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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In sec18 the protoxin was stable after a chase; but in sec7 and sec1 the protoxin was unstable, and in sec1 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and sec1 cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kex1 but unstable in kex2. Protoxin stability in kex1 kex2 double mutants indicated the order kex1 --> kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEX1- and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.
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PMID:Secretion of Saccharomyces cerevisiae killer toxin: processing of the glycosylated precursor. 635 2

Acetylation of cysteine S-conjugates of xenobiotics by microsomal N-acetyltransferase is the final step of detoxicative metabolism leading to mercapturic acid biosynthesis. To elucidate the subcellular site of N-acetylation and the effective mechanism by which the final metabolites are eliminated from the organisms, topological aspects and catalytic properties of microsomal N-acetyltransferase and mercapturic acid biosynthesis in vivo were investigated. Intravenous administration of radioactive S-benzyl-L-cysteine, a model compound of cysteine S-conjugates, resulted in rapid acetylation of the conjugate in liver and kidney to a similar extent. The acetylation was followed by a rapid excretion of the metabolite, a mercapturic acid, into the urine; about 60% of the injected dose appeared in urine within 60 min of administration. Limited proteolysis of microsomal vesicles obtained from liver and kidney by chymotrypsin or trypsin inactivated the transferase by 49-62% and 62-73%, respectively. Proteolytic inactivation of the transferase was not significantly affected by the presence of 0.04% sodium deoxycholate by which the vesicles became permeable to macromolecules due to its detergent action. To determine the sidedness of the active site of N-acetyltransferase on the microsomal membranes, two S-acetyldextran polymer derivatives (Mr 500 000) of cysteine and N-acetylcysteine which represent an nonpermeant substrate and product for this enzyme, respectively, were examined for their effects on the vesicle-associated enzyme activity. Both derivatives inhibited the transferase activity in a dose-dependent fashion; maximum inhibition of the enzyme activity was 40% by the former and 60% by the latter. Sulfobromophthalein strongly inhibited the enzyme activity and this inhibition was completely reversed by adding an equimolar amount of hepatic glutathione S-transferases (ligandins). In contrast to the strong inhibition by sulfobromophthalein itself, its glutathione S-conjugate did not inhibit the enzyme activity. These results indicate that the active site and the protease-sensitive domain(s) of the microsomal N-acetyltransferase are localized on the outer surface (cytoplasmic side) of endoplasmic reticulum and that the ligandin(s) might protect membranous N-acetyltransferase from inhibition by organic anions by binding them and catalyzing the conjugation with glutathione.
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PMID:Topological aspects of microsomal N-acetyltransferase, an enzyme responsible for the acetylation of cysteine S-conjugates of xenobiotics. 674 76

Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA-dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the interior of the polypeptide. a mechanism that explains the final transmembrane disposition of band 3 in the plasma membrane as resulting from the mode of its incorporation into the ER is presented.
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PMID:Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes. 732 13

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.
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PMID:Early events in the cellular formation of proparathyroid hormone. 737 10

Tubulin has been found to be synthesized on both membrane-bound and free polyribosomes prepared from brain. Cell-free studies indicate that tubulin made on rough microsomes is incorporated into the endoplasmic reticulum membrane as it is synthesized. This tubulin remains associated with the membrane after sedimentation and washing. The tubulin is not removed from the membrane after stripping ribosomes from the membranes in KCl-puromycin, followed by repeated washing by either sedimentation or flotation in 0.05 M-KCl. The membrane tubulin is partially susceptible to proteolysis by trypsin and chymotrypsin: beta-tubulin is more accessible to the proteases than in alpha-tubulin. Nonionic detergents extract mostly beta-tubulin from the microsomal membrane. Newly synthesized tubulin which has been extracted from microsomal membranes in 0.5% Nonidet P-40, coassembles and disassembles with carrier microtubule protein. The insertion of newly synthesized tubulin into endoplasmic reticulum membrane may be the first step in the incorporation of tubulin into the plasma membrane.
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PMID:Association of newly synthesized tubulin with brain microsomal membranes. 745 9

The mechanism by which secretory proteins are segregated within the cisternal space of microsomal vesicles was studied using dog pancreas mRNA which directs the synthesis of 14 well-characterized nonglycosylated pancreatic exocrine proteins. In the absence of microsomal membranes, each of the proteins was synthesized as larger polypeptide chains (presecretory proteins). 1,000-2,000 daltons larger than their authentic counterparts as judged by polyacrylamide gel electrophoresis in SDS. Conditions optimal for the study of reconstituted rough microsomes in the reticulocyte lysate system were examined in detail using mRNA and microsomal membranes isolated from dog pancreas. Functional reconstitution of rough microsomes was considerably more efficient in the presence of micrococcal nuclease- treated membranes than in the presence of EDTA-treated membranes. Analysis for segregation of nascent secretory proteins by microsomal vesicles, using post-translational incubation in the presence of trypsin and chymotrypsin, 50 mug/ml each, was shown to be inadequate, because of the disruption of vesicles by protease activity. Addition of 1-3 mM tetracaine or 1 mM dibucaine stabilized microsomal membranes incubated in the presence of trypsin and chymotrypsin at either 0 degrees or 22 degrees C. Each of the pancreatic presecretory proteins studied was correctly processed to authentic secretory proteins by nuclease-treated microsomal membranes, as judged by both one-dimensional and two-dimensional gel electophoresis. Post-translational addition of membranes did not result in either segregation or processing of nascent polypeptide chains. Post- translational proteolysis, carried out in the presence of 3 mM tetracaine, indicated that each of the 14 characterized dog pancreas secretory proteins was quantitatively segregated by nuclease-treated microsomal vesicles. Segregation of nascent secretory proteins was irreversible, since radioactive amylase, as well as the other labeled secretory proteins, remained quantitatively sequestered in microsomal vesicles during a 90-min incubation at 22 degrees C after the cessation of protein synthesis. Studies employing synchronized protein synthesis and delayed addition of membranes indicated that all pancreatic presecretory proteins contain amino terminal peptide extensions. These peptide extensions are shown to mediate the cotranslational binding of presecretory proteins to microsomal membranes and the transport of nascent secretory proteins to the vesicular space. The maximum chain lengths which, during synthesis, allow segregation of nascent polypeptide chains varied between 61 (pretrypsinogen 2 + 3) and 88 (preprocarboxypeptidase A1) amino acid residues among dog pancreas presecretory proteins. Reconstitution studies using homologous and heterologous mixtures of mRNA (dog, guinea pig, and rat pancreas; rat liver) and micrococcal nuclease-treated microsomal membranes (dog, guinea pig, and rat liver; dog pancreas), in the presence of placental ribonuclease inhibitor, suggest that the translocation mechanism described is common to the rough endoplasmic reticulum of all mammalian tissues.
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PMID:Mechanism of compartmentation of secretory proteins: transport of exocrine pancreatic proteins across the microsomal membrane. 746 18

The natural gene for bovine pancreatic trypsin inhibitor (BPTI) was expressed by in vitro transcription/translation systems as the 100-residue pre-proBPTI, with a signal peptide for translocation into the endoplasmic reticulum. Expression in the presence of microsomes defined the site of co-translational cleavage of the signal peptide. The resulting proBPTI in the microsomes consists of the 58 residues of mature BPTI, plus an additional 13 residues at the N terminus, including a cysteine residue at position -10, and seven residues at the C terminus. ProBPTI remained in the unfolded, reduced form within microsomes when synthesized under reducing conditions, but folded and formed disulphide bonds rapidly when the disulphide form of glutathione was added. Complete folding could occur within about one minute, even when residue Cys10 was replaced by Ser. The structure of proBPTI was determined by circular dichroism and two-dimensional NMR and found to be that of mature BPTI with flexible extensions on both termini. Its inhibition of the activity of alpha-chymotrypsin was indistinguishable from that of the mature protein. The extensions of the precursor appeared to play only very minor roles in refolding in vitro under conditions where folding and disulphide bond formation are coupled. Under pH and redox conditions thought to reflect those in vivo, complete folding and disulphide bond formation required several hours. Addition of protein disulphide isomerase to in vitro folding experiments caused substantial and similar increases in the rate of formation of the fully folded state for both mature BPTI and proBPTI; the half time for folding to the native state was reduced to approximately two minutes, which is comparable to that occurring in microsomes. The absence of substantial effects of the N and C-terminal extensions on the protein structure, inhibitor activity and refolding leaves their functional roles to be discovered.
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PMID:On the biosynthesis of bovine pancreatic trypsin inhibitor (BPTI). Structure, processing, folding and disulphide bond formation of the precursor in vitro and in microsomes. 769 Apr 7

The 94-kDa glucose-regulated protein (endoplasmin, grp94) is an abundant member of the 90-kDa molecular chaperone family in the endoplasmic reticulum. We have found earlier that the 50% homologous 90-kDa heat shock protein, hsp90, has ATP-binding site(s) and autophosphorylating activity (Csermely, P., and Kahn, C. R. (1991) J. Biol. Chem. 266, 4943-4950). In the present paper we demonstrate that highly purified grp94 is also able to autophosphorylate itself on serine and threonine residues. grp94 can be freed from the co-purifying casein kinase II by concanavalin A affinity chromatography, and its phosphorylation is unaffected by activators and inhibitors of numerous protein kinases known to associate with the homologous hsp90. The autophosphorylation persists in immunoprecipitates and in SDS-polyacrylamide gel-purified and renatured grp94. Autophosphorylation displays a monomolecular kinetics, is activated by micromolar calcium concentrations, has an extreme heat stability, and can utilize both ATP and GTP with relatively high km values of 243 +/- 14 microM and 116 +/- 23 microM, respectively. Sequence analysis of grp94 shows the presence of two ATP-binding sites. The major product of limited proteolysis of grp94 by chymotrypsin or papain is an N-terminal 85-kDa fragment that can bind to ATP-agarose but does not show autophosphorylation. Our data suggest that grp94 has an enzymatic function analogous in many respects to the similar activity of hsp70, hsp90, and grp78 (BiP). Autophosphorylation may participate in/regulate the complex formation of these proteins, so it may be involved in their chaperone function.
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PMID:Autophosphorylation of grp94 (endoplasmin). 789 Jul 76

One key problem in understanding the biosynthesis of collagens remains the assembly of the three alpha-chains. How and when are the different gene products selected, aligned, and folded into a triple helix? As the spatial arrangement during biosynthesis might be important, we concentrated on whether the rough endoplasmic reticular membrane is involved in this process. Microsomes were prepared from biosynthetically labeled chick tendon fibroblasts. Vesicles were spread as a monomolecular film which was then transferred over several compartments of a filmbalance containing fresh subphase. Fluorograms of the surface film showed that the monolayer contains procollagen chains. When the monolayer was transferred onto a chymotrypsin/trypsin-containing subphase, the gel bands of the proalpha-chains were shifted into the position of mature alpha-chains, indicating that only the propeptides were digested and the collagenous regions were protected due to triple helix formation. Our results suggest that newly synthesized proalpha-chains can associate as trimers and fold into a triple helical conformation while they are still associated with the membranes of the rough endoplasmic reticulum. These processes also occur when interchain disulfide linkage is inhibited, indicating that chain selection and registration is not dependent on formation of covalent bonds among the carboxyl propeptides.
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PMID:Triple helix formation of procollagen type I can occur at the rough endoplasmic reticulum membrane. 870 43

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.
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PMID:Proteasome activity limits the assembly of MHC class I molecules after IFN-gamma stimulation. 955 Mar 86


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