocytes.

We studied the endocytic transport of an 85-kDa lysosomal membrane glycoprotein (LGP85) from the cell surface to lysosomes in cultured rat hepatocytes. Fab' fragments of a monoclonal antibody against LGP85 (YA30 mAb) were conjugated with horseradish peroxidase (HRP) and then used as probes to monitor the endocytic transport of LGP85 from the plasma membrane to lysosomes. Continuous internalization and lysosomal transport of HRP-YA30 mAb Fab' occurred in the hepatic cells, resulting in its accumulation in the dense lysosomal fraction obtained from the cells on Percoll density centrifugation. The endocytic transport of HRP-YA30 mAb continued in the presence of the protein synthesis inhibitor, cycloheximide, indicating that LGP85 is cycled between the cell surface and lysosomes or endosomes, like other lysosomal membrane glycoproteins, lamp-1 and lamp-2, as reported previously [Akasaki et al. (1993) J. Biochem. 114, 598-604]. The half times (t1/2) of internalization and lysosomal transport of LGP85 were 32 min and 2.0 h, respectively. The kinetics of endocytic transport for LGP85 are very similar to those of lamp-1 and lamp-2. LGP85 possesses a short cytoplasmic tail whose amino acid sequence is quite different from those of lamp-1 and lamp-2. Therefore, these results suggested that continuous internalization from the cell surface and lysosomal transport of of endogenous LGP85 occur through a mechanism that can recognize this novel amino acid sequence, probably a Leu-Ile-containing motif, in normal hepatic cells of rat.

Identification and characterization of lysosomal enzymes involved in the proteolysis of phenobarbital-inducible cytochrome P450.

We have studied lysosomal proteases capable of degrading a major form of cytochrome P450 (CYP2B1) which was purified from the liver microsomes of phenobarbital-treated rats. After incubation of CYP2B1 with extracts of triton-filled lysosomes (tritosomes), its proteolysis was measured by a quantitative immunoblot procedure. A CYP2B1 protein band with an apparent molecular mass of 53 kilodaltons (kDa) was degraded to 42-, 38-, and 29-kDa polypeptides after a short period of incubation. These proteolytic fragments disappeared on prolonged incubation. One milligram of tritosomal protein contained enough activity to hydrolyze approximately 67.3 micrograms CYP2B1 protein/min at pH 4.5. Approximately 85% of the hydrolyzing activity was localized in a soluble fraction of the tritosomes. The degradation of CYP2B1 was effectively inhibited by pepstatin A, an aspartic protease inhibitor, but not by phenylmethanesulfonyl fluoride (PMSF), o-phenanthroline and leupeptin. CYP2B1-hydrolyzing activity was coeluted with cathepsin D when the soluble fraction was chromatographed by means of Ultrogel Ac44 gel filtration. Cathepsin D, purified from rat livers, was able to degrade CYP2B1 at pH 4.5 which corresponds to the intralysosomal pH. These results indicate that cathepsin D is responsible for the major CYP2B1-hydrolyzing enzyme activity in lysosomes.

Cycling of two endogenous lysosomal membrane proteins, lamp-2 and acid phosphatase, between the cell surface and lysosomes in cultured rat hepatocytes.

Our previous studies provided evidence that a 107-kDa major lysosomal membrane glycoprotein termed lamp-1 shuttles between lysosomes and the plasma membrane along the endocytic pathway in rat hepatic cells [Furuno et al. (1989) J. Biochem. 106, 708-716; Furuno et al. (1989) J. Biochem. 106, 717-722]. In the present study, we investigated the movement of a 96-kDa major lysosomal membrane glycoprotein, referred to as lamp-2, and lysosomal acid phosphatase (LAP) in the endocytic membrane transport system of cultured rat hepatocytes. Fab' fragments of anti-lamp-2 and anti-LAP antibodies conjugated with horseradish peroxidase (HRP) were used as probes to analyze quantitatively the transport of these two membrane proteins from the cell surface to lysosomes. After the addition of HRP-anti-lamp-2 and anti-LAP Fab' fragments to the culture medium, the delivery of the antibody conjugates to lysosomes was examined by cell fractionation on a Percoll density gradient. The amount of these HRP tracers in the lysosomal fraction became larger as the period of cell incubation was increased. Km values for uptake of HRP-anti-lamp-2, and LAP Fab' fragments were 0.74 and 0.62 microM, respectively, which were comparable to that of HRP-anti-lamp-1 Fab' (0.57 microM). The endocytic process of the two HRP-antibodies continued for an extended period in the cells exposed to the protein synthesis inhibitor, cycloheximide. Furthermore, we measured the transit times of HRP-anti-lamp-1, anti-lamp-2, and anti-LAP Fab' fragments from the cell surface to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of subtle differences in the surface structure of lysozymes by use of an immobilized Fab fragment.

A method was developed to evaluate the association constant at physiological pH (pH 7.5) between a lysozyme and the Fab fragment derived from anti-lysozyme monoclonal antibody 37-7, which was immobilized to the adsorbent for HPLC. Comparison of the association constants between lysozymes and the immobilized Fab fragment indicated that mAb 37-7 recognized the prominently exposed regions (hills and ridges) around His15 of hen lysozyme, but His15 itself was not directly involved in the binding with mAb 37-7. Moreover, the epitope was confirmed by the reactivity of His15 with monoiodoacetic acid in the presence of mAb 37-7. The association constant of 15-carboxymethylated histidine lysozyme (15CM lysozyme) with the immobilized Fab fragment was smaller by one-seventh than that of 15-carboxamidated histidine lysozyme, though the side chains introduced were almost identical in size. From the pH titration of 15CM lysozyme with 13C-enriched carboxyl group by use of 13C-NMR, the pKa of the introduced carboxyl group was evaluated to be 5.06. Since the carboxyl group was fully ionized under the conditions of measurement (pH 7.5), electrostatic repulsion was found to disturb severely the association between mAb 37-7 and hen lysozyme. Moreover, it was demonstrated that, because of the high reproducibility of measurement, the immobilized Fab fragment could detect subtle differences in the surface structure of lysozymes.

Isolation and characterization of a novel membrane glycoprotein of 85,000 molecular weight from rat liver lysosomes.

We have purified and characterized a novel glycoprotein (r-lamp-3) with an apparent molecular weight (Mr) of 85,000 from membranes of triton-filled lysosomes (tritosomes) by the use of immunoaffinity chromatography on a column of monoclonal antibody-Sepharose 4B. r-lamp-3 accounted for approximately 4% of the total proteins in tritosomal membranes. The isoelectric point (pI) of r-lamp-3 was 4.5 and it was shifted to 6.5 after neuraminidase treatment with its molecular weight decreased by about 7000. Pulse-chase experiments in cultured rat hepatocytes using [35S]methionine showed that r-lamp-3 was initially synthesized as a 77,000 polypeptide and processed to a mature protein with an Mr of 85,000. Upon treatment with endo-beta-N-acetylglucosaminidase H (Endo H), the precursor and mature forms were converted to 55,000 and 73,000 polypeptides, respectively. From the Mr reduction of the precursor form, we estimated the presence of 10--12 N-linked oligosaccharides/r-lamp-3 polypeptide. The data on enzymatic deglycosylation suggested that the mature form of r-lamp-3 contained the same numbers of high mannose-type and complex-type N-linked oligosaccharide chains.

Purification, some properties, and tissue distribution of a major lysosome-associated membrane glycoprotein (r-lamp-2) of rat liver.

We previously purified and characterized a major lysosomal membrane glycoprotein (r-lamp-1) from rat liver [Akasaki et al. (1990) Chem. Pharm. Bull. 38, 2766-2770]. The present study describes the purification of another major lysosomal membrane glycoprotein (r-lamp-2) from rat liver and compares the tissue distribution of r-lamp-1 and r-lamp-2 in rats. R-lamp-2 was purified to apparent electrophoretic homogeneity from rat liver by a simple method with a protein yield of approximately 4.0 micrograms/g wet weight of liver. The purification procedure includes: preparation of tritosomal membranes, extraction of tritosomal membranes with Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. R-lamp-2 exhibited an Mr of 96,000 on SDS-PAGE and had an acidic pI of less than 3.5. R-lamp-2 contained 52.3% carbohydrates. Its carbohydrate moieties were composed of numerous sialyl complex type N-linked oligosaccharides and small amounts of O-linked oligosaccharides. Both r-lamp-1 and r-lamp-2 were detected in all rat tissues examined by immunoblot analyses, while their apparent molecular weights differed among the tissues. Immunological quantitative analysis showed that the protein concentrations of r-lamp-2 were consistently lower than those of r-lamp-1 in all the tissues tested. There was a significant correlation with a regression coefficient of 0.86 in the tissue distribution between r-lamp-1 and r-lamp-2. A good correlation was also observed in the tissue distribution between acid phosphatase and r-lamp-2.(ABSTRACT TRUNCATED AT 250 WORDS)