scholarly journals A monoclonal antibody to kidney endopeptidase-24.11. Its application in immunoadsorbent purification of the enzyme and immunofluorescent microscopy of kidney and intestine

1983 ◽  
Vol 214 (2) ◽  
pp. 377-386 ◽  
Author(s):  
N S Gee ◽  
R Matsas ◽  
A J Kenny
Keyword(s):  
Monoclonal Antibody ◽  
Fluorescence Microscopy ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Specific Activity ◽  
Cell Types ◽  
Kidney Tubules ◽  
Pig Kidney ◽  
Endopeptidase 24.11 ◽  
Endopeptidase Activity

Hybridoma methodology has been used to produce a monoclonal antibody, GK 7C2, that binds specifically to microvillar endopeptidase-24.11 (EC 3.4.24.11). The antibody (an immunoglobulin G) was generated by fusion of mouse plasmacytoma cells with splenocytes from a Balb/c mouse immunized with pig kidney microvillar membranes. The identity of the antigen recognized by GK 7C2 was established by immuno-precipitation from detergent-solubilized pig kidney microvilli. The protein had an apparent Mr of 90 000 and contained endopeptidase activity sensitive to phosphoramidon. The identity was confirmed by immunoadsorbent purification of endopeptidase-24.11 by a column to which GK 7C2 had been attached. The endopeptidase, purified in a yield of 40%, was electrophoretically homogeneous and of specific activity comparable with that purified by other means. Fluorescence microscopy established that GK 7C2 bound specifically to the luminal membranes of kidney tubules and the intestinal mucosa. Thus endopeptidase-24.11 is located in the brush-border membranes of both cell types.

scholarly journals Studies on the phospholipases of rat intestinal mucosa

1970 ◽  
Vol 118 (2) ◽  
pp. 233-239 ◽  
Author(s):  
P. V. Subbaiah ◽  
J. Ganguly
Keyword(s):  
Fatty Acid ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Specific Activity ◽  
Sodium Deoxycholate ◽  
Phospholipase A ◽  
Phospholipase B ◽  
Ester Linkage ◽  
Hydrolysis Of ◽  
Soluble Fractions

1. Subcellular distribution and characteristics of different phospholipases of rat intestinal mucosa were studied. 2. The presence of free fatty acid was necessary for the maximal hydrolysis of lecithin (phosphatidylcholine), but there was no accumulation of lysolecithin (1 or 2-acylglycerophosphorylcholine);lysolecithin accumulated when the reaction was carried out in the presence of sodium deoxycholate and at or above pH8.0. 3. The fatty acid-activated phospholipase B as well as lysolecithinase showed optimum activity at pH6.5, whereas for the phospholipase A it was about pH8.6. 4. The bulk of the phospholipase A was present in the microsomal fraction, whereas the phospholipase B and lysolecithinase activities were distributed between the microsomal and soluble fractions of the mucosal homogenate. 5. Phospholipase A was equally distributed between the brush border and brush-border-free particulate fraction, with the brush border having highest specific activity, whereas the other two activities were distributed between the brush-border-free particulate and soluble fractions. 6. Various treatments showed marked differences between the phospholipase A and phospholipase B activities, but not between phospholipase B and lysolecithinase activities. 7. By using (β[1-14C]-oleoyl) lecithin it was shown that the mucosal phospholipase A was specific for the β-ester linkage of the lecithin molecule.

Proteins and enzymes of the brush-border membrane of mouse intestine: influence of organ culture on gel electrophoretic patterns

1982 ◽  
Vol 60 (4) ◽  
pp. 434-443 ◽  
Author(s):  
A. Berteloot ◽  
J. S. Hugon
Keyword(s):  
Membrane Proteins ◽  
Organ Culture ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Specific Activity ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Marker

Purifications of mouse intestinal brush-border membranes from control explants and scrapings of intestinal mucosa have been compared. Based on the specific activity of sucrase used as a specific marker of these membranes, higher purification factors were obtained with control explants (24.7 ± 0.9) as compared with scrapings of intestinal mucosa (14.8 ± 0.9). However, similar patterns of proteins and enzymes were obtained by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis after membrane solubilization by 2% SDS at room temperature. After 24 h of culture, higher molecular weight species of maltase–glucoamylase–isomaltase (band 4), alkaline phosphatase (bands 9–10), and trehalase (band 17) have been observed. Enzyme species appearing in the particulate fraction of culture media were, however, identical with those found at the brush-border membrane level in control explants, except for trehalase. These results are interpreted by considering the possible adsorption of serum components to brush-border membrane proteins. It thus appears that the membrane proteins and enzymes released in the media during organ culture are identical with those synthesized in the tissue in vitro or in vivo.

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 Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1679
Author(s):  
Vishnu Mohan ◽  
Jean P. Gaffney ◽  
Inna Solomonov ◽  
Maxim Levin ◽  
Mordehay Klepfish ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Drug Targets ◽  
Fas Ligand ◽  
Specific Activity ◽  
Matrix Metalloproteases ◽  
Ductal Adenocarcinoma ◽  
Enzyme Active Site ◽  
Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

scholarly journals Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 66
Author(s):  
Rashmita Pradhan ◽  
Phuong A. Ngo ◽  
Luz d. C. Martínez-Sánchez ◽  
Markus F. Neurath ◽  
Rocío López-Posadas
Keyword(s):  
Intestinal Mucosa ◽  
Rho Gtpases ◽  
Current Knowledge ◽  
Cell Types ◽  
Effector Proteins ◽  
Special Focus ◽  
Specific Cell ◽  
Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

Isolation and Characterization of a Monoclonal Antibody against Pig Kidney Sodium- and Potassium-Activated ATPase1

1985 ◽  
Vol 98 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Osamu URAYAMA ◽  
Hideaki NAGAMUNE ◽  
Makoto NAKAO ◽  
Yukichi HARA ◽  
Hiroyuki SUGIYAMA ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Pig Kidney ◽  
Isolation And Characterization ◽  
Sodium And Potassium

scholarly journals (Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit.

1989 ◽  
Vol 109 (3) ◽  
pp. 1057-1069 ◽  
Author(s):  
A Marxer ◽  
B Stieger ◽  
A Quaroni ◽  
M Kashgarian ◽  
H P Hauri
Keyword(s):  
Monoclonal Antibody ◽  
Small Intestine ◽  
Epithelial Cells ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Colon ◽  
Proximal Colon ◽  
Beta Subunit ◽  
Cell Pole

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

scholarly journals Certain mouse strains are deficient in a kidney brush-border metallo-endopeptidase activity

1983 ◽  
Vol 209 (1) ◽  
pp. 251-255 ◽  
Author(s):  
J S Bond ◽  
J D Shannon ◽  
R J Beynon
Keyword(s):  
Brush Border ◽  
Inbred Mice ◽  
Neutral Endopeptidase ◽  
Renal Tissue ◽  
Mouse Strains ◽  
Ph Optimum ◽  
Brush Border Enzymes ◽  
Endogenous Inhibitors ◽  
Endopeptidase Activity ◽  
Proteinase A

Preparations of microvilli from kidneys of BALB/c mice contain an alkaline metallo-endopeptidase, meprin (metallo-endopeptidase from renal tissue). Certain genealogically related inbred mice are markedly deficient in meprin activity. The meprin-deficient strains (CBA/J and C3H/HeJ) exhibit normal levels of other brush-border enzymes: alkaline phosphatase, aminopeptidase M and another proteinase, a phosphoramidon-sensitive neutral endopeptidase. Meprin deficiency cannot be attributed to a shift in pH optimum and is unlikely to be due to the presence of endogenous inhibitors.

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