Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin-resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.
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PMID:Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. 630 57

Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.
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PMID:Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity. 631 Nov 62

The presence of human blood group A determinants has been shown on the A+ rabbit intestinal brush border glycoproteins, particularly hydrolases. Sugar compositions of aminopeptidases N from A+ and A- rabbits were compatible with the presence in these molecules of eight N-linked glycans and of two O-linked glycans bearing the A determinants in A+ animals. The exact relative molecular masses of hydrophobic domain(s) of aminopeptidases N and A from pig and rabbit intestinal brush border have been determined by an isotopic dilution technique. The values obtained were compatible with the anchorage in the membrane of the monomeric rabbit enzymes, or of each subunit of the dimeric pig enzymes, by their N-terminal sequences, composed of 20-25 hydrophobic amino acids. This N-terminal hydrophobic sequence (14 residues) has been determined for rabbit aminopeptidase N. Short peptides containing approximately 60% hydrophobic amino acids have been extracted by chloroform-methanol from purified brush border and basolateral membranes of pig enterocytes. Their molecular properties were very similar to those of the aminopeptidase anchors released by trypsin treatment of detergent-extracted enzymes. However, several lines of evidence failed to support the assumption that these free hydrophobic peptides can be identified with anchors left inside the bilayer after proteolytic cleavage of surface hydrolases.
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PMID:Aminopeptidases and proteolipids of intestinal brush border. 634 98

Plasma membranes of mature rat astrocytes separated by differential centrifugation have been reported to be intact, based on electron microscopic examination of thin plastic sections. However, the effects of the separation procedure on the internal structure of the plasma membranes are unknown. The degree of membrane integrity is of concern to us since our goal is the separation of astrocytic plasma membranes and characterization of the specific intramembranous particle groups called assemblies. We have taken advantage of the astrocyte membrane-marker, the assembly, in order to monitor, by freeze-fracture, the identity of the separated astrocytes and the integrity of their cell membrane. Since some processes of an astrocyte contain assemblies whereas other processes of the same cell do not, it was also necessary to determine if processes with assemblies were separated by this technique. Astrocytic cell membranes were also examined to determine if trypsinization or the mechanical disruption steps of the separation affected the intramembranous particles. Freeze-fracture of the plasma membranes revealed that the particles were rearranged resulting in patches of clumped intramembranous particles and areas of bare membrane. The assemblies were rearranged rather than lost from the membrane since they could be identified among the clumped particles. More astrocytic plasma membranes contained non-clumped, normally distributed particles in the trypsin treated fractions. The non-trypsinized fractions had more damaged astrocytes with aggregated intramembranous particles and much more cellular debris. We interpret the findings for the non-trypsinized astrocytes as due to greater mechanical stress placed on the cells during tissue disruption. Trypsin treatment lessens this stress, thereby, tending to preserve the normal distribution of intramembranous particles.
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PMID:Freeze-fracture analysis of plasma membranes of isolated astrocytes from rat brain. 635 54

The importance of the red cell membrane sialoglycoproteins in the invasion of P. falciparum merozoites has been assessed. Human erythrocytes deficient in glycophorin A (En(a-)cells) or B (S-s-U-, S-s-U+ cells) showed significant resistance to invasion. Treatment of normal erythrocytes with trypsin and chymotrypsin also reduced invasion. These results indicate that determinants carried on glycophorins A, B and C play an essential role in the successful invasion into human red cells. Sugar components present on glycophorin, in particular N-acetyl glucosamine and N-acetyl galactosamine, as shown by specific sugar and antibody inhibition studies, appear to act as important determinants for attachment to the erythrocyte. This implicates a protein(s) on the merozoite surface membrane which has the properties of a lectin.
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PMID:Merozoites of P. falciparum require glycophorin for invasion into red cells. 637 Apr 71

The exhaustive extraction of microsomal estradiol receptor by Surfynol/dithiothreitol/trypsin in low ionic strength buffer was employed for distribution studies on non-stimulated porcine endometrium. It was found that more than half of the cytoplasmic receptor contents were of microsomal origin. "Empty" structures did not interfere with receptor analysis by agargel electrophoresis. The combined yields from homogenate fractions corresponded to those obtained from unfractionated homogenates. Freeze-fracturing of endometrium had a moderate receptor-solubilizing effect.
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PMID:Origin and quantification of cytoplasmic estradiol receptor in resting target cells. 666 99

Glycophorin, the major sialoglycoprotein from the human erythrocyte membrane, has been isolated and recombined with phosphatidylcholine and cholesterol. Sucrose density gradient analysis of the recombinants shows that it is possible not only to recombine this protein with phospholipid, but also with phospholipid-cholesterol mixtures. Surprisingly, by the same analysis, it was possible to make a recombinant with cholesterol and glycophorin, only, in the absence of added phospholipid. The accessibility of the protein to trypsin was ested in each of these recombinants. In all the recombinants which contained either phospholipid, or phospholipid and cholesterol, the protein was protected from extensive hydrolysis. This is consistent with closed vesicles and incorporation of the protein into the recombinant membrane. Extensive hydrolysis of the protein occurred in the cholesterol-glycophorin recombinant indicating some differences in structure. Freeze-fracture electron microscopy of the phospholipid and the phospholipid-cholesterol recombinants showed mostly unilamellar vesicles, 1000 to 5000 A in diameter. Intramembranous particles were observed on both fracture faces, and the fracture planes were those expected for phospholipid bilayers. The glycophorin-cholesterol recombinants also showed fracture planes consistent with bilayers, and revealed intramembranous particles. Pieces of membrane-like structures as well as apparent vesicular structures were observed. Finally in the recombinants of glycophorin with phospholipid and cholesterol, cholesterol is shown to reduce the population of the motionally restricted phospholipid headgroup environment, in proportion to the mole percent cholesterol content.
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PMID:Incorporation of the human erythrocyte sialoglycoprotein into recombined membranes containing cholesterol. 672 89

The presence of structures bridging the inner and outer acrosomal membranes of the equatorial segment of boar spermatozoa was clearly demonstrated in cells that have undergone a variety of treatment procedures to displace the electron-dense contents of the acrosome. En-face sections show bridges to be punctate and not linearly extensive as might be suggested by sections perpendicular to the flat plane of the head. About 4.5 x 10(5) bridges, each measuring 7 nm across and spaced 7 nm apart, are arrayed hexagonally in the equatorial segment, but bridges are not present within the principal segment of the acrosome. Short-term treatment with trypsin partially digests the bridges, but does not disrupt the spacing or strict parallel configuration of equatorial segment membranes. However, short-term treatment with pronase digests most bridges and effectively disrupts the typical configuration of the equatorial segment. Freeze-fracture of the cytoplasmic face of the acrosomal membranes of the equatorial segment reveals a pattern throughout the phospholipid layer of the membrane which is similar to the pattern of bridges present in en-face thin sections of the equatorial segments. The data suggest that numerous bridges link the inner and outer acrosomal membranes of the equatorial segment of the acrosome and they play a major, if not an exclusive, role in maintaining the close spacing and parallel arrangement of the membranes in this portion of the acrosome.
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PMID:On the presence of bridges linking the inner and outer acrosomal membranes of boar spermatozoa. 700 60

The progressive growth and eventual fusion of the atrioventricular (AV) endocardial cushions is of critical importance to normal embryonic heart development. Failure to do so would result in septal and AV valvular defects. A central feature in initial cushion growth is the migration of cushion tissue (CT) cells through an heterogeneous extracellular matrix (ECM) which has previously been shown (in particular hyaluronate) to modify migratory behavior. Attention was directed to migrating CT cells to determine if (1) their surfaces physically attach to or bind ECM and (2) are modified to suggest a morphological basis for cell:matrix interaction. The migratory appendages (filopodia) of CT cells maintained in organ culture attached both to collagenous microfibrils coated with polyanionic material and hyaluronate (HA) enriched ECM. The cell:matrix associations were of sufficient strength to restrain the cell from contracting following freezing procedures and were labile to mild trypsin treatment. HA enriched matrix persisted at the cell surface even after treatments which removed most free ECM, but was readily removed by hyaluronidase and trypsin digestion. Freeze fracture analyses revealed 16-18 nm particles elevated above the plane of the filopodial surface which closely interfaced with ECM components. These particles were variably distributed, ranging from almost homogenous dispersion to focalized clusters, but were absent on surrounding non-migratory (myocardial) cells. Results are consistent with a model in which cell attachment to its migratory substratum is mediated by polyanions (probably sulfated glycosaminoglycan and fucosylated glycoprotein) and detachment by hyaluronate.
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PMID:Endocardial cushion tissue development: structural analyses on the attachment of extracellular matrix to migrating mesenchymal cell surfaces. 703 67

In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.
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PMID:Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture. 710 88


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