scholarly journals Proteins of the kidney microvillar membrane. Structural and immunochemical properties of rat endopeptidase-2 and its immunohistochemical localization in tissues of rat and mouse

1989 ◽  
Vol 264 (2) ◽  
pp. 335-346 ◽  
Author(s):  
K Barnes ◽  
J Ingram ◽  
A J Kenny
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Immunohistochemical Localization ◽  
Mouse Kidney ◽  
Molecular Forms ◽  
Rat Tissues ◽  
Weak Staining ◽  
Mouse Tissues ◽  
Rabbit Polyclonal Antibody ◽  
Solubilized Form

The phosphoramidon-insensitive endopeptidase-2 in rat renal brush borders was investigated by immunochemical approaches with a rabbit polyclonal antibody raised to the purified enzyme released from the membrane by papain. An immunoaffinity column successfully purified the detergent-solubilized form of endopeptidase-2. This preparation had an apparent subunit Mr of 80,000, and did not show the two subunits, of Mr 80,000 and 74,000, consistently found in the papain-solubilized forms, indicating that the latter resulted from proteolysis by papain. SDS/polyacrylamide-gel electrophoresis of non-reduced samples of the enzyme revealed a band of Mr 220,000, confirming the presence of disulphide-bridged subunits. Treatment with endoglycosidases H and F generated smaller molecular forms, indicating that endopeptidase-2 contained about 30% asparagine-linked carbohydrate and that a few of these oligosaccharide chains were of the high-mannose type. Treatment with phosphatidylinositol-specific phospholipase indicated that the enzyme did not possess a glycolipid membrane anchor. A survey of rat tissues examined immunohistochemically and by immunoblotting revealed that only the kidney and intestinal tract expressed the antigen in significant amounts. Although some weak staining was seen in salivary glands and thyroid, other organs and tissues including brain and spinal cord were negative by both immunochemical techniques. In the kidney the antigen was confined to the lumen of the proximal tubule and was seen mainly in the population of juxtamedullary nephrons. In the gut, luminal staining was observed throughout its whole length, from duodenum to rectum. Excellent cross-reactivity of the antibody with Balb/c mouse tissues was observed. Immunohistochemistry of mouse kidney and gut revealed a distribution identical with that observed in the rat. Immunopurification of the detergent-solubilized mouse kidney antigen showed it to be a protein containing disulphide-linked subunits of Mr 90,000. It possessed endopeptidase-2-like activity, but was more efficient in hydrolysing azo-casein and less efficient in hydrolysing a model substrate than the rat enzyme. The close similarity between rat endopeptidase-2 and mouse meprin is further supported by these results.

Comparison of receptors for insulin-like growth factor II from various rat tissues

1987 ◽  
Vol 115 (1) ◽  
pp. 35-NP ◽  
Author(s):  
J. E. Taylor ◽  
C. D. Scott ◽  
R. C. Baxter
Keyword(s):  
Growth Factor ◽  
Ph Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subcutaneous Fat ◽  
Cross Reactivity ◽  
Rat Tissues ◽  
Insulin Like Growth Factor ◽  
Major Band ◽  
Factor Ii

ABSTRACT Receptors for insulin-like growth factor II (IGF-II) have been compared in solubilized microsomal membranes from rat lung, brain, kidney, heart, epididymal and subcutaneous fat, ovary, testis and adrenals. Highest binding/μg protein was seen with testicular membranes. Receptors from all tissues showed high affinity for human IGF-II (mean association constant = 65 litres/nmol) and a high degree of specificity (mean IGF-I cross-reactivity 0·3%; no cross-reactivity with insulin). Affinity labelling followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed binding was only to a type-II IGF receptor, with a major band seen at a molecular weight of about 230 000 in lung, brain, kidney and testis, and 240 000 in heart, fat and adrenal gland. All tissues showed broad or bimodal pH dependence of binding, with optima seen at about pH 6 and pH 9. Mild stimulation of IGF-II binding by low calcium concentrations (1–2 mmol/l) was seen in all tissues, although higher concentrations were inhibitory in the brain. It was concluded that IGF-II receptors from different rat tissues, when studied under uniform conditions, show similar binding affinities but differences in size and regulation which might be missed if receptors are examined in separate studies. J. Endocr. (1987) 115, 35–41

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Immunohistochemical localization of phosphoenolpyruvate carboxykinase in adult and developing mouse tissues.

1990 ◽  
Vol 38 (2) ◽  
pp. 171-178 ◽  
Author(s):  
D B Zimmer ◽  
M A Magnuson
Keyword(s):  
Nervous System ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Submaxillary Gland ◽  
Cell Types ◽  
Immunohistochemical Localization ◽  
Mouse Tissues ◽  
Multiple Cell ◽  
Cell Type Specific ◽  
Immunohistochemical Techniques ◽  
Developing Nervous System

We used immunohistochemical techniques to analyze the cell distribution of phosphoenolpyruvate carboxykinase (PEPCK) in adult and developing mouse tissues. PEPCK immunoreactivity was detected in many tissues, including some that had not been previously reported to contain PEPCK enzyme activity (bladder, stomach, ovary, vagina, parotid gland, submaxillary gland, and eye). In some multicellular tissues, PEPCK immunoreactivity was observed in multiple cell types. Several tissues (spleen, thyroid, and submaxillary gland) contained no detectable PEPCK immunoreactivity. During development, PEPCK immunoreactivity was associated with the developing nervous system and somites in 15-day embryos. At prenatal day 18, PEPCK immunoreactivity was detected only in the nervous system. At prenatal day 20, PEPCK immunoreactivity was observed in many of the tissues that contain PEPCK in the adult, with the exception of liver, lung, and stomach. PEPCK immunoreactivity was detected in liver at postnatal day 1, lung at postnatal day 7, and stomach after postnatal day 21. The only tissue in which PEPCK immunoreactivity decreased during development was the pancreas, where PEPCK immunoreactivity was detected at prenatal day 20 and was present until postnatal day 21. These results suggest that PEPCK expression is cell-type specific, more widespread than previously thought, and differentially expressed during development.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Immunohistochemical localization of copper-zinc and manganese superoxide dismutases in rat tissues.

1989 ◽  
Vol 22 (3) ◽  
pp. 351-365 ◽  
Author(s):  
KAZUSHIGE DOBASHI ◽  
KOHTARO ASAYAMA ◽  
KIYOHIKO KATO ◽  
MAKIO KOBAYASHI ◽  
AKIRA KAWAOI
Keyword(s):  
Immunohistochemical Localization ◽  
Superoxide Dismutases ◽  
Rat Tissues ◽  
Manganese Superoxide ◽  
Copper Zinc ◽  
Manganese Superoxide Dismutases

ELECTROPHORETIC EVIDENCE FOR ISOZYMES OF ARYLAMIDASE ("AMINOPEPTIDASE") IN THE RAT. I. RENAL, SERUM AND URINARY ENZYMES SPLITTING dl-ALANYL-2-NAPHTHYLAMIDE AND l-LEUCYL-2-NAPHTHYLAMIDE

1964 ◽  
Vol 12 (12) ◽  
pp. 869-874 ◽  
Author(s):  
BENITO MONIS
Keyword(s):  
Present Report ◽  
Molecular Forms ◽  
Supernatant Fraction ◽  
Blood Levels ◽  
Rat Tissues ◽  
Bilateral Nephrectomy ◽  
Rat Urine ◽  
Urinary Enzyme ◽  
Renal Ablation ◽  
Starting Point

Bilateral nephrectomy elicited no changes of serum blood levels of arylamidase assayed with two chromogenic substrates (l-leucyl-2-naphthylamide and dl-alanyl-2-naphthylamide) two days after the operation. Since rat urine contains similar enzymes it was postulated that the urinary enzyme is of renal origin and distinct from serum arylamidase. For this purpose, electrophoretic studies were undertaken. Starch block (for quantitative determinations) and starch gel for zymograms of arylamidase were used. It was shown that both procedures demonstrated the identity of urinary and renal arylamidase, which was distinct from the serum enzyme. The renal and urinary enzyme showed two distinct isozymes: one remaining at the origin and the other migrating somewhat less than the isozyme in serum. It is postulated that the isozyme remaining at the origin corresponds to a membrane-bound form, whereas the electrophoretically-mobile isozyme is found in the cellular supernatant fraction. The arylamidase from serum was represented by a single enzyme which migrated farthest towards the anode. Histochemical procedures for the demonstration of arylamidase activity in tissues at the light microscopic level permit the localization of enzyme(s) that can hydrolyze synthetic chromogenic naphthylamides containing l-leucyl and dl-alanyl groups (1). Rat kidneys have a high concentration of histochemically demonstrable arylamidase in the proximal convoluted tubules (2). Blood levels of arylamidase in the normal adult rat vary within a narrow range (3). The observation of arylamidase activity in rat urine raised several questions that led to the studies which are the basis of the present report. The relationship of serum and urinary arylamidase was the starting point of this investigation. It was speculated that if the urinary enzyme had its origin in the serum, bilateral nephrectomy should alter blood levels of the enzyme. If no such changes occurred, this would suggest that the urinary enzyme was released from the proximal convoluted tubule. In fact, total renal ablation led to no significant variation of serum arylamidase activity. It was therefore postulated that urinary arylamidase originated in the kidneys and that both are distinct from serum arylamidase. To test this hypothesis, zone electrophoretic studies were undertaken using two different supporting media. It will be shown that distinct molecular forms or isozymes of arylamidase can be separated from rat tissues and fluids by differential electrophoretic mobility. The electrophoretic identity of urinary and renal arylamidase, both of which are distinct from the serum arylamidase, is demonstrated in this study.

scholarly journals Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Snatashree Mohanty ◽  
M. Makesh ◽  
K. V. Rajendran ◽  
P. P. Suresh Babu ◽  
Deepika Anand ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Catla Catla ◽  
Cirrhinus Mrigala ◽  
Indian Major Carps ◽  
Sds Page ◽  
Igg1 Isotype ◽  
Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

scholarly journals IMMUNOHISTOCHEMICAL LOCALIZATION OF CATALASE IN MAMMALIAN TISSUES

1969 ◽  
Vol 17 (1) ◽  
pp. 30-35 ◽  
Author(s):  
SHIGERU MORIKAWA ◽  
TAKAYUKI HARADA
Keyword(s):  
Fluorescent Antibody ◽  
Bovine Liver ◽  
Cross Reactivity ◽  
Immunohistochemical Localization ◽  
Hepatic Cells ◽  
Surface Active Agents ◽  
Erythrocyte Catalase ◽  
Mammalian Tissues ◽  
Splenic Cells ◽  
Proximal Tubuli

The distribution of catalase was investigated in bovine tissues using fluorescent antibody techniques. The immunochemical properties of liver catalase were also examined. Two distinct components of liver catalase in immune system were found. Both of them possessed common antigenicity with erythrocyte catalase. No cross-reactivity was observed by immunodiffusion between catalase and the other hemoproteins such as lactoperoxidase, cytochrome c and hemoglobins. Bovine liver, pancreas, kidney, spleen and peripheral blood were examined. Catalase was located mainly in the cytoplasm of hepatic cells, acinar cells of the pancreas, epithelia of proximal tubuli, splenic cells scattered in the red pulp and some leukocytes. It was not found in any nucleus. Intracorpuscular catalase could be revealed in the erythrocytes treated with surface-active agents but not in frozen sections.

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