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The GDP-Mannose:dolichyl-PP-tetrasaccharide Mannosyltransferase (MTaseIII), which catalyzes the transfer of mannose from GDP-mannose to the Man2(GlcNac)2-PP-Dolichol acceptor glycolipidwas solubilized from bovine mammary gland microsomes.The purification protocol involved the following sequence of steps: ammonium sulfate precipitation, adsorption chromatography on hydroxylapatite column,glycerol linear gradient centrifugation, hydrophobic chromatography on Phenyl- Sepharose affinity matrix.

The main objective of the study are :
1. To study the tissue localization and tissue – specificity of the mannosyltransferase in mammals.
2. To study intracellular distribution of the enzyme.
3. To study acceptor substrate specificity using polyprenols of various carbon chain length and terminal saturation and unsaturation.
4. To optimize the soubilization of the membrane bound enzymes and partially purify it.
5. To determine the properties and kinetic parameters of the enzyme catalyzed reaction.
6. To study the metal ion requirements.

The precursor lipid linked oligosaccharide is assembled in the endoplasmic reticulum ,by the sequential addition of monosaccharide to the lipid carrier dolichyl phosphate catalysed by various glycosyltransferase (5-9). It is now well established that the first seven sugar residues (two N-acetylglucosaminyl and five maanosyl) are derived from the corresponding sugar nucleotides(UDP-GlcNacand GDP-Mannose), while the next four mannosyl (Man5-9) and three terminal glycosyl residues come from dolichylphosphomannose (Dol-P-Man) and dolichylphosphoglucose(Dol-P-Glc), respectively. Although the complete sequence of reaction and structures of all lipid linked oligosaccharides intermediate of n –glycosylation pathway, popularly known as dolichol cycle have been worked out (1), the regulation and enzymology of this pathway still remains as the unsolved challenging problem. This report describes the purification of the mannosyltransferase that catalyzes the transfer of mannose to Man2 GlcNAc2-PP-dolichol from GDP-Mannose(MTaseIII) from bovine mammary gland microsomes.

Materials: GDP-Mannose,UDP –GlcNAc,dolichyl phosphate(Dol-P),β mercaptoethanol, Nonident P-40,dithiothreitol ,hydroxylapatite, DEAE-cellulose (DE-52) ,Phenyl Sepharose ,Affinity matrix GDP-Hexanolamine-Sepharose. The lactating bovine mammary gland tissue was obtained from local slaughter house.

Preparation of the particulate Enzymes from Bovine mammary Gland: All operations were performed at 0^о-40 ^оC. The lactating swine mammary gland tissue (500gm) was used to prepare the crude microsomal pellet as described (8).  The membrane pellet was resuspended in 50 mm Tris HCL and,pH7.2,containing 20% glycerol,2 mMEDTA and 0.04% β-mercaptoethanol (BufferA)to obtain a protein concentration of approximately 10 mg/ml.This membrane suspension was used for the enzyme purification studied and referred to as crude microsomal fraction.
Purification of Mannosyltransferase
Step1. Preparation of Bovine Mammary Gland Microsomal Detergent Extract:
To 25 ml  of microsomal fraction (225mg protein) was added dropwise with gentle stirring,5 ml of 2%(w/v)of Nonident P-40,so that the detergent concentration was 0.5%and protein detergent ratio wads 3:2.After incubation on ice for 15-20 minutes the lysate was diluted with an equal volume of dilution buffer (50mM Tris-HCL,pH 7.2,containing 20%  glycerol and 0.04% β mercaptoethanol,Buffer B)and the insoluble material was removed by centrifugation at26000xg for 60 min. The clear supernatant liquid ,which contained the solubilized mannosyltransferase ,was carefully removed with the help of Pasteur pipette. This enzyme fraction was fully active for about one monthwhen stored at-70C or about a week at 0C.
Step 2 :Ammonium sulfate precipitation:
An appropriate amount of ammonium sulfate was slowly added to the detergent solubilized enzyme with continuous gentle stirring to 35 % saturation. The mixture was allowed to stand for 1 hour on ice before removing the insoluble residues by centrifugation at 100,000xg for 30 minute and the precipitate so obtained was dissolved with minimum volume of suspension buffer (25mM sodium phosphate buffer,pH 6.85,containing 20% glycerol,0.1%NP-40% β-mercaptoethanol,Buffer C).
Step 3. Hydroxylapatite Column Chromatography: The Ammonium sulfate fraction was dialyzed 6h against 250 volumes of the equilibration buffer (10mM sodium phosphate buffer,pH6.85, containing 12% glycerol, 0.1%NP-40% and 0.04%   β-mercaptoethanol; Buffer D) used to equilibrate the hydroxylapatite column.Materials:GDP-Mannose,UDP –GlcNAc,dolichyl phosphate(Dol-P),β mercaptoethanol, Nonident P-40,dithiothreitol ,hydroxylapatite, DEAE-cellulose (DE-52) ,Phenyl Sepharose ,Affinity matrix GDP-Hexanolamine-Sepharose. The lactating bovine mammary gland tissue was obtained from local slaughter house.
Preparation of the particulate Enzymes from Bovine mammary Gland: All operations were performed at 0^о-40 ^оC. The lactating swine mammary gland tissue (500gm) was used to prepare the crude microsomal pellet as described (8).  The membrane pellet was resuspended in 50 mm Tris HCL and,pH7.2,containing 20% glycerol,2 mMEDTA and 0.04% β-mercaptoethanol (BufferA)to obtain a protein concentration of approximately 10 mg/ml.This membrane suspension was used for the enzyme purification studied and referred to as crude microsomal fraction.
Purification of Mannosyltransferase
Step1. Preparation of Bovine Mammary Gland Microsomal Detergent Extract:
To 25 ml  of microsomal fraction (225mg protein) was added dropwise with gentle stirring,5 ml of 2%(w/v)of Nonident P-40,so that the detergent concentration was 0.5%and protein detergent ratio wads 3:2.After incubation on ice for 15-20 minutes the lysate was diluted with an equal volume of dilution buffer (50mM Tris-HCL,pH 7.2,containing 20%  glycerol and 0.04% β mercaptoethanol,Buffer B)and the insoluble material was removed by centrifugation at26000xg for 60 min. The clear supernatant liquid ,which contained the solubilized mannosyltransferase ,was carefully removed with the help of Pasteur pipette. This enzyme fraction was fully active for about one monthwhen stored at-70C or about a week at 0C.
Step 2 :Ammonium sulfate precipitation:
An appropriate amount of ammonium sulfate was slowly added to the detergent solubilized enzyme with continuous gentle stirring to 35 % saturation. The mixture was allowed to stand for 1 hour on ice before removing the insoluble residues by centrifugation at 100,000xg for 30 minute and the precipitate so obtained was dissolved with minimum volume of suspension buffer (25mM sodium phosphate buffer,pH 6.85,containing 20% glycerol,0.1%NP-40% β-mercaptoethanol,Buffer C).
Step 3. Hydroxylapatite Column Chromatography: The Ammonium sulfate fraction was dialyzed 6h against 250 volumes of the equilibration buffer (10mM sodium phosphate buffer,pH6.85, containing 12% glycerol, 0.1%NP-40% and 0.04%   β-mercaptoethanol; Buffer D) used to equilibrate the hydroxylapatite column.The dialyzed protein fraction (24 mg) was then loaded on the hydroxylapaptite column (1.5x1.2 cm) at the rate of 0.1 ml/min.The column was then washed with three column volumes of equilibration buffer (Buffer D ) and the adsorbed proteins were eluted with a linear gradient (10-400mM)of sodium phosphate buffer containing 12% glycerol ,0.1%NP-40 and 0.04%,βmercaptoethanol ,at a flow rate of 0.5ml/min.1.5ml fractions were collected and an aliquot (0.05ml) from every other tube was assayed for the mannosyltransferase activity and the protein content. Only peak fractions containing majorityof mannosyltransferase activity was pooled and used for further purification of the enzyme.

Step 4. Glycerol Density Gradient Centrifugation: The pooled fraction corresponding to the enzyme peak from the hydroxylapatite column (800µl) were loaded on top of 12-30% linear glycerol gradient in 9 ml of 170mM sodium phosphate buffer ,pH6.85,containing 12%glycerol, 0.1%NP-40 and 0.04% β mercaptoethanol (BufferE) with 1 ml cushion of 50%glycerol (v/v) in the same buffer and centrifuged in SW41-Tirotor at 37,000 rpm for 17.5h.After centrifugation ,25 fractions of 430µl each were collected starting from the bottom of the gradient and aliquot of 0.05ml from each tube was assayed for the enzyme activity and protein content.Peak fractions from six gradient tubes showing the maximum enzyme activity were pooled,concentrated to 2 ml by Centricon using Amicon membrane  filter 10,and immediately used for further purification.

Step5.Phenyl Sepharose Chromatography:  The peak fraction containing the mannosyltransferase activity from the previous step was bought to 0.2M ammonium sulfate concentration by the addition of appropriate amount of solid ammonium sulfate and applied to a 0.5x7cm column of Phenyl Sepharose pre-equillibrated with 0.2 M ammonium sulfate in Buffer E.The column was washed with 25ml of the equilibration solution followed by 64 ml of Buffer C,without ammonium sulfate.Fractions (1.5ml) were collected at a flow rate of 0.15 ml/min and an aliquot of 0.05µl from each fraction was assayed for the enzyme activity and the protein. The activity fraction were pooled and immediately used for the next step of purification.

Step6. GDP-Hexolamine –Sepharose Affinity Chromatography: The enzyme fraction from the Phenyl-Sepharose column was added to 1ml of GDP-Hexanolamine-Sepharose affinity matrix, previously equilibrated with Buffer E, in a small Pasteur pipette and gently mixed in a rotary disc.The column was then washed with 16ml of the equilibration buffer followed by batch elution with 10mM GDP-mannose in the equilibration buffer. One ml fraction was collected and aliquot (0.05ml) from each fraction was used for further measurement of enzyme activity and the protein content.

The mannosyltransferase,which catalyzes the transfer of the third mannose residue to Man2GlcNAc2-PP-Dolichol from GDP-Mannose, referred to as MTase III,was purified from microsomes of lactating swine mammary gland using the purification scheme described under “Experimental Procedures”. The purification data are summarized in the shown table. MTase III was efficiently solubilized from the microsomal membrane fraction releasing about 90% of the total initial activity with 2.5- fold purification. The ammonium sulfate precipitation at 35% saturation resulted in further purification by 1.7 –fold. At this stage the overall yield and purification was 59% and 4.2 fold, respectively.

The next step of adsorption chromatography on hydroxylapatite column showed that 70% of the total activity was eluted under major peak at 170mM gradient of sodium phosphate. After this step, purification increased 15 –fold, giving an overall purification of 60-fold with 51% yield. An overall purification of 143 fold with a yield of 6.8%was achieved .the chromatography of phenyl sepharose column purified the enzyme by 940-fold.The last step in the purification scheme was the affinity chromatography on GDP-hexanolamine-Sepharose column, which also removed the inactive protein slightly improved the specific activity .The total purification scheme,as summarized in the table resulted in 950-fold purification of MTase III with final yield of about 1%. The characterization of the enzyme is still to be done, hence the search must continue.

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