Jenny Fichmann | Mission College (original) (raw)

Papers by Jenny Fichmann

Research paper thumbnail of 2-D Protein Gel Electrophoresis: An Overview

Humana Press eBooks, Nov 14, 2003

ABSTRACT Two-dimensional electrophoresis (2-D) of proteins used to be an art practiced by a few r... more ABSTRACT Two-dimensional electrophoresis (2-D) of proteins used to be an art practiced by a few researchers, and their worldwide meetings could be held in a side room of a medium-sized hotel. With the rapidly growing volume of sequence data produced by the genome projects and the development of new mass spectrometry methods, high-resolution protein analysis has become an important tool in molecular biology. High-resolution 2-D can reveal virtually all proteins present in a cell or tissue at any given time, including those with posttranslational modifications and rapid turnover rates. With the new analytical tools and the genomic and protein database networks, large-scale studies can be performed on the actual gene products or proteins, in their precursor, mature, and modified forms. This task has lately been called the proteome project. The proteome projects are the necessary complement to genome analysis, and aim to identify and characterize all proteins expressed by an organism or a tissue (1).

Research paper thumbnail of Two-dimensional electrophoresis : The basic separation method in proteome analysis

American biotechnology laboratory, 1998

Research paper thumbnail of Advantages of Immobilized pH Gradients

2-D Proteome Analysis Protocols

Research paper thumbnail of Pyrophosphate-Driven Proton Transport by Microsomal Membranes of Corn Coleoptiles

Research paper thumbnail of Functional Expression of Falcipain, a Plasmodium falciparum Cysteine Proteinase, Supports Its Role as a Malarial Hemoglobinase

Research paper thumbnail of 2-D protein gel electrophoresis. An overview

Methods in molecular biology (Clifton, N.J.), 1999

Research paper thumbnail of The Use of Antisense mRNA to lnhibit the Tonoplast H+ ATPase in Carrot

Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacu... more Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35s promoter. Bafilomycin-sensitive ATP- ase, H+-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of

Research paper thumbnail of Immunological comparison of the coated vesicle H+-ATPases of plants and animals

Protoplasma, 1989

Polyclonal antibodies raised against the tonoplast and plasma membrane H +-ATPases of corn were u... more Polyclonal antibodies raised against the tonoplast and plasma membrane H +-ATPases of corn were used to probe Western blots lof clathrin-coated vesicles purified from zucchini hypocotyls and bovine brain. Antibodies to two tonoplast H +-ATPase subunits recognized their respective polypeptides in purified zucchini coated vesicle preparations. Antibody raised against the plasma membrane H +-ATPase failed to cross-react with zucchini coated vesicles, even though it recognized a 100kDa polypeptide in purified zucchini plasma membrane preparations. These results suggest that zucchini coated vesicles contain a vacuolar-type rather than a plasma membrane-type H+-ATPase. Antibody to the corn 70 kDa subunit also cross-reacted with a 70kDa band in bovine brain coated vesicle preparations. Surprisingly, the corn plasma membrane H +-ATPase antibody recognized a 100kDa polypeptide in the bovine brain coated vesicle preparation. However, at least half of the 100 kDa antigen could be dissociated from the membrane by treatment with 2.0 M urea, indicating that it may represent one of the 100-110 kDa coat assembly proteins. A comparable protein appears to be absent from zucchini coated vesicles.

Research paper thumbnail of Immunological comparison of the coated vesicle H+-ATPases of plants and animals

Protoplasma, 1989

Polyclonal antibodies raised against the tonoplast and plasma membrane H +-ATPases of corn were u... more Polyclonal antibodies raised against the tonoplast and plasma membrane H +-ATPases of corn were used to probe Western blots lof clathrin-coated vesicles purified from zucchini hypocotyls and bovine brain. Antibodies to two tonoplast H +-ATPase subunits recognized their respective polypeptides in purified zucchini coated vesicle preparations. Antibody raised against the plasma membrane H +-ATPase failed to cross-react with zucchini coated vesicles, even though it recognized a 100kDa polypeptide in purified zucchini plasma membrane preparations. These results suggest that zucchini coated vesicles contain a vacuolar-type rather than a plasma membrane-type H+-ATPase. Antibody to the corn 70 kDa subunit also cross-reacted with a 70kDa band in bovine brain coated vesicle preparations. Surprisingly, the corn plasma membrane H +-ATPase antibody recognized a 100kDa polypeptide in the bovine brain coated vesicle preparation. However, at least half of the 100 kDa antigen could be dissociated from the membrane by treatment with 2.0 M urea, indicating that it may represent one of the 100-110 kDa coat assembly proteins. A comparable protein appears to be absent from zucchini coated vesicles.

Research paper thumbnail of Functional Expression of Falcipain, aPlasmodium falciparum Cysteine Proteinase, Supports Its Role as a Malarial Hemoglobinase

Erythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasi... more Erythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis. We have previously shown that a Plasmodium falciparum trophozoite cysteine proteinase, now termed falcipain, is required for hemoglobin degradation, and we have hypothesized that this proteinase is responsible for initial cleavages of hemoglobin. To further evaluate the biological role of falcipain, we expressed the enzyme in bacterial and viral expression systems. After expression in the baculovirus system, falcipain was enzymatically active and had biochemical properties very similar to those of the native proteinase. Recombinant falcipain rapidly hydrolyzed both denatured and native hemoglobin. Hemoglobin hydrolysis was blocked by cysteine proteinase inhibitors but not by inhibitors of other classes of proteinases. Our results support our hypothesis that falcipain is a critical malarial hemoglobinase that is responsible for both initial cleavages of hemoglobin and the subsequent hydrolysis of globin into small peptides.

Research paper thumbnail of Running Preparative Carrier Ampholyte and Immobilized pH Gradient IEF Gels for 2-D

2-D Proteome Analysis Protocols, 1998

Page 1. Running pH Gradient IEF Gels for 2-D 211 23 211 From: Methods in Molecular Biology, Vol. ... more Page 1. Running pH Gradient IEF Gels for 2-D 211 23 211 From: Methods in Molecular Biology, Vol. 112: 2-D Proteome Analysis Protocols Edited by: AJ Link © Humana Press Inc., Totowa, NJ Running Preparative Carrier Ampholyte ...

Research paper thumbnail of The Use of Antisense mRNA to Inhibit the Tonoplast H+ ATPase in Carrot

THE PLANT CELL ONLINE, 1992

Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar... more Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35s promoter. Bafilomycin-sensitive ATPase, H+-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of the A subunit was inhibited in the tonoplast fraction, but not in the Golgi fraction. 'RHo-dimensional protein gel blots of total microsomes of wild-type and control transformant cell tines revealed two major immunoreactive polypeptides in the acidlc pl range. In contrast, highly purified tonoplast membranes contained only the less acidic polypeptide. Because the less acidic polypeptide was preferentially diminished in the two antisense cell lines, we infer that the antisense constructs specifically blocked expression of a tonoplast-specific isoform of the V-ATPase A subunit in carbt. Regenerated plants containing the antisense constructs exhibited altered leaf morphologies and reduced cell expansion. The altered phenotype was correlated with the presence of the antisense construct.

Research paper thumbnail of Pyrophosphate-Driven Proton Transport by Microsomal Membranes of Corn Coleoptiles

PLANT PHYSIOLOGY, 1985

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose den... more Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATPdriven H"-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H-transport. At saturating (3 millimolar) concentrations of Mg2":ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H'transport, but had no effect on ATP-driven transport. Moreover, PPidependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO3, unlike the ATPdependent Hf-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound HW-translocating pyrophosphatases. Both potassium and a permanent anion (NO3-> Cl-), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N'-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.

Research paper thumbnail of Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells

Electrophoresis, 1997

Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a pow... more Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-y-(IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokineregulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.

Research paper thumbnail of Immunological comparison of the coated vesicle H+-ATPases of plants and animals

Protoplasma, 1989

Polyclonal antibodies raised against the tonoplast and plasma membrane H + -ATPases of corn were ... more Polyclonal antibodies raised against the tonoplast and plasma membrane H + -ATPases of corn were used to probe Western blots lof clathrin-coated vesicles purified from zucchini hypocotyls and bovine brain. Antibodies to two tonoplast H +-ATPase subunits recognized their respective polypeptides in purified zucchini coated vesicle preparations. Antibody raised against the plasma membrane H +-ATPase failed to cross-react with zucchini coated vesicles, even though it recognized a 100kDa polypeptide in purified zucchini plasma membrane preparations. These results suggest that zucchini coated vesicles contain a vacuolar-type rather than a plasma membrane-type H+-ATPase. Antibody to the corn 70 kDa subunit also cross-reacted with a 70kDa band in bovine brain coated vesicle preparations. Surprisingly, the corn plasma membrane H + -ATPase antibody recognized a 100kDa polypeptide in the bovine brain coated vesicle preparation. However, at least half of the 100 kDa antigen could be dissociated from the membrane by treatment with 2.0 M urea, indicating that it may represent one of the 100-110 kDa coat assembly proteins. A comparable protein appears to be absent from zucchini coated vesicles.

Research paper thumbnail of 2-D Protein Gel Electrophoresis: An Overview

Humana Press eBooks, Nov 14, 2003

ABSTRACT Two-dimensional electrophoresis (2-D) of proteins used to be an art practiced by a few r... more ABSTRACT Two-dimensional electrophoresis (2-D) of proteins used to be an art practiced by a few researchers, and their worldwide meetings could be held in a side room of a medium-sized hotel. With the rapidly growing volume of sequence data produced by the genome projects and the development of new mass spectrometry methods, high-resolution protein analysis has become an important tool in molecular biology. High-resolution 2-D can reveal virtually all proteins present in a cell or tissue at any given time, including those with posttranslational modifications and rapid turnover rates. With the new analytical tools and the genomic and protein database networks, large-scale studies can be performed on the actual gene products or proteins, in their precursor, mature, and modified forms. This task has lately been called the proteome project. The proteome projects are the necessary complement to genome analysis, and aim to identify and characterize all proteins expressed by an organism or a tissue (1).

Research paper thumbnail of Two-dimensional electrophoresis : The basic separation method in proteome analysis

American biotechnology laboratory, 1998

Research paper thumbnail of Advantages of Immobilized pH Gradients

2-D Proteome Analysis Protocols

Research paper thumbnail of Pyrophosphate-Driven Proton Transport by Microsomal Membranes of Corn Coleoptiles

Research paper thumbnail of Functional Expression of Falcipain, a Plasmodium falciparum Cysteine Proteinase, Supports Its Role as a Malarial Hemoglobinase

Research paper thumbnail of 2-D protein gel electrophoresis. An overview

Methods in molecular biology (Clifton, N.J.), 1999

Research paper thumbnail of The Use of Antisense mRNA to lnhibit the Tonoplast H+ ATPase in Carrot

Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacu... more Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35s promoter. Bafilomycin-sensitive ATP- ase, H+-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of

Research paper thumbnail of Immunological comparison of the coated vesicle H+-ATPases of plants and animals

Protoplasma, 1989

Polyclonal antibodies raised against the tonoplast and plasma membrane H +-ATPases of corn were u... more Polyclonal antibodies raised against the tonoplast and plasma membrane H +-ATPases of corn were used to probe Western blots lof clathrin-coated vesicles purified from zucchini hypocotyls and bovine brain. Antibodies to two tonoplast H +-ATPase subunits recognized their respective polypeptides in purified zucchini coated vesicle preparations. Antibody raised against the plasma membrane H +-ATPase failed to cross-react with zucchini coated vesicles, even though it recognized a 100kDa polypeptide in purified zucchini plasma membrane preparations. These results suggest that zucchini coated vesicles contain a vacuolar-type rather than a plasma membrane-type H+-ATPase. Antibody to the corn 70 kDa subunit also cross-reacted with a 70kDa band in bovine brain coated vesicle preparations. Surprisingly, the corn plasma membrane H +-ATPase antibody recognized a 100kDa polypeptide in the bovine brain coated vesicle preparation. However, at least half of the 100 kDa antigen could be dissociated from the membrane by treatment with 2.0 M urea, indicating that it may represent one of the 100-110 kDa coat assembly proteins. A comparable protein appears to be absent from zucchini coated vesicles.

Research paper thumbnail of Immunological comparison of the coated vesicle H+-ATPases of plants and animals

Protoplasma, 1989

Polyclonal antibodies raised against the tonoplast and plasma membrane H +-ATPases of corn were u... more Polyclonal antibodies raised against the tonoplast and plasma membrane H +-ATPases of corn were used to probe Western blots lof clathrin-coated vesicles purified from zucchini hypocotyls and bovine brain. Antibodies to two tonoplast H +-ATPase subunits recognized their respective polypeptides in purified zucchini coated vesicle preparations. Antibody raised against the plasma membrane H +-ATPase failed to cross-react with zucchini coated vesicles, even though it recognized a 100kDa polypeptide in purified zucchini plasma membrane preparations. These results suggest that zucchini coated vesicles contain a vacuolar-type rather than a plasma membrane-type H+-ATPase. Antibody to the corn 70 kDa subunit also cross-reacted with a 70kDa band in bovine brain coated vesicle preparations. Surprisingly, the corn plasma membrane H +-ATPase antibody recognized a 100kDa polypeptide in the bovine brain coated vesicle preparation. However, at least half of the 100 kDa antigen could be dissociated from the membrane by treatment with 2.0 M urea, indicating that it may represent one of the 100-110 kDa coat assembly proteins. A comparable protein appears to be absent from zucchini coated vesicles.

Research paper thumbnail of Functional Expression of Falcipain, aPlasmodium falciparum Cysteine Proteinase, Supports Its Role as a Malarial Hemoglobinase

Erythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasi... more Erythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis. We have previously shown that a Plasmodium falciparum trophozoite cysteine proteinase, now termed falcipain, is required for hemoglobin degradation, and we have hypothesized that this proteinase is responsible for initial cleavages of hemoglobin. To further evaluate the biological role of falcipain, we expressed the enzyme in bacterial and viral expression systems. After expression in the baculovirus system, falcipain was enzymatically active and had biochemical properties very similar to those of the native proteinase. Recombinant falcipain rapidly hydrolyzed both denatured and native hemoglobin. Hemoglobin hydrolysis was blocked by cysteine proteinase inhibitors but not by inhibitors of other classes of proteinases. Our results support our hypothesis that falcipain is a critical malarial hemoglobinase that is responsible for both initial cleavages of hemoglobin and the subsequent hydrolysis of globin into small peptides.

Research paper thumbnail of Running Preparative Carrier Ampholyte and Immobilized pH Gradient IEF Gels for 2-D

2-D Proteome Analysis Protocols, 1998

Page 1. Running pH Gradient IEF Gels for 2-D 211 23 211 From: Methods in Molecular Biology, Vol. ... more Page 1. Running pH Gradient IEF Gels for 2-D 211 23 211 From: Methods in Molecular Biology, Vol. 112: 2-D Proteome Analysis Protocols Edited by: AJ Link © Humana Press Inc., Totowa, NJ Running Preparative Carrier Ampholyte ...

Research paper thumbnail of The Use of Antisense mRNA to Inhibit the Tonoplast H+ ATPase in Carrot

THE PLANT CELL ONLINE, 1992

Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar... more Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35s promoter. Bafilomycin-sensitive ATPase, H+-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of the A subunit was inhibited in the tonoplast fraction, but not in the Golgi fraction. 'RHo-dimensional protein gel blots of total microsomes of wild-type and control transformant cell tines revealed two major immunoreactive polypeptides in the acidlc pl range. In contrast, highly purified tonoplast membranes contained only the less acidic polypeptide. Because the less acidic polypeptide was preferentially diminished in the two antisense cell lines, we infer that the antisense constructs specifically blocked expression of a tonoplast-specific isoform of the V-ATPase A subunit in carbt. Regenerated plants containing the antisense constructs exhibited altered leaf morphologies and reduced cell expansion. The altered phenotype was correlated with the presence of the antisense construct.

Research paper thumbnail of Pyrophosphate-Driven Proton Transport by Microsomal Membranes of Corn Coleoptiles

PLANT PHYSIOLOGY, 1985

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose den... more Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATPdriven H"-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H-transport. At saturating (3 millimolar) concentrations of Mg2":ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H'transport, but had no effect on ATP-driven transport. Moreover, PPidependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO3, unlike the ATPdependent Hf-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound HW-translocating pyrophosphatases. Both potassium and a permanent anion (NO3-> Cl-), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N'-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.

Research paper thumbnail of Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells

Electrophoresis, 1997

Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a pow... more Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-y-(IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokineregulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.

Research paper thumbnail of Immunological comparison of the coated vesicle H+-ATPases of plants and animals

Protoplasma, 1989

Polyclonal antibodies raised against the tonoplast and plasma membrane H + -ATPases of corn were ... more Polyclonal antibodies raised against the tonoplast and plasma membrane H + -ATPases of corn were used to probe Western blots lof clathrin-coated vesicles purified from zucchini hypocotyls and bovine brain. Antibodies to two tonoplast H +-ATPase subunits recognized their respective polypeptides in purified zucchini coated vesicle preparations. Antibody raised against the plasma membrane H +-ATPase failed to cross-react with zucchini coated vesicles, even though it recognized a 100kDa polypeptide in purified zucchini plasma membrane preparations. These results suggest that zucchini coated vesicles contain a vacuolar-type rather than a plasma membrane-type H+-ATPase. Antibody to the corn 70 kDa subunit also cross-reacted with a 70kDa band in bovine brain coated vesicle preparations. Surprisingly, the corn plasma membrane H + -ATPase antibody recognized a 100kDa polypeptide in the bovine brain coated vesicle preparation. However, at least half of the 100 kDa antigen could be dissociated from the membrane by treatment with 2.0 M urea, indicating that it may represent one of the 100-110 kDa coat assembly proteins. A comparable protein appears to be absent from zucchini coated vesicles.