Identification of protease isozymes after analytical isoelectric focusing using fluorogenic substrates impregnated into cellulose membranes.

Smith, Robert E.

doi:10.1177/32.12.6389692

Cited by 39 publications

(18 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cathepsin B is known to have isozymes (41). For example, a minor cathepsin B isozyme with a different carbohydrate side chain, and at least one different amino acid residue (serine instead of cysteine at position 117) has been reported for porcine spleen (42).…”

Section: Discussionmentioning

confidence: 99%

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

Fong¹,

Calhoun²,

Hsieh³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonudeotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this done encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with w880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome.Proteinases are present in all forms of living organisms. The events of general and limited proteolyses have been attributed to actions of these enzymes. In general proteolysis, proteinases digest nutrient proteins and participate in the turnover of cellular proteins, whereas in limited proteolysis, proteinases modify substrate proteins and alter the properties and physiological functions of these proteins. Thus, limited proteolysis can play regulatory roles during cell growth and differentiation (1,2).Based on the amino acid residues at their active sites, proteinases are classified into serine, cysteine, aspartate, and metalloproteinases. Members of the cysteine proteinase family have wide phylogenetic distribution, and examples include papain from the papaya plant and cathepsin B from mammalian tissues. Cathepsin B is a lysosomal enzyme that is functional during intracellular protein catabolism and may also be involved in the proteolytic processing of protein precursors such as proinsulin (3)(4)(5). Cathepsin B has been implicated in several disease states including muscular dystrophy (6), rheumatoid arthritis (7), and tumor metastasis (8-11). We and others have demonstrated that cathepsin B-like proteinases may also be involved during differentiation of the cellular slime mold Dictyostelium discoideum (12, 13).To further investigate the role of cathepsin B in cellular functions, we have selected the molecular genetic approach. Here we report our results on the isolation and characterization of a cDNA clone for human cathepsin B.MATERIALS AND METHODS Synthesis of Oligodeoxyribonucleotides. A mixture of 17-nucleotide-long oligonucleotides encoding a portion of the rat liver cathepsin B protein sequence was synthesized by using a solid-phase phosphotriester method (14, 15). For amino acids specified by more than one codon, all possible nucleotides were inserted at the ambiguous positions. The synthesis was performed with a Vega Model 280 Automated Polynucleotide Synthesizer by a...

show abstract

Section: Discussionmentioning

confidence: 99%

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

Fong¹,

Calhoun²,

Hsieh³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The problem of endproduct diffusion was addressed by adsorbing the fluorogenic substrate onto a matrix that trapped the cleaved fluorescent product (Smith 1984). This concept was adapted into a commercially available product sold to detect electrophoretically separated enzymes by a fluorescent reaction product.…”

Section: Discussionmentioning

confidence: 99%

Use of Enzyme Overlay Membranes to Survey Proteinase Activity in Frozen Sections: Cathepsin-like and Plasmin-like Activity in Regenerating Newt Limbs

Day

Neufeld

1997

J Histochem Cytochem.

View full text Add to dashboard Cite

SUMMARYWe present a method that permits extremely simple and rapid screening of proteolytic enzyme activity in sectioned tissues. Enzyme overlay membranes (EOMs) are custom-made membranes designed to fluoresce at sites of specific proteolytic enzyme activity after separation of proteins by gel electrophoresis. EOMs, selected to detect either plasmin-like or cathepsin B-like activity, have been used in a novel way to document the distribution of enzyme activity in frozen sectioned tissues. When moistened membranes were placed in contact with sectioned regenerating newt limbs, a fluorescent pattern of enzyme activity was generated. In limbs at 3 hr post amputation, cathepsin B-like activity was prominent across the amputation site but plasmin-like activity was distributed in dermal and deeper proximal tissues, suggesting different roles for these two classes of enzymes. EOM enzymology in situ (EEI) on frozen sectioned tissues may be a widely useful technique to display distribution and level of activity of proteolytic enzymes in various systems. (J Histochem Cytochem 45:779-783, 1997)

show abstract

“…Assay of DPPIV activity DPPIV was identified after SDS/PAGE in unfixed gels by its enzymic activity using an overlay technique (Smith, 1984) in which cellulose acetate strips impregnated with the fluorogenic substrate Ala-Pro-amino-4-trifluoromethylcoumarin (Ala-Pro-AFC) (Enzyme Systems Products, Livermore, CA, U.S.A.) were placed over the gels, incubated 30 min at 37°C, and then observed and photographed under u.v. light (Piazza et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady-state RNA levels and an altered protein. Use as a liver-cell transplantation model

Thompson

Hixson

Callanan

et al. 1991

Biochemical Journal

100

View full text Add to dashboard Cite

Dipeptidyl peptidase IV (DPPIV) is a serine exoproteinase expressed at high levels in epithelial cells of kidney, liver and small intestine. Recently Watanabe, Kohima & Fujimoto [(1987) Experientia 43, 400-401] and Gossrau et al. [(1990) Histochem. J. 22, 172-173] reported that Fischer 344 rats are deficient in this enzyme. We have examined DPPIV expression in Fischer 344 rats available from U.S. and German suppliers and find that livers of the U.S. Fischer rats, in contrast with their German counterparts, express active DPPIV (D+). Northern analysis of liver RNA showed comparable levels of 3.4 kb and 5.6 kb DPPIV transcripts in both D+ rats from the U.S. and German (D-) rats. Monoclonal antibody (MAb) 236.3 to DPPIV immunoprecipitated at 150 kDa enzymically active (105 kDa, denatured) protein from surface-labelled D+ hepatocytes and reacted with canalicular and sinusoidal membranes (as shown by immunofluorescence microscopy). MAb 236.3 failed to immunoprecipitate a labelled peptide from D- cell extract or to stain D- liver sections. Polyclonal antibody (PAb) specific for DPPIV immunoprecipitated an enzymically active peptide from D+ hepatocyte extracts and a smaller, inactive peptide from D- hepatocyte extracts. Peptide maps of DPPIV immunoprecipitated from D+ extracts with MAb 236.3 and PAb were identical, but differed from that of the D- hepatocyte component recognized by PAb. The molecular basis of the DPPIV deficiency in the D- rats thus appears to be the translation of an enzymically inactive protein missing the epitope recognized by MAb 236.3. We have exploited these D- rats as hosts for syngeneic transplantation of liver cells from D+ Fischer rats. DPPIV expression is stable in the transplanted cells and allows them to be readily distinguished from the surrounding D- tissue.

show abstract

Identification of protease isozymes after analytical isoelectric focusing using fluorogenic substrates impregnated into cellulose membranes.

Cited by 39 publications

References 19 publications

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

Use of Enzyme Overlay Membranes to Survey Proteinase Activity in Frozen Sections: Cathepsin-like and Plasmin-like Activity in Regenerating Newt Limbs

A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady-state RNA levels and an altered protein. Use as a liver-cell transplantation model

Contact Info

Product

Resources

About