scholarly journals A novel metalloproteinase originally isolated from brain myelin membranes is present in many tissues

1990 ◽  
Vol 268 (1) ◽  
pp. 245-248 ◽  
Author(s):  
A Chantry ◽  
P Glynn
Keyword(s):  
Monoclonal Antibody ◽  
Bovine Brain ◽  
Enzymic Activity ◽  
Immunoreactive Band

A monoclonal antibody, CG4, was raised to a novel 60 kDa metalloproteinase purified from a bovine brain myelin glycoprotein fraction. Glycoproteins extracted from both myelin and nine different bovine tissues showed the 60 kDa CG4-immunoreactive band by immunoblotting in amounts that broadly paralleled enzymic activity of this metalloproteinase and varied relatively little among the tissues.

G-proteins in mammalian gametes: an immunocytochemical study

1988 ◽  
Vol 91 (1) ◽  
pp. 21-31
Author(s):  
N.B. Garty ◽  
D. Galiani ◽  
A. Aharonheim ◽  
Y.K. Ho ◽  
D.M. Phillips ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Cumulus Cells ◽  
Bovine Brain ◽  
Regulatory Proteins ◽  
Immunocytochemical Study ◽  
Cell Cytoplasm ◽  
Sperm Tail ◽  
Immunoreactive Material

The presence of transductory GTP(G)-regulatory proteins in mammalian gametes has been examined by indirect fluorescence immunocytochemistry. Using rabbit antisera to bovine rod beta gamma-transducin (RA beta gamma T), bovine rod holotransducin (AS-1), bovine rod alpha-transducin (RA alpha T), synthetic bovine rod alpha-transducin C-terminal decapeptide (AS-6), bovine brain alpha 39Go (RA alpha 39), and two mouse monoclonal antibodies raised against frog retinal transducin (4A), and rat brain beta-tubulin, we demonstrated the presence of corresponding immunoreactive material in both rat oocytes and bovine ejaculated sperm. Immunostaining in the oocyte was evenly distributed on the oolemma, excluding the cell cytoplasm and zona pellucida. Immunoreactive material was also present in the cumulus cells that encapsulate the oocyte. In contrast, the immunofluorescence corresponding to transductory G-proteins was confined in sperm to functionally defined regions in the head and tail, in a manner specific for each antibody. While RA beta gamma T, AS-1 and RA alpha 39 all stained the entire acrosome, AS-6 and RA alpha T stained only the acrosomal tip. Monoclonal antibody 4A stained the midpiece exclusively and anti-rat betaq-tubulin (a structural G-protein) stained the full length of the sperm tail. The existence of several G-protein types in mammalian gametes suggests their possible involvement in the regulation of various effector systems, in a manner reminiscent of somatic cells. The unique situation in sperm, where different G-proteins show distinct and specific patterns of distribution, further suggests their association with various effector systems in discrete functional domains.

A novel component of the axonal cortical cytoskeleton, A60, defined by a monoclonal antibody

1989 ◽  
Vol 94 (3) ◽  
pp. 489-500
Author(s):  
D.A. Rayner ◽  
A.J. Baines
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filament ◽  
Subcellular Fractionation ◽  
Bovine Brain ◽  
Cytoskeletal Proteins ◽  
Intermediate Filament Protein ◽  
Brain Membranes ◽  
Associated Proteins ◽  
The Central Nervous System ◽  
Cortical Cytoskeleton

A Mr 60,000 protein of the axonal cortical cytoplasm, which is recognized by a novel monoclonal antibody, is described. The antibody, DR1, was produced by immunizing mice with a soluble extract of bovine brain membranes that is enriched in known membrane cytoskeletal proteins. DR1 recognizes a Mr 60,000 protein in this extract. Immunofluorescence and subcellular fractionation reveal that the protein is primarily located in axons, where it appears to form a thick lining to the axolemma. Operationally, this Mr 60,000 protein is defined as a cytoskeleton-associated peripheral membrane protein. It is solubilized from brain membranes only under harsh conditions (0.1 M-NaOH), but not with KI (0.8 M) or Triton X-100 (1%). It is present at higher levels in the central nervous system than in peripheral nerves that have been examined. The Mr 60,000 protein copurifies with neurofilaments through cycles of assembly and disassembly. It does not appear to react with the anti-IFA antibody, suggesting that it is not a member of the intermediate filament class of proteins. This Mr 60,000 protein, which we designate A60, is distinct from other known neurofilament-associated proteins, including the Mr 60,000 protein alpha-internexin and the Mr 58,000 intermediate filament protein peripherin. A60 is suggested as being a previously unrecognized component of the axonal cortical cytoskeleton.

scholarly journals A monoclonal antibody distinguishes two types of phosphatidylinositol 4-kinase

1991 ◽  
Vol 273 (1) ◽  
pp. 63-66 ◽  
Author(s):  
G C Endemann ◽  
A Graziani ◽  
L C Cantley
Keyword(s):  
Monoclonal Antibody ◽  
Rat Brain ◽  
Rat Liver ◽  
Bovine Brain ◽  
Human Erythrocytes ◽  
Gene Products ◽  
Type Ii ◽  
Type Iii ◽  
Distinct Gene ◽  
Phosphatidylinositol 4

A monoclonal antibody has been developed against the type II PtdIns 4-kinase from bovine brain. This antibody, 4C5G, causes greater than 90% inhibition of the type II PtdIns 4-kinase from bovine brain, rat brain and human erythrocytes. However, it fails to inhibit type III PtdIns 4-kinase from bovine brain or PtdIns 3-kinase from rat liver. These results suggest that type II and type III PtdIns 4-kinases are distinct gene products, and that 4C5G will be useful in studying the function of the type II PtdIns 4-kinase.

scholarly journals An aromatic, but not a basic, residue is involved in the toxicity of group-II phospholipase A2 neurotoxins

1999 ◽  
Vol 341 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Jože PUNGERĆAR ◽  
Igor KRIAJ ◽  
Ning-Sheng LIANG ◽  
Franc GUBENŠEK
Keyword(s):  
Bovine Brain ◽  
Enzymic Activity ◽  
Aliphatic Chain ◽  
Wild Type ◽  
Basic Residue ◽  
Enzyme Active Site ◽  
Single Mutant ◽  
Adsorption Surface ◽  
Interfacial Adsorption ◽  
Triton X

Ammodytoxins (Atxs) A, B and C are basic phospholipase A2s from Vipera ammodytes ammodytes snake venom, and they exhibit presynaptic toxicity. The most toxic is AtxA, followed by AtxC, its naturally occurring F124 → I/K128 → E mutant, which is 17 times less toxic. Two mutants of AtxA have been produced in bacteria and characterized. The specific enzymic activity of the K128 → E mutant on mixed phosphatidylcholine/Triton X-100 micelles is similar to that of the wild type. The K108 → N/K111 → N mutant, however, possesses 160% of the wild-type activity. Replacement of the two basic residues by uncharged, polar residues on the opposite side of the protein to the enzyme active site and interfacial adsorption surface results in increased enzymic activity at the water/lipid aggregate interface, due to a redistribution of electrostatic charge. The binding affinity of the double mutant for the specific acceptor in bovine brain was similar to that of AtxA, whereas the affinity of the single mutant was similar to that of AtxC, which was slightly weaker than that of AtxA. Interestingly, the substitution of any of these three basic surface residues did not significantly change the lethal potency of AtxA. Since the single mutant AtxA(K128 → E) is equivalent to the AtxC(I124 → F) mutant, this indicates that the residue at position 124 is important for presynaptic toxicity of Atxs. The more than 10-fold lower toxicity of AtxC, compared with AtxA, is a consequence of the substitution of Phe-124 (aromatic ring) with Ile (aliphatic chain). Exposed aromatic residues in the C-terminal region may also be important for the neurotoxicity of other similar toxins.

scholarly journals Purification of aromatic l-amino acid decarboxylase from bovine brain with a monoclonal antibody

1988 ◽  
Vol 252 (2) ◽  
pp. 331-335 ◽  
Author(s):  
I Nishigaki ◽  
H Ichinose ◽  
K Tamai ◽  
T Nagatsu
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Adrenal Medulla ◽  
Ph Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Bovine Brain ◽  
Acid Decarboxylase ◽  
Amino Acid Decarboxylase ◽  
Bovine Adrenal Medulla

Aromatic L-amino acid decarboxylase was purified from bovine brain for the first time by affinity chromatography using a monoclonal antibody to the enzyme, and it was compared with the decarboxylase purified from bovine adrenal medulla by the same procedure. The monoclonal antibody was produced from a hybridoma established for the enzyme highly purified from bovine adrenal medulla. The Mr values of brain and adrenal-medulla enzyme were both estimated to be approx. 100,000 by gel-permeation chromatography. SDS/polyacrylamide-gel electrophoresis revealed a single band with an apparent Mr of 50,000. Western immunoblot analysis showed that the antibody recognized each enzyme. With regard to substrate specificity, pH-dependence and effect of pyridoxal 5′-phosphate as a cofactor, both enzymes were similar.

scholarly journals Human monoclonal antibody recognizing liver-type aldolase B

1986 ◽  
Vol 240 (3) ◽  
pp. 847-856 ◽  
Author(s):  
N Hibi ◽  
S Arii ◽  
T Iizumi ◽  
T Nemoto ◽  
T M Chu
Keyword(s):  
Monoclonal Antibody ◽  
Human Monoclonal Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tetrameric Structure ◽  
Liver And Kidney ◽  
Aldolase B ◽  
Antigen Activity

A human hybridoma clone (4E3) has been established by fusing lymphocytes from a lymph node taken from a breast cancer patient and human lymphoblastoid cells, LICR-LON-HMy2, by the poly(ethylene glycol) method. 4E3 has been stabilized and continued to secrete IgMk antibody into culture medium (greater than 10 micrograms/ml) for over 1 year. The following characteristics of the antigen strongly suggested that 4E3 recognizes liver-type aldolase B (EC 4.1.2.13): the Mr of the native molecule is 160,000 and that of the subunit is 40,000, and thus it has a tetrameric structure of identical subunits; the antigen is abundant in the liver and kidney of human, mouse and rabbit, and is localized by immunohistochemical methods in the cytoplasm of hepatocytes and in the proximal tubules of the kidney; the antigen is precipitable by 50-80% saturation with (NH4)2SO4; the antigen shows charge-dependent heterogeneity on DEAE-cellulose chromatography. To confirm this notion, aldolase B was purified to homogeneity from the liver of human, mouse and rabbit by phosphocellulose chromatography. During the chromatographic purification, the antigen activity as assayed by enzyme-linked immunosorbent assay (e.l.i.s.a.) was superimposed on the enzymic activity of aldolase. Furthermore, monoclonal antibody 4E3 strongly reacted with purified aldolase B in SDS/polyacrylamide-gel electrophoresis followed by Western blotting and also in e.l.i.s.a. using microplates coated with purified enzyme. The reaction between aldolase B and 4E3 activated the human complement system as assessed by the attachment of C3 to the immune complex of aldolase B and 4E3.

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 Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate.

1987 ◽  
Vol 105 (6) ◽  
pp. 2551-2558 ◽  
Author(s):  
G H Frost ◽  
W C Thompson ◽  
D H Carney
Keyword(s):  
Monoclonal Antibody ◽  
Cell Proliferation ◽  
Dna Synthesis ◽  
Human Fibroblasts ◽  
Enzymic Activity ◽  
Thrombin Receptor ◽  
High Affinity ◽  
Thrombin Receptors ◽  
Affinity Interaction

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I-thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.

scholarly journals Radioimmunoassay of Human Pancreatic Amylase with Monoclonal Antibody

1989 ◽  
Vol 26 (5) ◽  
pp. 416-421 ◽  
Author(s):  
Chiyo Fujita ◽  
Chihiro Kasai ◽  
Hiromi Kosuge ◽  
Kenji Ogata ◽  
Ichiyo Oshima ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Acute Pancreatitis ◽  
Normal Subjects ◽  
Enzymic Activity ◽  
Pancreatic Amylase ◽  
Normal Range ◽  
Salivary Amylase ◽  
Serum Samples ◽  
The Mean ◽  
Mean Concentration

A monoclonal antibody (E-21) was obtained that specifically binds to human pancreatic amylase and shows negligible cross-reaction with human salivary amylase. Using this antibody a radioimmunoassay was developed for pancreatic amylase in human serum. The assay was shown to be sensitive (detectable up to 7 mg/L), reproducible, and specific for pancreatic amylase. In normal subjects, the mean concentration of serum pancreatic amylase determined by this method was 36·3 mg/L with a 95% confidence range of 16·5 to 79·2 mg/L. A good correlation was observed between the concentrations of immunoreactive pancreatic amylase (IR-PA) and enzymatic activities in 20 serum samples ( r = 0·97). The concentration of serum IR-PA was below the detectable limit in pancreatectomised patients, and was greatly increased in patients with acute pancreatitis; the latter was accompanied by parallel changes in total enzymic activity. In patients with mumps, the serum IR-PA level was within the normal range whereas the total enzymic activity was elevated.

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