Release of the variable surface coat glycoprotein from Trypanosoma brucei requires the cleavage of a phosphate ester

The membrane-bound and released forms of the variant surface coat glycoprotein from Trypanosoma brucei have been purified to homogeneity by a new rapid method in the absence of detergents. The conversion of the membrane-bound form to the released form has been found to consist of the cleavage of a phosphodiester bond, distal to the phosphate, linking the protein to a phospholipid. We suggest that this linkage constitutes the normal mode of attachment of the protein to the outer leaflet of the plasma membrane.     

Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C.

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.     

Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160.

The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.     

Optimization of expression and the purification by organic extraction of the integral membrane protein EmrE

EmrE is a member of the small multidrug resistance family of proteins and functions as a efflux transporter of lipophilic cations. This integral membrane protein is composed of 110 amino acids and is very hydrophobic with small loops exposed to the aqueous environment. This protein has been purified in a variety of ways including extraction with chloroform:methanol mixtures. This study explored culture growth and induction conditions, the parameters of organic solvent extraction, running conditions of a lipophilic column for lipid removal, as well as solubilization conditions. Optimal expression and purification protocols are crucial to the characterization goals of this protein. The experiments presented here led to an improvement in the yield of pure EmrE obtained by organic solvent extraction methods at a level of 0.9+/-0.2mg/L of culture. This is on the order of a 60% improvement over previous reports.     

Colvalent linkage of phospholipid to myelin basic protein: identification of phosphatidylinositol bisphosphate as the attached phospholipid

Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.     

Preparative electrophoretic method for the purification of a hydrophobic membrane protein: subunit c of the mitochondrial ATP synthase from rat liver.

A method is described for the purification of subunit c of ATP synthase from rat liver mitochondria. After sample preparation and solvent extraction, the protein was purified to homogeneity by a single-step preparative electrophoretic procedure, using aqueous buffer and containing lithium dodecyl sulfate. The subunit is an extremely hydrophobic and insoluble protein and all solubilization attempts, using a variety of detergents, were unsuccessful except for lithium dodecyl sulfate. Buffer exchange and FPLC gel filtration removed the detergent from the purified sample, leaving the protein in a soluble form. The mammalian protein is composed of 75 amino acid residues, with a molecular mass of 7602 Da and is classified as a proteolipid. Subunit c accounts for 25 and 85% of the intralysosomal accumulation, within neurons, of storage material in juvenile and late-infantile forms of Batten's disease, respectively. This purification procedure allows access to a continuous supply of pure subunit c from a conventional source such as rat liver and preserves precious autopsy materials. The protein could be used as substrate in future proteolytic studies involving pepstatin-insensitive lysosomal proteases and for raising of more specific antibodies. The procedure could also be adapted/modified and used as a model for purifying other extremely insoluble proteins.     

Characterization and biosynthesis of soluble and membrane-bound carbonic anhydrase in brain

Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, and in vitro biosynthesis. A procedure for the rapid purification of both membrane-bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2 hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed with p-nitrophenyl acetate. The membrane-bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane-bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane-bound form. The failure to observe any precursor-product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase-like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms. In vitro synthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance are discussed.     

Delta 6-desaturase activity in liver microsomes of rats fed diets enriched with cholesterol and/or omega 3 fatty acids.

A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91% and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80,000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and O-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25,000 of the 80,000-Mr glycoprotein.     

Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme

We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.     

Purification of haemagglutinin and neuraminidase from influenza virus strain 3QB and isolation of a peptide from an antigenic region of haemagglutinin

Methods were developed for the purification of the surface, membrane-bound glycoproteins haemagglutinin and neuraminidase of influenza virus strain 3QB, in antigenically active forms. The methods employed in the purification included selective removal of the neuraminidase with the proteinase, bromelain, and subsequent disruption of the residual virus particle with the detergent Sarkosyl to release the haemagglutinin. Using techniques for proteolytic digestion of intact, native proteins an antigenically active peptide was isolated from the purified haemagglutinin, the surface glycoprotein against which the major antigenic response is directed. The amino acid composition of this peptide was determined. This was a 16-residue peptide with amino-terminal isoleucine and composition Ile1 Val1 Asx2 Thr1 Ser2 Glx2 Pro1 Gly3 Ala1 Leu1 Lys1.     

Isolation and partial characterization of the heterophile antigen of infectious mononucleosis from bovine erythrocytes

The heterophile antigen (Paul-Bunnell antigen, PBA) of infectious mononucleosis was isolated by extraction of an aqueous suspension of bovine erythrocyte stromata with chloroform-methanol (2:1). The upper aqueous layer contained gangliosides, PBA, and a high-molecular-weight glycoprotein. PBA and gangliosides were separated from the high-molecular-weight glycoprotein by extraction of lyophilized upper layer with chloroform-methanol solvents. Separation of PBA from gangliosides was carried out by chromatography on DEAE-cellulose with chloroform-methanol solvents. PBA appeared to be a minor glycoprotein component of the erythrocyte membrane and had both hydrophobic and hydrophilic properties. It was soluble in either organic or aqueous solvents. On SDS-polyacrylamide gel electrophoresis, it migrated as a single component that stained for protein with Coomassie blue, for carbohydrate with periodic acid-Schiff reagent, and for lipid with oil red 0; it had an apparent molecular weight of 26,000. It was composed of 62% protein with major amino acids: glutamic acid, proline, glycine, isoleucine, leucine, and threonine (158, 116, 98, 90, 85, and 82 residues per 1,000 residues, respectively). Carbohydrate content was 9.2% with major sugar constituents: sialic acid, galactosamine, and galactose. Serologic activity of PBA was destroyed by pronase but not by trypsin.     

Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.     

Improved purification of brine-shrimp (Artemia saline) (Na+ + K+)-activated adenosine triphosphatase and amino-acid and carbohydrate analyses of the isolated subunits.

Purification of the (Na+ + K+)-activated ATPase has been improved 2-fold the respect to both purity and yield over the previous method [Peterson, Ewing, Hootman & Conte (1978) J. Biol. Chem. 253, 4762-4770] by using Lubrol WX and non-denaturing concentrations of sodium dodecyl sulphate (SDS). The enzyme was purified 200-fold over the homogenate. The preparation had a specific activity of about 600 mumol of Pi/h per mg of protein, and was about 60% pure according to quantification of Coomassie Blue-stained SDS/polyacrylamide gels. The yield of purified enzyme was about 10 mg of protein per 100g of dry brine-shrimp (Artemia salina) cysts. The method is highly suitable for purification either on a small scale (10-25g of dry cysts) or on a large scale (900g of dry cysts) and methods are described for both. The large (Na+ + K+)-activated ATPase subunit (alpha-subunit) was isolated in pure form by SDS-gel filtration on Bio-Gel A 1.5m. The small subunit (beta-subunit) was eluted with other contaminating proteins on the Bio-Gel column, but was isolated in pure form by extraction from SDS/polyacrylamide gels. The amino acid and carbohydrate compositions of both subunits are reported. The alpha-subunit contained 5.2% carbohydrate by weight, and the beta-subunit 9.2%. Sialic acid was absent from both subunits.     

Purification and analysis of lung and plasma angiotensin I-converting enzyme by high-performance liquid chromatography

We purified angiotensin I-converting enzyme (ACE) from pig and human lung and plasma for comparison of some physicochemical properties between the endothelial membrane-bound form and the soluble form of the enzyme. After affinity chromatography on Sepharose CL-4B/lisinopril, gel-filtration HPLC on Superose 12 achieved homogeneity for both forms as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Whatever the source of ACE, the molecular weight was 300 +/- 40 kDa after calibration of Superose 12 with standard globular proteins and 172 +/- 4 kDa by SDS-PAGE, with or without reduction, a result suggesting interactions between the glycopolypeptide chain and the chromatographic gel possibly related to the overall shape and sugar content of the enzyme. Ion-exchange HPLC analysis on TSK-DEAE showed that the membrane-bound and soluble forms of ACE are not isoenzymes, although isoelectrofocusing did show that the isoelectric point of soluble ACE was lower than those of tissue ACE, suggesting a different glycosylation. No significant difference between porcine and human ACE appeared. HPLC methods seem to be of particular interest for the purification of ACE with a high yield and for the analysis of its putative differently glycosylated isoforms.     

Solubilization, purification, and characterization of a membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line

The release of free arachidonic acid from membrane phospholipids is believed to be the rate-controlling step in the production of the prostaglandins, leukotrienes, and related metabolites in inflammatory cells such as the macrophage. We have previously identified several different phospholipases in the macrophage-like cell line P388D1 potentially capable of controlling arachidonic acid release. Among them, a membrane-bound, alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular interest because of the likelihood that the regulatory enzyme has these properties. This phospholipase A2 has now been solubilized from the membrane fraction with octyl glucoside and partially purified. The first two steps in this purification are butanol extractions that yield a lyophilized, stable preparation of phospholipase A2 lacking other phospholipase activities. This phospholipase A2 shows considerably more activity when assayed in the presence of glycerol, regardless of whether the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated vesicles or mixed micelles with the nonionic surfactant Triton X-100. Glycerol (70%) increases both the Vmax and the Km with both substrate forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater for vesicles than for mixed micelles. The lyophilized preparation of the enzyme is routinely purified about 60-fold and is suitable for evaluating phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps in the purification are acetonitrile extraction followed by high performance liquid chromatography on an Aquapore BU-300 column and a Superose 12 column. This yields a 2500-fold purification of the membrane-bound phospholipase A2 with a 25% recovery and a specific activity of about 800 nmol min-1 mg-1 toward 100 microM dipalmitoylphosphatidylcholine in mixed micelles. When this material was subjected to analysis on a Superose 12 sizing column, the molecular mass of the active fraction was approximately 18,000 daltons.     

Interactions of KB-cell glycoproteins with an adenovirus capsid protein.

Glycoprotein material extracted from human KB cells with a flurocarbon, trichlorotrifluoroethane (Arklone P), into a waste-soluble fraction binds to fibre, a structural protein of the adenovirus type-5 capsid. The fibre-binding glycoprotein(s) were purified by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on a fibre-Sepharose support. The purification procedure also includes a trypsinization step which eliminates the bulk of contaminating KB cell proteins present in the aqueous fraction without appreciably affecting the activity of the fibre-binding glycoprotein(s). Some comparison is made of the membrane-bound receptors for adenovirus and the water-soluble fibre-binding glycoprotein(s).     

Purification of phospholamban from bovine cardiac muscle with organic solvents

Phospholamban (PLB), an integral membrane protein of cardiac sarcoplasmic reticulum, was extracted from bovine cardiac muscle with an acidic chloroform/methanol mixture. A combination of gel permeation and ion-exchange chromatographies in organic solvents allowed the purification of PLB. The intensive use of organic solvents throughout the isolation yielded a highly purified and intact protein that can be phosphorylated by cAMP protein kinase. The ease of purification and the high yield obtained (2.5 mg/100 g of fresh tissue) show that organic solvents can be very useful in the extraction and purification of hydrophobic membrane proteins.     

Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins

Trypanosoma brucei variant surface glycoproteins are apparently synthesized with a hydrophobic carboxyl-terminal peptide that is cleaved and replaced by a complex glycosylphosphatidylinositol membrane anchor within 1 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated core glycolipid that would be transferred en bloc to the variant surface glycoprotein polypeptide. We report the purification and chemical characterization of a glycolipid from T. brucei that has properties consistent with a role as a variant surface glycoprotein glycolipid donor. This candidate glycolipid precursor has been defined by thin-layer chromatography of extracts of trypanosomes metabolically labeled with radioactive myristic acid, ethanolamine, glucosamine, mannose, and phosphate and by enzymatic, chemical, and gas chromatographic-mass spectrometric analysis. Mild alkali released 100% of the myristic acid, and reaction with phospholipase A2 released 50%. Nitrous acid deamination generated dimyristylphosphatidylinositol, and periodate oxidation released phosphatidic acid. Treatment of purified glycolipid with phosphatidylinositol-specific phospholipase C released dimyristylglycerol and a water-soluble glycan that was sized on Bio-Gel P-4 columns. The candidate precursor contained mannose, myristic acid, phosphate, and ethanolamine with an unsubstituted amino group, but not galactose.     

Purification and identification of inactive forms of repressible and constitutive acid phosphatase in yeast

Acid phosphatase (EC 3.1.3.2, orthophosphoric-monoester phosphohydrolase, (acid optimum)) from the budding yeast Saccharomyces cerevisiae was purified from repressed and depressed cells. Without Triton X-100 in the extraction buffer only the constitutive or repressible active enzyme eluted from a Sepharose CL-6B column, the last step of the purification procedure. When Triton X-100 was included in the extraction buffer, an additional protein peak eluted prior to the active acid phosphatase. The material from this new peak, a glycoprotein, had no acid phosphatase activity but cross-reacted with antibodies raised against repressible acid phosphatase. The tryptic fingerprints of the inactive proteins are very similar to the ones of the corresponding active enzymes. We conclude that this new glycoprotein represents an inactive form of repressible and constitutive acid phosphatase. The fact that inactive acid phosphatase can be recovered only in the presence of Triton X-100 indicates that it is membrane-bound.