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1.
Int Immunopharmacol ; 3(8): 1217-24, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12860177

ABSTRACT

Synthetic lipopeptides derived from the N-terminus of bacterial lipoprotein constitute potent immunoadjuvants for parenteral and mucosal immunization. When combined with tetanus toxoid (TT) or gliadin as antigens, the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (P(3)CSK(4)) markedly enhanced the specific antibody levels. Lipopeptides also act as macrophage/monocyte activators: P(3)CSK(4) induced nitric oxide release from bone marrow-derived macrophages (BMDM) of LPS responder and nonresponder mice. The antitumoral effect of the lipopeptide was demonstrated by a strong cytostatic activity of the lipopeptide-treated macrophages against the murine B-cell lymphoma cell line Abelson 8-1. The chemically well-defined lipopeptides can be synthesized with high purity and reproducibility and constitute ideal agents to be combined with antigens/vaccines or antitumor treatment.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antibody Formation/drug effects , Lipoproteins/pharmacology , Adjuvants, Immunologic/administration & dosage , Animals , Antibody Formation/immunology , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Division/drug effects , Cytotoxicity, Immunologic , Female , Gliadin/immunology , Lipoproteins/administration & dosage , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/pathology , Macrophage Activation/drug effects , Macrophage Activation/immunology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred BALB C , Nitric Oxide/metabolism , Tetanus Toxoid/administration & dosage , Tetanus Toxoid/immunology , Tumor Cells, Cultured
2.
FEMS Immunol Med Microbiol ; 37(2-3): 193-200, 2003 Jul 15.
Article in English | MEDLINE | ID: mdl-12832125

ABSTRACT

Synthetic lipopeptides derived from the bacterial cell wall component lipoprotein activate B-lymphocytes and macrophages/monocytes in vitro. In vivo they constitute potent immunoadjuvants for a broad range of different antigens and species comparable or superior to Freund's adjuvant. Here, we demonstrate that P(3)CSK(4), representing a highly active lipopentapeptide derivative in vitro, significantly enhances and accelerates the humoral immune response to tetanus toxoid. P(3)CSK(4) could substitute for up to 90% of the antigen without any decrease in the specific IgG level, and the presence of the lipopeptide resulted in a prolonged production of specific IgG in time. Investigations using P(3)CSK(4) as an adjuvant in genetic immunization confirmed earlier data demonstrating that lipopeptides constitute adjuvants for low-immunogenic DNA constructs and/or for application routes resulting in weak immune responses. We monitored a lipopeptide-dependent shift from a Th1-type to Th2-type response, when DNA immunization was followed by i.p. administration of the lipopeptide adjuvant.


Subject(s)
Adjuvants, Immunologic , Antibodies, Bacterial/blood , Lipoproteins/administration & dosage , Tetanus Toxoid/immunology , Vaccines, DNA/immunology , Animals , Biolistics , Female , Immunization , Lipoproteins/chemistry , Lipoproteins/genetics , Lipoproteins/immunology , Mice , Mice, Inbred BALB C , Tetanus Toxoid/administration & dosage , Vaccines, DNA/administration & dosage
3.
Int Immunopharmacol ; 3(4): 523-39, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12689657

ABSTRACT

Vaccination with the antiallergic drug Histaglobin is used to treat a broad range of human allergic diseases including bronchial asthma, allergic rhinitis, and atopic dermatitis. In order to further elucidate its functional activity, Histaglobin was investigated in an in vivo mouse allergy model. Mice were sensitized with ovalbumin either prior to or after Histaglobin treatment, and its antiallergic potential was evaluated. Ovalbumin-sensitized mice exhibited increased serum levels of IL-4, tumor necrosis factor alpha (TNF-alpha), and an increase of total and ovalbumin-specific IgE; total and ovalbumin-specific IgG levels were also elevated. Subsequent administration (therapeutic treatment) of Histaglobin resulted in a decrease of total and specific serum IgE levels; total and specific IgG1 serum levels were reduced by more than 50% and 45%, respectively; the mice displayed a down-regulation of IL-4 and TNF-alpha serum levels and showed increased levels of IFN-gamma and IgG2a. Mice pretreated with Histaglobin, prior to ovalbumin sensitization (prophylactic treatment), were found to be widely unresponsive to ovalbumin. They exhibited higher serum levels of IFN-gamma and IgG2a (total and specific) compared to saline-treated control mice. The inhibitory effects were still observed 1 month post-immunization. Our data, indicating a Histaglobin-induced modulation of the Th1/Th2 balance in favour of Th1, correspond with the well-known antiallergic activity of Histaglobin observed in patients.


Subject(s)
Anti-Allergic Agents/therapeutic use , Histamine/therapeutic use , Hypersensitivity/drug therapy , Th1 Cells/immunology , Th2 Cells/immunology , gamma-Globulins/therapeutic use , Animals , Disease Models, Animal , Drug Combinations , Female , Hypersensitivity/blood , Hypersensitivity/immunology , Immunoglobulin E/blood , Interleukin-4/blood , Mice , Mice, Inbred BALB C , Ovalbumin/immunology , Tumor Necrosis Factor-alpha/metabolism
4.
Int Immunopharmacol ; 2(8): 1065-77, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12349944

ABSTRACT

Bacteria-derived synthetic lipoproteins constitute potent macrophage activators in vivo and are effective stimuli, enhancing the immune response especially with respect to low or non-immunogenic compounds. N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), exhibiting one of the most effective lipopeptide derivatives, represents a highly efficient immunoadjuvant in parenteral, oral, nasal and genetic immunization either in combination with or after covalent linkage to antigen. In order to further elucidate its molecular mode of action with respect to the transcriptional level, we focused our investigations on the P3CSK4-induced modulation of gene transcription. We could show that P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. P3CSK4 activates the expression of tumor suppressor protein p53 (p53), c-rel, inhibitor of nuclear factor kappa B (NFkappaB) alpha (IkappaB alpha), type 2 (inducible) nitric oxide (NO) synthase (iNOS), CD40-LR, intercellular adhesion molecule-1 (ICAM-1) and interleukin 1/6/15 (IL-1/6/15). We detected no activation of heat shock protein (HSP) 27, 60, 84 and 86, osmotic stress protein 94 (Osp 94), IL-12, extracellular signal-regulated protein kinase 1 (ERK1), p38 mitogen activated protein (MAP)-kinase (p38), c-Jun NH2-terminal kinase (JNK), signal transducer and activator of transcription 1 (STAT1), CD14 and caspase genes. Furthermore, we monitored inhibition of STAT6, Janus kinase 3 (Jak3) and cyclin D1/D3 gene transcription after stimulating bone marrow-derived macrophages (BMDM) with lipopeptide. In addition, we monitored significant differences after lipopeptide and lipopolysaccharide (LPS) stimulation of bone marrow-derived murine macrophages. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines.


Subject(s)
Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , Lipoproteins/pharmacology , Transcription, Genetic/drug effects , Adjuvants, Immunologic/pharmacology , Animals , Female , Gene Expression Regulation/immunology , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Inbred BALB C , Transcription, Genetic/immunology
5.
Immunology ; 103(1): 49-60, 2001 May.
Article in English | MEDLINE | ID: mdl-11380692

ABSTRACT

Synthetic lipopeptides based on bacterial lipoprotein are efficient activators for monocytes/macrophages inducing the release of interleukin (IL)-1, IL-6, tumour necrosis factor-alpha (TNF-alpha), reactive oxygen/nitrogen intermediates, and the translocation of nuclear factor kappaB (NFkappaB). In this report we investigate the signal transduction pathways involved in leucocyte activation by the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4). We show that P3CSK4 activates mitogen-activated protein (MAP)-kinases ERK1/2 and MAP kinase (MAPK)-kinases MEK1/2 in bone-marrow-derived macrophages (BMDM) and in the macrophage cell line RAW 264.7. Additionally, we could detect differences between the P3CSK4 and lipopolysaccharide (LPS)-induced phosphorylation of MAP kinases: Different levels in phosphorylation were found both in kinetics and dose-response using RAW 264.7 cells or BMDM from BALB/c and LPS responder mice (C57BL/10ScSn) or LPS non-responder mice (C57BL/10ScCr). The lipopeptide activated the MAPK-signalling cascade in both LPS responder and non-responder macrophages, whereas LPS induced the MAPK signalling pathway only in macrophages derived from LPS responder mice. An approximately 70% decrease of lipopeptide induced NFkappaB translocation and an about 50% reduction of nitric oxide (NO) release was observed in the presence of anti-CD14. These data correspond to the reduction of phosphorylation of ERK1/2 after stimulation with P3CSK4 in the presence of anti-CD14 antibodies. Inhibition of MEK1/2 by PD98059 completely reduced the lipopeptide-induced phosphorylation of ERK1/2 indicating that MEK1/2 are solely responsible for the phosphorylation of the downstream-located MAP kinases ERK1/2.


Subject(s)
Drosophila Proteins , Lipoproteins/immunology , Macrophage Activation/immunology , Mitogen-Activated Protein Kinase 1/immunology , Mitogen-Activated Protein Kinases/immunology , Signal Transduction/immunology , Animals , Bone Marrow/immunology , Cell Line , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Lipopolysaccharide Receptors/immunology , Lipopolysaccharides/immunology , Membrane Glycoproteins/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/genetics , Nitric Oxide/metabolism , Phosphorylation , Receptors, Cell Surface/immunology , Toll-Like Receptor 4 , Toll-Like Receptors , Translocation, Genetic
6.
Eur J Med Res ; 6(1): 10-20, 2001 Jan 29.
Article in English | MEDLINE | ID: mdl-11313186

ABSTRACT

Lactate dehydrogenase catalyzes the final step in glycolysis, the interconversion of pyruvate and lactate. The tetrameric enzyme is composed of one or two subunits (H and/or M) resulting in five isoenzyme forms: LDH-H4, -H3M1, -H2M2, -H1M3, and -M4. The relative distribution of the LDH isoenzymes is tissue dependent and a significant marker for the diagnosis of hepatoma of the liver, myocardial infarction, muscular dystrophy, and a wide variety of other acute and chronic diseases to be detected by alterations of the LDH isoenzyme pattern in serum. Immunochemical approaches to the routine determination of LDH depend on isoenzyme specific antibodies. Since the H- and M-subunits for human LDH are highly homologous, LDH isoenzyme specific antibodies for immunochemical monitoring are hard to generate. Here we present data on the generation and characterization of LDH isoenzyme-specific mono- and polyclonal antibodies in different species in the presence of lipopeptide adjuvants. Western-Blot and ELISA analysis showed that antisera and monoclonal antibodies recognize their homologous antigens with high specificity and are therefore suitable for immunochemical monitoring of the LDH isoenzymes H4 and M4. In addition, they can be used for the determination of LDH isoenzyme specific activity which is an essential prerequisite for online amperometric immunosensor monitoring.


Subject(s)
Adjuvants, Immunologic , Antibodies/immunology , Immunoassay/methods , L-Lactate Dehydrogenase/immunology , Lipoproteins/immunology , Animals , Antibodies/metabolism , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Humans , Immunoblotting , Isoenzymes/immunology , Isoenzymes/metabolism , L-Lactate Dehydrogenase/metabolism , Lipoproteins/metabolism , Mice , Mice, Inbred BALB C , Rabbits
7.
Int J Immunopharmacol ; 22(10): 755-63, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10963848

ABSTRACT

The transcription factor NF-kappaB is the central regulator for the expression of various genes involved in inflammation, infection and immune response including the genes for IL-1beta, TNF-alpha, IL-6 and leukocyte adhesion molecules. Here, we show that the anti-allergic drug histaglobin down-regulates the release of IL-1beta, TNF-alpha, IL-6 and IL-10 in human peripheral blood mononuclear cell cultures. This down-regulatory effect becomes even more pronounced when the cultures are simultaneously activated with the T-lymphocyte mitogen phytohemagglutinin (PHA) or with the B-lymphocyte and macrophage activator lipopeptide (P(3)CSK(4)). We also demonstrate that histaglobin inhibits the nuclear translocation of NF-kappaB in response to TNF-alpha or lipopolysaccharide (LPS) in bone marrow-derived macrophages of Balb/c mice. The inhibitory effect of histaglobin on NF-kappaB activation and cytokine release might be responsible for its anti-allergic effect as demonstrated in clinical studies.


Subject(s)
Anti-Allergic Agents/pharmacology , Cytokines/biosynthesis , Histamine/pharmacology , NF-kappa B/metabolism , Translocation, Genetic/drug effects , gamma-Globulins/pharmacology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cells, Cultured , Down-Regulation/drug effects , Drug Combinations , Fluorescent Antibody Technique , Humans , Indicators and Reagents , Interleukin-1/biosynthesis , Interleukin-10/biosynthesis , Interleukin-6/biosynthesis , Monocytes/drug effects , Monocytes/metabolism , Tumor Necrosis Factor-alpha/biosynthesis
8.
Eur J Med Res ; 5(3): 101-9, 2000 Mar 27.
Article in English | MEDLINE | ID: mdl-10756163

ABSTRACT

The bacterial extract OM-89 used for the prevention and treatment of recurrent urinary tract infections constitutes an effective immunostimulant in vitro and in vivo. Here we demonstrate that OM-89 shows mitogenic properties towards murine spleen cell cultures from LPS responder and non-responder mice. In macrophages the extract induces the translocation of NF-kappaB into the cell nucleus and RNI (radical nitrogen intermediates) release, which could be attributed to single fractions of the extract. Our findings on the in vitro immunostimulatory effect of OM-89, as well as its immunogenic and adjuvant properties, are of importance for understanding its therapeutic efficacy as demonstrated in clinical studies.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antigens, Bacterial/pharmacology , Lymphocytes/immunology , Macrophages/immunology , Animals , Bone Marrow Cells/cytology , Cell Line , Cells, Cultured , Escherichia coli , Female , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Lymphocyte Activation , Lymphocytes/drug effects , Macrophages/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , NF-kappa B/metabolism , Nitric Oxide/physiology , Recombinant Proteins , Salmonella , Spleen/immunology
9.
Int J Immunopharmacol ; 22(5): 339-53, 2000 May.
Article in English | MEDLINE | ID: mdl-10708882

ABSTRACT

The preparation of antibodies against the liver toxin microcystin, as described here, is of major importance for its detection and purification in food and water, and for a therapeutic approach to neutralize the toxin by passive immunization. Microcystin-LR (MLR) and microcystin-RR (MRR) were purified from cyanobacterial cell materials by extraction, Sephadex LH-20-, ODS silica gel-, ionic exchange and RP-HPLC-chromatography. In order to reduce the toxicity for parenteral administration, microcystins were coupled by the carbodiimide method to poly-L-lysine (PLL(50.000)). Mice and rabbits were immunized with the conjugates in the presence of two lipopeptide immunoadjuvants (P(3)CSK(4) and P(3)CS-T(h)). High MLR-specific antibody levels were observed after parenteral coadministration of antigen and lipopeptides, whereas no anti-MLR antibodies were obtained with free microcystin or the microcystin-PLL(50.000)-conjugate in the absence of lipopeptide. In oral immunization, coadministration of antigen and adjuvants resulted in an accelerated development of anti MLR-specific antibodies and high antibody levels. Using the antisera, we could detect different microcystins and nodularin down to a concentration range of 10-50 ng/ml by a competitive inhibition ELISA; detection of microcystins in crude cell preparations was also possible. Furthermore, microcystins from different sources could be detected and discriminated from cyclic cyanopeptolines.


Subject(s)
Peptides, Cyclic/immunology , Administration, Oral , Animals , Antibody Formation , Enzyme-Linked Immunosorbent Assay , Female , Immune Sera/immunology , Immunization , Marine Toxins , Mice , Mice, Inbred BALB C , Microcystins , Peptides, Cyclic/isolation & purification , Rabbits
10.
Int J Immunopharmacol ; 22(12): 1093-102, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11137616

ABSTRACT

Synthetic lipopeptides derived from the N-terminus of bacterial lipoprotein constitute potent macrophage activators and polyclonal B-lymphocyte stimulators. They are also efficient immunoadjuvants in parenteral, oral and nasal immunization either in combination with or after covalent linkage to an antigen. Here we show how alterations in the molecular structure influence their biological properties indicating P3CSK4 as one of the most active members of a lipopentapeptide fatty acid library. This compound resulted in a most pronounced macrophage stimulation as indicated by NO release, activation of NFkappaB translocation, and enhancement of tyrosine protein phosphorylation. Furthermore, P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. Finally we have evidence that P3CSK4 constitutes an effective adjuvant for DNA immunizations, especially increasing weak humoral immune responses. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines.


Subject(s)
Adjuvants, Immunologic/pharmacology , Bacteria/chemistry , Immunity/genetics , Lipoproteins/pharmacology , Macrophage Activation/drug effects , Antibodies/analysis , DNA/drug effects , DNA/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Immunity/drug effects , In Vitro Techniques , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , NF-kappa B/immunology , Nitric Oxide/metabolism , Phosphorylation , Protein-Tyrosine Kinases/metabolism
11.
Int J Immunopharmacol ; 19(5): 277-87, 1997 May.
Article in English | MEDLINE | ID: mdl-9439766

ABSTRACT

To generate conventional or monoclonal antibodies for the serological detection of drugs, antibiotics, toxins and other low molecular mass substances, a suitable and effective adjuvant is needed. Lipopeptides derived from a major component of the bacterial cell wall constitute potent nontoxic and nonpyrogenic immunoadjuvants when mixed with conventional antigens. Here we demonstrate that the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl- serine (P3CS) coupled to a Th-cell epitope (P3CS-Th) can efficiently enhance the specific immune response against low molecular weight compounds in different species. In the presence of the synthetic lipopeptide P3CS-Th, the peptides which are per se non-immunogenic stimulated a specific humoral immune response in mice after intraperitoneal application. Mixtures containing adjuvants without the Th sequence showed no significant antibody induction. A marked enhancement of the humoral immune response was obtained with the low molecular mass antigens Iturin AL, Herbicolin A and Microcystin (MLR) coupled to poly-l-lysin (MLR-PLL), in rabbits and in chickens. Lipopeptide-Th cell epitope conjugates also constituted adjuvants for the in vitro immunization of either human mononuclear cells or mouse B-cells with MLR-PLL; after fusion of the immunized cultures with the heteromyeloma cell lines CB-F7 or the mouse myeloma cell line SP 2/0, respectively, we observed a significantly increased yield of antibody secreting hybridomas.


Subject(s)
Adjuvants, Immunologic/physiology , Epitopes, T-Lymphocyte/metabolism , Lipoproteins/metabolism , Peptides/immunology , Animals , Antibody Formation , Antibody Specificity , Antigen-Antibody Reactions , Chickens , Dipeptides/immunology , Enzyme Inhibitors/immunology , Epitopes, T-Lymphocyte/immunology , Humans , Immunization , Leukocytes, Mononuclear/immunology , Lipoproteins/immunology , Lymphocyte Culture Test, Mixed , Mice , Microcystins , Peptides, Cyclic/immunology , Phosphoprotein Phosphatases/antagonists & inhibitors , Rabbits
12.
FEMS Immunol Med Microbiol ; 17(4): 225-34, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9143880

ABSTRACT

Lipopeptides of bacterial origin constitute potent immunoadjuvants when combined with antigens. After the immunization with lipopeptides covalently coupled to non-immunogenic low-molecular-mass antigens or haptens, a hapten-specific humoral immune response can often be obtained. The response against synthetically prepared melittin fragments was further enhanced by the additional introduction of a T helper (Th)-cell epitope into the lipopeptide-hapten conjugate. The Th-cell epitope applied, which is presented by the MHC class II molecule of the BALB/c (H-2d) haplotype, consisted of a synthetic 16-amino-acid oligopeptide derived from sperm whale myoglobin. The immune-enhancing effect was most pronounced for the melittin-derived peptide fragments [Mel(1-16)] and [Mel(17-26)-CONH2]. Antibodies obtained after 3 immunizations with the conjugates recognized the synthetic as well as the native melittin molecule. Our results show that it is possible to markedly enhance a weak hapten-specific immune response by coupling the haptens to a lipopeptide conjugated to a haplotype-specific T helper-cell epitope. The novel conjugates are well suited for the optimization of immunization procedures, and for the development of novel synthetic vaccines.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antibody Formation , Epitopes/immunology , Haptens/pharmacology , Melitten/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Mice , Mice, Inbred BALB C
13.
Int J Immunopharmacol ; 19(9-10): 547-50, 1997.
Article in English | MEDLINE | ID: mdl-9637352

ABSTRACT

We investigated the immunostimulatory properties of synthetically prepared lipopeptides derived from the cell wall of Gram negative bacteria. These compounds constitute potent macrophage activators and polyclonal B-lymphocyte stimulators. They are also immunoadjuvants in parenteral or oral immunization. By coupling the lipopeptides to haptens or low molecular weight antigens which are not immunogenic per se, highly immunogenic conjugates can be prepared. Lipopeptide antigen conjugates as synthetic vaccines give protection by enhancing the antibody-mediated immune response, and they stimulate the cellular immune response in vivo by priming of cytotoxic T-cells.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Gram-Negative Bacteria/immunology , Oligopeptides/administration & dosage , Administration, Oral , Animals , Antigens/administration & dosage , Bacterial Vaccines/administration & dosage , Cell Wall/immunology , Epitopes, T-Lymphocyte/administration & dosage , Foot-and-Mouth Disease/prevention & control , Freund's Adjuvant/administration & dosage , Guinea Pigs , Immunization , Lipopeptides , Mice , Oligopeptides/chemistry , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Viral Vaccines/administration & dosage , Virus Diseases/immunology , Virus Diseases/prevention & control
14.
Arch Microbiol ; 155(3): 217-20, 1991.
Article in English | MEDLINE | ID: mdl-2048932

ABSTRACT

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.


Subject(s)
Saccharomyces cerevisiae/enzymology , Trehalase/analysis , Autoradiography , Blotting, Western , Saccharomyces cerevisiae/ultrastructure , Trehalase/biosynthesis , Trehalase/chemistry , Vacuoles/enzymology
15.
J Biol Chem ; 263(17): 8537-43, 1988 Jun 15.
Article in English | MEDLINE | ID: mdl-3286651

ABSTRACT

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.


Subject(s)
Saccharomyces cerevisiae/genetics , Trehalase/isolation & purification , Amino Acids/analysis , Cross Reactions , Hydrogen-Ion Concentration , Immunosorbent Techniques , Molecular Weight , Saccharomyces cerevisiae/enzymology
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