Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mechanical and enzymatic methods of disaggregating tumors were studied with the goals of (1) minimizing cell losses while (2) maintaining functional and surface membrane markers needed to objectively identify inflammatory cells (IC)1 in resultant suspensions. Application of the principles and methods described makes accurate estimation of the percentage of each IC type present in neoplasms possible for the first time. Compared to purely mechanical means of disaggregating tumors, all enzyme mixtures tested markedly increased yields of viable cells/g neoplasm. Best results were obtained with a combination of collagenase and a protease of broader substrate range (alpha chymotrypsin, papain, pronase or trypsin). The combination of enzymes that gave the highest yields with the least effect on inflammatory cell markers was trypsin, collagenase and DNAse (TCD). Because mechanical injury appeared to be the greatest single cause of cell loss (the enzymes themselves had little direct effect), potential sources were identified and either eliminated or minimized. With TCD, depending on the tumor system, cell recovery (measured as DNA recovered in cell suspensions) was as high as 50% and yields were as much as 6.9 X 10(8) viable cells/g tumor. Complete disaggregation was not required to obtain representative IC populations from tumor fragments. Neutrophils, eosinophils and mast cells from disaggregated neoplasms were counted in Giemsa stained cytocentrifuge preparations based on their unique morphologic appearances. Macrophages were identified by their capacity to phagocytose zymosan, a function which proved highly resistant to the effect of enzymes. Flourescent microscopic identification of brain associated thymus antigen (BATA) allowed quantification of T lymphocytes, since this marker was virtually unchanged by enzyme exposure. Surface immunoglobulin (Ig) was stripped from B lymphocytes most rapidly by pronase and chymotrypsin, slowly by trypsin and papain, and not at all by collagenase. Ig positive cells therefore could be quantified in suspensions generated by collagenase or very short (20 min) exposure of fragments to trypsin.
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PMID:Inflammatory cells in solid murine neoplasms. I. Tumor disaggregation and identification of constituent inflammatory cells. 18 47

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.
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PMID:Proteases induce secretion of collagenase and plasminogen activator by fibroblasts. 20 72

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.
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PMID:Cartilage proteoglycan aggregates. Electron-microscopic studies of native and fragmented molecules. 21 57

In contrast to intact human erythrocytes, human erythrocyte ghosts can be agglutinated but not fused by Sendai virus. Membrane fusion can, however, be induced in virus-agglutinated erythrocyte ghosts by addition of proteolytic enzymes such as trypsin, papain, or Pronase. When erythrocyte ghosts were reacted with antispectrin antiserum, the antiserum inhibited both the induction of fusion and the proteolysis of the membrane spectrin. The correlation between the membrane fusion process and the membrane cytoskeleton is discussed.
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PMID:Involvement of spectrin in membrane fusion: induction of fusion in human erythrocyte ghosts by proteolytic enzymes and its inhibition by antispectrin antibody. 21 96

Experiments demonstrating the existence of receptors for iron-saturated transferrin on both B and T lymphoblastoid cell lines of human origin are described. Binding of 125I-labeled transferrin is rapid, saturable and reversible. It can be specifically inhibited by unlabeled transferrin but not by other proteins. The number of receptors on T cell lines determined by Scatchard analysis is almost double the number on B cell lines but the binding affinities are equal. The putative transferrin receptor can be removed from the cell by the proteolytic enzymes papain and trypsin, and is re-expressed during overnight incubation at 37 degrees C. Resynthesis is inhibited by puromycin. The receptor can be solubilized by deoxycholate, and retains transferrin binding capacity when non-covalently attached to an amphipathic matrix consisting of deoxycholate-coupled poly(L-lysyl) Agarose.
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PMID:Transferrin receptors on human B and T lymphoblastoid cell lines. 21 39

A possible use of papain and trypsin immobilized on Biogel P-100 for purification of DNA-containing sheep smallpox viruses was studied. It was shown that the enzymes depolymerize tissue proteins, resulting in a 90% purification of the viruses without causing any considerable morphological changes in the virion structure. It was concluded that fixed proteinases can be effectively used at the final stage of purification of tissue virus-containing suspensions.
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PMID:[Use of immobilized proteinases for purification of sheep smallpox virus]. 22 69

HLA-A and -B antigens are phosphorylated in transformed lymphoblastoid cells and peripheral blood lymphocytes, both incubated with 32Pi. The phosphate group is attached to HLA-A and -B heavy chain (p44) as identified by immunoprecipitation with anti-beta2-microglobulin IgG, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, isoelectric focusing, and susceptibility to limited proteolysis by papain and trypsin. The site(s) of phosphorylation is identified as a serine residue(s) located in the hydrophilic carboxy terminus of the p44 chain. HLA antigens are also phosphorylated in isolated membranes from transformed lymphoblastoid cells that are incubated with [gamma32P]ATP. The phosphorylation of the carboxy terminus of HLA-A and -B antigens in vivo is good evidence that this portion of the molecule is intracellular. Furthermore, this modification suggests a general way in which interactions between membrane proteins and cytoskeletal elements may be regulated.
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PMID:Phosphorylation in vivo and in vitro of human histocompatibility antigens (HLA-A and HLA-B) in the carboxy-terminal intracellular domain. 28 20

G-banding of chromosome metaphase preparations derived from haemic cells of healthy individuals and from patients with acute myeloid leukaemia was performed with the aid of trypsin, papain, and pretreatment of the chromosome spreads with emulphogene before proteolytic digestion. Papain digestion revealed more distinguishable bands than did trypsin digestion. Pretreatment of the chromosome spreads with emulphogene greatly enhanced the number of distinguishable bands for both enzymes. The combination of pretreatment with emulphogene and digestion with papain revealed optimal numbers of bands for individual chromosomes essentially identical with those agreed at the Paris Conference 1971. The use of the emulphogene-papain technique appears also to offer an advantage in the banding of chromosomes from leukaemic cells.
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PMID:A comparative study of chromosome G-banding using trypsin, papain, and pretreatment with emulphogene. 28 44

A myeloma IgD immunoglobulin (designated WAH) that was present in high concentration in plasma ( approximately 3.5 g/dl) was purified in >90% yield by a two-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 years without the addition of a proteolytic inhibitor, no "spontaneous" degradation was apparent and the isolated IgD remained structurally intact. However, the purified IgD showed extreme susceptibility in vitro to various proteolytic enzymes; e.g., Fab(delta) (M(r) approximately 47,000) and Fc(delta) (M(r) approximately 80,000) fragments were generated quantitatively after only 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage, and cyanogen bromide fragmentation, several series of well defined fragments corresponding to the different regions and domains of the IgD molecule were generated. These fragments are useful for physical, chemical, and immunological studies, as well as for the sequence determination of the IgD delta chain. A model of the IgD molecule was derived from such studies and from overlapping of the series of fragments. The possible existence of an extra constant domain in the delta chain appears unlikely in view of our finding of an extended hinge region of about 50 residues which can be cleaved off the amino terminus of the papain Fc(delta) by brief treatment with trypsin. In addition to a distinct stretch of carbohydrate attachment sites, the delta-chain hinge region contains a segment unusually rich in electrical charge. This charged segment is responsible for the lability of IgD to spontaneous degradation and may be related to its biological role as a B lymphocyte receptor.
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PMID:Structural studies of human IgD: isolation by a two-step purification procedure and characterization by chemical and enzymatic fragmentation. 29 45

The cloning potential of PHA-treated T cells is significantly enhanced when lymphocyte culture fluid (LCF) from mitogen-treated lymphocytes is added to the soft agar culture system. The mitogens seem to stimulate the release of a lymphocyte colony enhancing factor (LCEF) into the culture medium. A study of the physico-chemical properties of the LCEF revealed that it is a nondialyzable, heat-labile molecule which migrates in the haptoglobin (2--2) post-transferrin region in acrylamide electrophoresis. It is stable to RNase and DNase but labile to papain and trypsin. The LCEF was partially purified from the crude LCF using a sequence of techniques--ammonium sulphate precipitation, DEAE-cellulose and Bio-Gel P-150 chromatography and disc electrophoresis. The mol. wt of the purified LCEF, determined from gel filtration chromatography, was 90,000--110,000.
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PMID:Partial purification and characterization of the human lymphocyte colony enhancing factor (LCEF). 30 93


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