scholarly journals A monoclonal antibody to bovine brain inositol monophosphatase. Immunoaffinity purification of the brain and kidney enzymes and evidence for their structural identity

1989 ◽  
Vol 264 (3) ◽  
pp. 793-798 ◽  
Author(s):  
N S Gee ◽  
S Howell ◽  
G Ryan ◽  
C I Ragan
Keyword(s):  
Monoclonal Antibody ◽  
Specific Activity ◽  
Binding Capacity ◽  
Bovine Brain ◽  
High Yield ◽  
Brain Extract ◽  
Kidney Cortex ◽  
Inositol Monophosphatase ◽  
Hydrolysis Of ◽  
The Brain

A monoclonal IgG2b(K) antibody, G-2A4, has been generated against bovine brain myo-inositol monophosphatase (EC 3.1.3.25). The identity of the antigen recognized by the antibody was established by using e.l.i.s.a. and Western blotting procedures, and by immunoprecipitation of enzyme activity from crude brain supernatant. In addition, the hydrolysis of Ins1P by crude brain extract was inhibited by up to 83% by the pure antibody. Under identical conditions, the hydrolysis of Ins(1,4)P2 was unaffected. An immunoadsorbent column containing monoclonal antibody G-2A4 covalently attached to CNBr-activated Sepharose 4B has been used for rapid purification of the brain enzyme. Elution conditions have been optimized to allow isolation of the enzyme in high yield (54%) with full retention of column-binding capacity. The enzyme was electrophoretically homogeneous, Mr 30,000 and of higher specific activity than that purified conventionally. Chromatography of the pure enzyme on high resolution ion-exchange columns revealed some charge heterogeneity, possibly indicative of some type of post-translational modification. The immunoadsorbent column has also been used to purify the bovine kidney cortex enzyme to homogeneity. Partial proteolytic fragmentation patterns of the brain and kidney enzymes using endoprotease glu-C were identical, suggesting that they are almost certainly products of the same gene.

THE HYDROLYSIS OF MONOPHOSPHOINOSITIDE BY EXTRACTS OF BRAIN

1967 ◽  
Vol 45 (6) ◽  
pp. 853-861 ◽  
Author(s):  
W. Thompson
Keyword(s):  
Fatty Acids ◽  
Enzyme Activity ◽  
Calcium Ions ◽  
Brain Extract ◽  
Monovalent Cations ◽  
High Concentration ◽  
Diffusible Factor ◽  
Hydrolysis Of ◽  
The Brain ◽  
Long Chain

The hydrolysis of monophosphoinositide by soluble extracts from rat brain is described. Diglyceride and inositol monophosphate are liberated along with a small amount of free fatty acids. Hydrolysis of the lipid is optimal at pH 5.4 in acetate buffer. The reaction is stimulated by calcium ions or by high concentration of monovalent cations and, to a less extent, by long-chain cationic amphipathic compounds. Enzyme activity is lost on dialysis of the brain extract and can be restored by diffusible factor(s). Some differences in the conditions for hydrolysis of mono- and tri-phosphoinositides are noted.

scholarly journals Tropomyosin-like properties of clathrin light chains allow a rapid, high-yield purification.

1983 ◽  
Vol 96 (3) ◽  
pp. 911-914 ◽  
Author(s):  
F M Brodsky ◽  
N J Holmes ◽  
P Parham
Keyword(s):  
Monoclonal Antibody ◽  
High Pressure ◽  
Liquid Chromatography ◽  
High Pressure Liquid Chromatography ◽  
Bovine Brain ◽  
Size Exclusion ◽  
High Yield ◽  
Light Chains ◽  
Heat Denaturation ◽  
Immunoaffinity Column

The light chains (LCa and LCb) of bovine brain clathrin are resistant to heat denaturation by boiling, a property shared by tropomyosin (Bailey, K., 1948, Biochem. J., 43:271-281). Light chains were partially purified by boiling and centrifugation of a Tris-extract of crude membranes prepared from bovine brains (Keen, J. H., M. C. Willingham, and I. H. Pastan, 1979, Cell., 16:303-312). Contaminant polypeptides were then removed by size-exclusion high-pressure liquid chromatography. The purified light chains were separated from each other by using an immunoaffinity column prepared from a monoclonal antibody CVC.7 specific for LCa and not LCb.

scholarly journals Purification and properties of myo-inositol-1-phosphatase from bovine brain

1988 ◽  
Vol 253 (2) ◽  
pp. 387-394 ◽  
Author(s):  
P V Attwood ◽  
J B Ducep ◽  
M C Chanal
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bovine Brain ◽  
Native Enzyme ◽  
Hydroxyl Groups ◽  
Inositol 1 ◽  
Substrate Structure ◽  
Purification And Properties ◽  
Phosphorylated Compounds ◽  
Hydrolysis Of

myo-Inositol-1-phosphatase from bovine brain was purified over 2000-fold. The native enzyme has a Mr of 59,000, and on SDS/polyacrylamide-gel electrophoresis the subunit Mr was 31,000. Thus the native enzyme is a dimer of two apparently identical subunits. The enzyme, purified to a specific activity of more than 300 units/mg of protein (1 unit of enzyme activity corresponds to the release of 1 mumol of Pi/h at 37 degrees C), catalysed the hydrolysis of a variety of phosphorylated compounds, the best one, in terms of V/Km, being D-myo-inositol 1-phosphate. Kinetic constants of compounds tested, including both isomers of glycerophosphate and two deoxy forms of beta-glycerophosphate, were measured. They show the importance of the two hydroxyl groups which are adjacent to the phosphate in myo-inositol 1-phosphate. With a wide variety of substrates Li+ was found to be an uncompetitive inhibitor whose Ki varied with substrate structure.

scholarly journals Limited proteolysis and ‘in vitro’ mutagenesis of bovine brain inositol monophosphatase identifies an N-terminal region important for activity

1990 ◽  
Vol 272 (2) ◽  
pp. 465-468 ◽  
Author(s):  
P Whiting ◽  
N S Gee ◽  
J Potter ◽  
S Howell ◽  
C I Ragan
Keyword(s):  
Monoclonal Antibody ◽  
Cleavage Site ◽  
Limited Proteolysis ◽  
Bovine Brain ◽  
Terminal Region ◽  
In Vitro Mutagenesis ◽  
Inositol Monophosphatase ◽  
Prolonged Incubation ◽  
High Concentrations

Bovine brain inositol monophosphatase is rapidly cleaved by endoprotease lys-C at a single site in the absence of SDS. Further sites are revealed only after prolonged incubation with high concentrations of protease. The initial cleavage occurs near one end of the enzyme, generating an N-terminally-derived 36-residue peptide, which is blocked, and a large 28 kDa fragment bearing a free N-terminus. The start sequence of this fragment was found to be Xaa-Ser-Pro-Ala-Asp-Leu-Val, consistent with the cDNA sequence, and Lys-36-Ser-37 was identified as the cleavage site. The activity of the cleaved enzyme was markedly decreased to 3% of that of the native enzyme, although its dimeric structure was preserved. The 36-residue peptide was not covalently associated with the large fragment after proteolytic cleavage, although the possibility of non-covalent association could not be excluded. Finally, the epitope for the inhibitory monoclonal antibody G-2A4 [Gee, Howell, Ryan & Ragan (1989) Biochem J. 264. 793-798] was found to lie proximal to the endoprotease lys-C cleavage site. In vitro mutagenesis further mapped the epitope for monoclonal antibody G-2A4 to residues around Cys-8 of the enzyme. These results suggest that the N-terminal region of the enzyme is important for activity.

The entry into the brain of large molecules derived from dietary protein

1978 ◽  
Vol 200 (1139) ◽  
pp. 175-192 ◽  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Immune Serum ◽  
The Body ◽  
Brain Extract ◽  
Blood Contamination ◽  
Adult Rats ◽  
Large Molecules ◽  
Derivatives Of ◽  
The Brain

When bovine IgG, haemoglobin, or α-gliadin extracted from wheat, labelled with radio-iodine, are fed by stomach tube to suckling or adult rats, significant amounts of protein-bound radioactivity are found in extracts of whole brain. These amounts are greatly in excess of what can be accounted for by blood contamination: the specific activity of the brain is of the order of that of other tissues of the body, suggesting that it is permeated with protein derivatives of the fed material. This is borne out by differential centrifugation of brain macerates which shows sub­stantial protein-bound radioactivity in the cell sap fraction. Ultracentrifugation studies reveal the presence of high molecular mass material in the cell sap, some of greater sedimentation velocity than the original protein fed, suggesting complexing of derivatives of this with native components of the brain. This is confirmed by gel filtration studies. Immunological studies on the brain extract show that a high proportion of the protein-bound radioactivity of the brain retains the ability to be precipitated by specific immune serum, when α-gliadin or bovine IgG has been fed.

scholarly journals Purification of endopeptidase-24.11 (‘enkephalinase’) from pig brain by immunoadsorbent chromatography

1983 ◽  
Vol 215 (3) ◽  
pp. 519-523 ◽  
Author(s):  
J M Relton ◽  
N S Gee ◽  
R Matsas ◽  
A J Turner ◽  
A J Kenny
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Deae Cellulose ◽  
Single Step ◽  
Endopeptidase 24.11 ◽  
Pig Brain ◽  
Endopeptidase Activity ◽  
Hydrolysis Of ◽  
The Brain ◽  
Subunit Size

Membrane preparations from striatum of pig brain contain endopeptidase activity towards iodoinsulin B-chain. Only 50% of the hydrolysis of insulin B-chain is inhibitable by phosphoramidon, and DEAE-cellulose chromatography can resolve the phosphoramidon-sensitive and -insensitive activities. The former activity (now designated ‘endopeptidase-24.11’) is responsible for hydrolysis of [D-Ala2,Leu5]enkephalin and is identical with an enzyme in brain that has previously been referred to as ‘enkephalinase’. Pig striatal endopeptidase-24.11 has now been purified to homogeneity in a single step by immunoadsorbent chromatography using a monoclonal antibody. The overall purification was 23 000-fold, with a yield of 30%. The brain enzyme appears to be identical with kidney endopeptidase-24.11 in amino acid composition as well as by immunological and kinetic criteria. However, it differs slightly in apparent subunit size (Mr = 87 000), attributable to differences in glycosylation.

scholarly journals Enzymatic depolymerization of wheat straw polysaccharides

2021 ◽  
Vol 939 (1) ◽  
pp. 012005
Author(s):  
Zh Makhatov ◽  
N Alibayev ◽  
Z Konarbayeva ◽  
B Makhatov ◽  
A Makhatova ◽  
...  
Keyword(s):  
Wheat Straw ◽  
Specific Activity ◽  
Maximum Yield ◽  
High Yield ◽  
Cellulolytic Enzymes ◽  
Cultivation Conditions ◽  
Glucose Yield ◽  
Complex Preparation ◽  
Hydrolysis Of ◽  
Enzymatic Depolymerization

Abstract The purpose of this study is to develop a technology for enzymatic processing for depolymerization of polysaccharides in wheat straw to obtain the maximum yield of glucose and sorbitol. Cellulolytic enzymes endo-1,4-β-glucanase (EC 3.2.1.4) and cellobiose (1,3-β-glucosidase) (CF 3.2.1.21) were isolated and studied in local strains Tr. viride 121, which are grown under deep cultivation conditions. A technology has been developed for obtaining a complex preparation “Cellozyme G20x” with a high yield and specific activity of cellulase, xylanase, β-glucanase and pectinase, and a scheme for purification from cellulases by precipitation, ultrafiltration, and freeze drying is not inferior in efficiency to commercial preparations. The physicochemical properties of the preparation “Cellozyme G20x” have been studied, the optimal parameters of the action and stability of the enzyme preparation have been established. The efficiency of Cellozyme G20x for hydrolysis of straw polysaccharides was 35-40% in terms of glucose yield.

scholarly journals The purification and properties of myo-inositol monophosphatase from bovine brain

1988 ◽  
Vol 249 (3) ◽  
pp. 883-889 ◽  
Author(s):  
N S Gee ◽  
C I Ragan ◽  
K J Watling ◽  
S Aspley ◽  
R G Jackson ◽  
...  
Keyword(s):  
Bovine Brain ◽  
Small Extent ◽  
Inositol Monophosphatase ◽  
Inositol 1 ◽  
Bivalent Cations ◽  
Purification And Properties ◽  
Hydrolysis Of

1. An inositol monophosphatase was purified to homogeneity from bovine brain. 2. The enzyme is a dimer of subunit Mr 29,000. 3. The enzyme hydrolyses both enantiomers of myo-inositol 1-phosphate and both enantiomers of myo-inositol 4-phosphate, but has no activity towards inositol bisphosphates, inositol trisphosphates or inositol 1,3,4,5-tetrakisphosphate. 4. Several non-inositol-containing monophosphates are also substrates. 5. The enzyme requires Mg2+ for activity, and Zn2+ supports activity to a small extent. 6. Other bivalent cations (including Zn2+) are inhibitors, competitive with Mg2+. 7. Phosphate, but not inositol, is an inhibitor competitive with substrate. 8. Li+ inhibits hydrolysis of inositol 1-phosphate and inositol 4-phosphate uncompetitively with different apparent Ki values (1.0 mM and 0.26 mM respectively).

scholarly journals Purification and some properties of arylsulphatases A and B from rabbit kidney cortex

1977 ◽  
Vol 165 (1) ◽  
pp. 127-134 ◽  
Author(s):  
J J Helwig ◽  
A A Farooqui ◽  
C Bollack ◽  
P Mandel
Keyword(s):  
Ascorbic Acid ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Chromatography Method ◽  
Arylsulphatase A ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N–acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate.

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