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

We have used purified, (125)I-labeled human transcobalamin II (TC II), saturated with cobalamin (Cbl), to study the uptake process for the TC II-Cbl complex by intact normal cultured human skin fibroblasts. We have also investigated the possibility that a defect in one step of this process underlies that inborn error of Cbl metabolism-designated cbl C-in which mutant cells are unable to retain Cbl intracellularly or convert it to its coenzyme forms. TC II-Cbl binding at 4 degrees C reached a plateau after 3-4 hr; 95% of the bound (125)I was releasable with trypsin. Binding of TC II-Cbl at 4 degrees C could be inhibited by human and rabbit TC II-Cbl and human TC II devoid of Cbl but not by other Cbl-binding proteins, albumin, or free Cbl. Specific binding reached saturation at congruent with5 ng TC II/ml (0.13 nM) and could be inhibited by ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'- tetraacetic acid. At 37 degrees C, the TC II-Cbl complex was internalized as shown by a progressive decrease in the trypsin-releasable fraction of bound (125)I. After 2 h at 37 degrees C, increasing amounts of acid-soluble (125)I were found in the incubation medium indicating that the labeled TC II was being degraded. Chloroquine, an inhibitor of lysosomal proteolysis, prevented this degradation. The binding, internalization, and degradation of TC II-Cbl by cbl C cells was indistingusihable from that by control cells. Our studies provide additional support for the concepts: (a) that the TC II-Cbl complex binds to a specific cell surface receptor through a site on the TC II; (b) that the interaction between the receptor and TC II is calcium dependent; (c) that the TC II-Cbl is internalized via endocytosis; (d) that the degradation of TC II and release of Cbl from the complex occurs in lysosomes. We also conclude that the defect in cbl C must reside at some step beyond this receptor-mediated uptake process.
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PMID:Binding and uptake of transcobalamin II by human fibroblasts. 61 8

Studies were designed to evaluate the binding of binding of vitamin B12 to cell membrane preparations from human placenta. The transcobalamin II-vitamin B12 complex (TCII-B12), which has a much greater affinity for the membranes than vitamin B12 alone, binds to a single saturable binding site with an approximate Ka = 7.2 mM-1. The binding requires a divalent cation and is temperature-dependent. Free TCII can compete with TCII-B12 for the binding site but has somewhat less affinity than does TCII-B12. Rat TCII-B12 has an affinity constant that is less than one-fifth that of human TCII-B12; human TCI-B12, bovine TCII-B12, hog intrinsic factor-B12 (IF-B12), and human IF-B12 do not bind to the membranes. Pretreating the membranes with trypsin causes a marked decrease in subsequent binding; this suggests the binding site includes a relatively exposed membrane protein. These data suggest that a specific cell surface receptor for the TCII-B12 complex exists in placenta. This TCII-B12 receptor can be solubilized with Triton X-100.
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PMID:A saturable high affinity binding site for transcobalamin II-vitamin B12 complexes in human placental membrane preparations. 83 Jun 65

Binding of cobalamin (Cbl) was compared in liver and kidney plasma membranes prepared from rat and human tissues. Single, high-affinity, saturable (200 pmol/l), binding sites for TC II-Cbl were found in all tissues; by contrast no receptors were present for free cobalamin, for which only non-specific adsorption occurred. Binding constants for TC II-CNCbl determined for liver and kidney plasma membranes were of a similar magnitude. Mean values for Ka (litre/nmol) were 16.7 (rat liver), 18.8 (rat kidney), 8.0 (human liver) and 7.5 (human kidney). Results for binding TC II-OHCbl instead of TC II-CNCbl showed no difference in Ka and Bmax. values, although the non-specific adsorption was decreased to a third. Competitive inhibition results showed that the receptors are specific for the TC II molecule and that binding is unaffected either by the cobalamin moiety or by the presence of free cobalamin. Degradation of the receptor protein molecules by trypsin (10 micrograms/ml) resulted in 90% inhibition of binding. It is concluded that differences between liver and kidney in cobalamin uptake and accumulation cannot be attributed to differences in their TC II receptors.
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PMID:A comparison of cobalamin binding by liver and kidney in rat and man. 298 31

The accumulation of the large and hydrophilic IgG, TC II-B12 and B12 molecules is demonstrated for the first time in a human placental system which has metabolic and physiological functions. A trypsin-sensitive component is present in the human term placental uptake of TC II-B12, for which a placental membrane receptor has been previously identified; this component is absent for the accumulation of free B12, which has no known receptor. Analyses of the cytosol and incubation media indicate degradation, binding and release of TC II-B12 and B12 as TC II-B12, free B12 and TC I-like complexes. It is suggested that the human placental tissue slice be used for studies involving the binding, uptake and processing of macromolecules as exemplified by TC II-B12.
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PMID:Macromolecule transfer in the human trophoblast: transcobalamin II-vitamin B12 uptake. 696 54