Concanavalin A-Sepharose Column Research Articles

Thermal Inactivation of Myrosinase from White Mustard Seeds

Myrosinases (thioglucosidases) catalyze the hydrolysis of a class of compounds called glucosinolates, of which the aglycones show various biological functions. It is often necessary to minimize the loss of myrosinase activity during thermal processing of cruciferous vegetables. Myrosinase was isolated from a popular spice, white mustard (Sinapis alba), and its thermal inactivation kinetics was investigated. The enzyme was extracted from white mustard seeds and purified by a sequential processes of ammonium sulfate fractionation, Concanavalin A-Sepharose column chromatography, and gel permeation chromatography. At least three isozymes were revealed by Concanavalin A-Sepharose column chromatography. The purity of the major myrosinase was examined by native polyacrylamide gel electrophoresis and on-gel activity staining with methyl red. The molecular weight of the major enzyme was estimated to be 171 kDa. When the consecutive step model was used for the thermal inactivation of the major myrosinase, its inactivation energy was 44.388 kJ/mol for the early stage of destruction and 32.019 kJ/mol for the late stage of destruction. When the distinct two enzymes model was used, the inactivation energy was 77.772 kJ/mol for the labile enzyme and 95.145 kJ/mol for the stable enzyme. The thermal inactivation energies lie within energy range causing nutrient destruction on heating.

Purification of three major forms of β-hCG from urine and production of polyclonal antibodies against them

Objectives This study has been conducted to develop a proper and relatively simple method for purification of three major forms of β-hCG from urine to use as clinical calibrators and also to produce anti-β-hCG polyclonal antibodies against these purified forms. Design and methods Major β-hCG forms from urine of pregnant women were purified using an ultrafiltration method followed by three successive steps: 1) affinity chromatography on a Concanavalin A-Sepharose column, 2) ion exchange chromatography on a DEAE-Sephacel column and 3) preparative gel electrophoresis. Specific polyclonal antibodies against the purified forms were produced by immunizing rats. We also extracted β-hCG from urine using trypsin affinity chromatography. Results Three β-hCG forms with the molecular mass (MW) of 28, 32 and 35 kDa were purified and identified by ELISA method using monoclonal antibodies against two distinct epitopes (β2 and β11) on β-hCG. The titer of prepared antibodies was comparable with common standard anti-β-hCG antibodies. Also we could purify a 37 kDa form of β-hCG by trypsin affinity chromatography. Conclusions Here we proposed two different proper methods for purification of major forms of β-hCG from urine. We also found that three prepared β-hCG forms expose the β11 epitope and have an hCGβcf epitope expressed. Specific antibodies against purified β-hCG forms had comparable titers and could be used in diagnostic kits.

Isolation of β -glucan from the cell wall of Saccharomyces cerevisiae

β-Glucan, one of the major cell wall components of Saccharomyces cerevisiae (S. cerevisiae), has been found to enhance immune functions. At present study, we developed an optimal procedure to extract and purify β-glucan. At first, yeast cells were grown in sabouraud dextrose agar and then cultured in yeast extract–peptone–glucose (YPG) broth. After incubation, cells were harvested, washed and disrupted by means of sonication method. The obtained cell walls were used to prepare alkali-soluble β-glucan (glucan-S1). In this regard, 2% sodium hydroxide (NaOH) and 3% acetic acid were used in alkaline–acid extraction, respectively. This preparation contained 2.4% protein. In the next step, DEAE sephacel chromatography was used to remove remaining proteins (glucan-S2). Subsequently this preparation was applied into concanavalin-A sepharose column to remove manann. Finally, β-glucan free of mannoprotein complexes was prepared (glucan-S3).

Recycling of Dolichyl Monophosphate to the Cytoplasmic Leaflet of the Endoplasmic Reticulum after the Cleavage of Dolichyl Pyrophosphate on the Lumenal Monolayer

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.

Role of Glycoproteins Isolated from Epicoccum purpurascens in Host-Pathogen Interaction

Objective: Attachment to host matrix is an important provisory step for the institution of any fungal infection. The present study investigates the role of glycoproteins of Epicoccum purpurascens in host-fungal adherence. Methods:Epicoccum spore-mycelial extract was fractionated on a concanavalin A-Sepharose column. Three glycoproteins of 12, 17 and 33 kDa (Epi p 1) were electroeluted and checked for hemagglutination and hemagglutination inhibition. The monosaccharide content of the highly potent protein Epi p 1 was determined by high-performance anion exchange chromatography and pulsed amperometric detection. The interaction of Epi p 1 with mannose-binding lectin (MBL) leading to the activation of the complement system was studied by immunoblot, ELISA and ELISA inhibition techniques. Immunoblot and immunoblot inhibition were carried out with culture filtrate to determine the nature of Epi p 1. Results: 33 (Epi p 1)-, 17- and 12-kDa proteins were 58, 46 and 38 times more potent than crude extract in hemagglutination activity (HA). The HA of Epi p 1 was inhibited by N-acetyl glucosamine, glucose and laminin. Epi p 1 had a high mannose content, showed MBL binding in ion-dependent manner and caused complement activation. The protein was detected in culture filtrate and thus seems to play a significant role in fungal invasion. Conclusion: Epi p 1, an allergenic glycoprotein of E. purpurascens, is involved in host-fungal interactions through MBL.

Acid and alkaline phosphatase activities of a fraction isolated from Parawixia bistriata spider venom

This present study describes the isolation of a high molecular weight fraction (F1) from the venom of the social spider Parawixia bistriata, by gel filtration and also its subfractions by further purification with affinity chromatography on a Concanavalin A-Sepharose column. Acid and an alkaline phosphatase activities were found in fractions. The effects of pH, temperature and metallic ions on these activities were evaluated. Optimal temperature for both enzymes was 55 °C and optimal pH was 5.0 and 8.5 for the acid and alkaline phosphatase activities, respectively. As ZnCl 2 inhibited enzymatic activities and the chelating agent ethylenediaminetetracetic acid (EDTA) raised the basal phosphatase activities, it was speculated that the venom itself could contain Zn ++; this was confirmed with the use of an atomic absorption flame spectrometer. In conclusion, the high molecular weight components of the spider venom of P. bistriata have acid and alkaline phosphatase activities, which may reflect the presence of at least two different enzymes.

Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Alpha1-antichymotrypsin-like proteins I and II purified from bovine adrenal medulla are enriched in chromaffin granules and inhibit the proenkephalin processing enzyme "prohormone thiol protease".

Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K(i,app) of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.

Isolation of isolectins from Vitis vinifera L. Cv. Chardonnay grape berries.

A lectin fraction from Chardonnay grape juice has been isolated by affinity chromatography on a column of p-aminophenyl beta-D-glucoside-derivatized agarose. The lectin fractions agglutinate rabbit and human erythrocytes without serological specificity. None of the usual monosaccharides, glycosides, or glycoproteins inhibit the hemagglutinating activity. Erythroagglutination is only inhibited by nitrophenyl glycosides, p-nitrophenyl beta-D-glucoside being the strongest inhibitor. In SDS-PAGE in the presence of 2-mercaptoethanol and gel filtration HPLC, the lectin fraction gave a single band or peak corresponding to M(r) 13.2-11.9 kDa, thus indicating it to be a monomer. Three bands were observed by isoelectric focusing with pI values of 4.1, 4. 4, and 4.9. The isolectins seem to be glycoproteins since they are bound on a concanavalin A-Sepharose column.

Isolation and characterization of biologically active lectin from Korean mistletoe, Viscum album var. Coloratum.

A mistletoe lectin was isolated from water extracts of Korean mistletoe, a subspecies of Viscum album, grown on Quercus mongolica using CM-Sepharose chromatography followed by an affinity chromatography on a concanavalin A-Sepharose column. The compound proved to be a mistletoe lectin II with D-galactose and N-acetyl-D-galactosamine specificity. Matrix-assisted laser desorption time-of-flight mass spectroscopy showed it to have an average molecular mass of 62.7 kDa and to consist of two subunits of 30.6 kDa and 32.5 kDa. It was a basic protein with isoelectric points of 9.4 and 9.6 by capillary isoelectric focusing and was cytotoxic to Molt4 cell.

Acetylcholinesterase at high catalytic efficiency and substrate specificity in the optic lobe of Eledone moschata (Cephalopoda: Octopoda): biochemical characterization and histochemical localization

In the optic lobe of the cephalopod mollusc Eledone moschata, two acetylcholinesterase forms I and II were detected, both showing a marked active site specificity with differently sized substrates. Catalytic efficiency ( k cat \\K m ) of the prevailing form II is similar to that of acetylcholinesterases from vertebrate nervous system. Enzyme forms I and II were co-purified from a high-salt-Triton X-100 soluble extract of optic lobe by consecutive a.nity chromatographies on procainamide- and concanavalin A-Sepharose columns and then separately obtained by preparative density gradient centrifugation. According to gel-filtration chromatography, sedimentation analysis and SDS-PAGE, the major form II is an amphiphilic globular dimer (135 136 kDa, 6.3 7.4 S) of monomers (66 kDa) S-S linked between terminal segments. Phosphatidylinositol anchors give cell membrane insertion, self-aggregation and detergent (Triton X-100, Brij 97) interaction. Form I, characterized only in part owing to its small amount, showed molecular size (129 kDa) and sedimentation coe.cient (7.5 S) similar to those of form II; it is likely to be attached to the cell membrane by electrostatic interactions. Both forms behaved similarly with various inhibitors and underwent excess-substrate inhibition. The results obtained suggest a common origin of both form I and II from a single gene. The former could be a degradation product of the prevailing one (II), which is likely to be functional in cholinergic synapses.

Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector.

Biologically active recombinant porcine FSH (rec-pFSH) free from the cognate pituitary glycoprotein hormone LH was produced. It was synthesized by a baculovirus vector-insect cell system using two cDNAs encoding the glycoprotein alpha and FSH beta subunits. Its antigenicity was the same as that of pFSH prepared from the pituitary. Glycosylation of rec-pFSH was shown by tunicamycin treatment but the molecular mass of each subunit was lower than that of pituitary-derived FSH, because of the absence of trimming of terminal sugars in insect cells. Rec-pFSH was secreted into the culture medium at about 1 mg/l and purified in six fractions, because of the heterogeneity of the sugar group, by S-Sepharose and concanavalin A-Sepharose column chromatography. The biological activity of rec-pFSH was examined by measuring its effect on progesterone secretion from porcine granulosa cells and germinal vesicle breakdown (GVBD) of porcine oocytes. It showed adequate activity with respect to progesterone secretion, although some fractions rich in the sugar group showed lower activity than that of pituitary-derived FSH. It exhibited higher GVBD activity than that of pituitary-derived FSH at concentrations as low as 1 ng/ml. These results demonstrate that the baculovirus vector-insect cell system can provide biologically active rec-pFSH.

Major proteins of yam bean tubers

The tuberous roots of the Mexican yam bean, jicama, ( Pachyrhizus erosus L. Urban) contained large quantities of two acidic glycoproteins which accounted for more than 70% of the total soluble proteins (about 3 g per 100 g of tuber on a dry weight basis). The two major proteins, tentatively named YBG1 and YBG2, had apparent M r s of 28 000 and 26 000, respectively, by SDS-PAGE. A third protein named YBP22 which accounted for 2–5% of the total soluble proteins had an M r of 22 000. YBG1 and YBG2 exhibited great similarity on the basis of their amino acid composition and had identical N-terminal amino acid sequences. The first 23 amino acids in the N-terminal region of YBG2 were DDLPDYVDWRDYGAVTRIKNQGQ which showed strong homology with the papain class of cysteine proteases. YBG1 and YBG2 were found to bind to a Concanavalin A-Sepharose column and were also stained positively by a sensitive glycoprotein stain. Both glycoproteins exhibited cysteine proteolytic activity. In contrast, YBP22 showed sequence homology with several known protease inhibitors, and a polyclonal antibody raised against this protein cross reacted with soybean trypsin inhibitor.

Interaction of guanosine 5′-triphosphate binding protein Gq from Sepia officinalis with illuminated rhodopsin bound to concanavalin A

The heterotrimeric guanosine 5′-triphosphate (GTP)-binding protein Gq was suggested to couple the light receptor rhodopsin with the effector phospholipase C in visual cells of invertebrates. We indirectly linked Gq from Sepia officinalis to a concanavalin A-sepharose column via rhodopsin. Rhodopsin had been solubilized previously with 10 mM n-dodecyl-β-maltoside from the purified photosensory membrane under illumination. All three subunits of the Gq were released almost pure by elution with 100 μM GTP. The α and β subunits were identified by specific antipeptide antisera. The α subunit has a relative molecular mass of 46 kDa, and the β subunit of 35 kDa. The γ subunit corresponds to a 9 kDa polypeptide owing to the molecular mass, which is similar to the G γ subunit of squid. The use of specific antibodies shows that neither actin nor G-protein related to transducin were in the fractions eluted with GTP or α-methyl mannoside. We demonstrate that all three subunits of Gq were associated with rhodopsin of invertebrates. Such use of a lectin column might be useful for further investigations of the interaction of rhodopsin and Gq.

Purification and properties of human urinary β- d-mannosidase

Two forms of the lysosomal enzyme β-mannosidase were identified and purified from human urine. The purification strategy employed allowed sufficient quantities of both forms to be obtained for subunit analysis and for further characterizations. The two β-mannosidases were identified as β-mannosidase B and A, in order of their elution from an ion-exchange column. In all samples examined, the A form was predominant, and the B/A ratio was consistently 0.14. The two forms displayed the same optimum pH (i.e., 4.3) and both were retained by a Concanavalin-A Sepharose column, but showed different isoelectric points, molecular masses and subunit compositions. Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of pure β-mannosidases B and A suggest that active protein B (160 kDa) consists of three subunits, one 75 kDa and two 49 kDa subunits. Protein A is smaller and appears to be composed of three subunits of 75 kDa, 49 kDa and 37 kDa. Two forms of β-mannosidase, exhibiting a chromatographic behaviour comparable to the urinary forms, were also detected in human kidney. Nevertheless, in this tissue their relative distribution was different, the B/A ratio being 19.

Stimulation of prenylated flavanone production by mannans and acidic polysaccharides in callus culture of Sophora flavescens

The effect of polysaccharides on the production of prenylated flavanones sophoraflavanone G and lehmannin in Sophora flavescens callus culture was investigated. The production of these flavanones was stimulated up to 5 times by the addition of 2 mg/ml yeast extract. The active component was partially purified as a mannan ( c 92% of mannose and c 5% glucose) by DEAE-cellulose and Concanavalin A-Sepharose column chromatographies. Acidic polysaccharides also stimulated the production of prenylated flavanones. The effect of pectin was higher than that of pectinic acid from the same source. Other polysaccharides such as cellulose, β-1,3-glucan and chitosan did not influence the productivity.

Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis)

Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M.

Heterogeneity of the prolactin receptor in the rat mammary gland and liver during various physiological states.

This work was undertaken to determine variations in the 125I-labelled ovine prolactin (oPRL) binding in rat liver and mammary gland membranes, and to study the molecular forms of prolactin receptor in different physiological situations. Prolactin binding was determined using 125I-labelled oPRL in the 100,000 g pellet. 125I-Labelled oPRL was cross-linked to receptors in membranes from rat liver and mammary gland and subjected to SDS-PAGE under reducing conditions, followed by autoradiography of dried gels. In the mammary gland, the specific binding of oPRL to membranes, expressed as mean +/- S.E.M. fmol/mg protein increased from 1.36 +/- 0.24 on the day of dioestrus to 3.26 +/- 0.60 on the day of oestrus. It remained very low during pregnancy but increased during lactation to reach 4.78 +/- 0.99. In the liver, the specific binding of oPRL to membranes was higher than in the mammary gland on the days of dioestrus 1, dioestrus 2 and oestrus, but not on the day of pro-oestrus. It increased until day 14 of pregnancy when the specific binding of 125I-labelled oPRL was 17.01 +/- 0.30. Cross-linking revealed different molecular forms in the mammary glands and the liver. In the mammary gland we observed four prolactin-binding forms of 80, 50, 40 and 16 kDa, all of which were specific for prolactin. The 80, 50 and 40 kDa forms were also able to bind to a Concanavalin A-Sepharose column, indicating that these binding forms were glycosylated while the smaller one (16 kDa) appeared to be unglycosylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of targeting proteinase for rat alpha 1-macroglobulin in vivo. Mast-cell tryptase is a major component of the alpha 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes.

The alpha 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes was purified by a series of column chromatographic steps on concanavalin A-Sepharose, Sephacryl S-300, DEAE-cellulose and TSK gel DEAE-5PW columns. The complex contained no detectable alpha 2-macroglobulin. Studies on the substrate specificity indicated that the complex had tryptase-like activities towards various synthetic substrates, but no elastase, chymotrypsin, cathepsin-B and cathepsin-L activities. The proteinase activity was completely inhibited by di-isopropyl fluorophosphate, leupeptin and antipain, indicating that the proteinase bound to alpha 1-macroglobulin is a serine proteinase. Two protein bands (62 and 59 kDa) of the complex were labelled with [3H]diisopropyl fluorophosphate and both bands cross-reacted with anti-(mast-cell tryptase)antibody. These results suggest that mast-cell tryptase is a major targeting proteinase for alpha 1-macroglobulin in vivo. The main alpha-macroglobulin-proteinase complex in the adjuvant-treated rats was also the alpha 1-macroglobulin-tryptase complex, even though the plasma level of alpha 2-macroglobulin was elevated.

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Partial purification of the alpha-1,6-mannosyltransferase that initiates outer chain synthesis.

The alpha-1,6-mannosyltransferase (alpha-1,6-ManT) that initiates outer chain synthesis in Saccharomyces cerevisiae was partially purified along with an alpha-1,2-mannosyltransferase (alpha-1,2-ManT) that acts on alpha-methylmannoside. The enzymes were solubilized by extracting a 145,000 g pellet of S.cerevisiae mnn1 mutant with 1% Triton X-100. The extract was then passed through a concanavalin A-Sepharose column and the bound material was eluted with alpha-methylmannoside. After exhaustive dialysis, the fractions containing both mannosyltransferase activities were chromatographed on DEAE-Trisacryl which removed approximately 90% of the alpha-1,2-ManT. The fractions containing alpha-1,6-ManT and residual alpha-1,2-ManT were further purified by sequential chromatography on Sephacryl S-200 and CM-Trisacryl. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of individual fractions eluted from Sephacryl S-200 and from CM-Trisacryl, followed by silver staining of the gels, showed two major bands whose intensity corresponded to the enzyme activities. A protein band of approximately 62 kDa corresponded to the alpha-1,6-ManT and another band of approximately 66 kDa, which was eluted from the Sephacryl S-200 column slightly earlier, corresponded to the alpha-1,2-ManT.