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)

We report the novel properties of a 33 KDa cellular protein rapidly phosphorylated by stimulation of growth by IL3 in IL3 dependent lines. Although pp33 is readily soluble in SDS, SDS-solubilised pp33 is insoluble in non-ionic detergents and is excluded from electrophoretic analysis (IEF, NEPHGE) employing such detergents. Native pp33 is not extracted by non-ionic detergents with or without cation chelation. pp33 is concentrated in a cell fraction containing endoplasmic reticulum where it is associated with a specific trypsin-sensitive degredative enzyme, active at 4 degrees. Its unusual characteristics and kinetics of phosphorylation suggest pp33 may be a novel molecule, explain its absence in studies elsewhere where non-ionic detergent extraction has been exclusively used and suggest it is intimately related to the signal transduced by IL3.
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PMID:Novel characteristics of a 33KDa protein (pp33) rapidly phosphorylated in IL3 dependent cells by stimulation with IL3. 280 17

Co-translational insertion of liver microsomal cytochrome P-450 into the endoplasmic reticulum membrane is mediated by the signal recognition particle (SRP) and the presence in the cytochrome molecule of a signal sequence that can be recognized by SRP has been postulated. To locate this signal sequence, six hybrid cDNAs were constructed in which various segments of a cDNA for a rabbit liver cytochrome P-450 are fused with a cDNA or its fragment encoding yeast porin (an outer mitochondrial membrane protein) or with a cDNA for pre-interleukin 2 (a secretory protein) from which the 5'-terminal portion encoding most of its signal sequence had been removed. These hybrid cDNAs were inserted into an SP-6 transcription vector and transcribed in vitro. The mRNAs thus synthesized were translated in a cell-free system in the presence of rough microsomes. It was thus found that only those chimeric proteins containing (at their amino-terminal end) the amino-terminal cytochrome P-450 segments consisting of greater than or equal to 29 amino acid residues were co-translationally inserted into the membrane in an SRP-dependent fashion. These proteins were, however, neither processed nor translocated across the membrane. These findings, coupled with the observation that the major portion of these proteins, when inserted into the membrane, was degraded by trypsin, led to the conclusion that a short amino-terminal segment (less than 29 residues) of the cytochrome P-450 functions not only as an insertion signal but also as a stop-transfer sequence. This segment is, therefore, similar to the internal signal of type II plasma membrane proteins, but differs from the latter in the topogenic function.
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PMID:A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence. 282 91

The simian rotavirus SA11 genome segment 10 codes for a nonstructural glycoprotein, NS28, that has been hypothesized to be involved in budding of viral particles into the endoplasmic reticulum (ER) membrane. Previous studies had suggested that NS28 is an integral membrane protein of the ER, possibly a transmembrane protein. We have examined the topography of NS28 inserted in microsomal membranes following cell-free translation of genome segment 10 transcripts. These transcripts were obtained either by hybrid selection of mRNA synthesized by the endogenous viral RNA polymerase or by in vitro transcription of genome segment 10 cDNA using SP6 polymerase. Full-length and truncated gene 10 transcripts were translated in a cell-free system supplemented with dog pancreatic microsomes. The existence of a cytoplasmic domain of the translation product was demonstrated by protease protection experiments. An 18,000 (18K) mol wt glycosylated polypeptide was protected from digestion with proteinase K and trypsin, whereas chymotrypsin digestion yielded a 23K mol wt glycosylated polypeptide. Correlation of these biochemical data with the known sequence of NS28 suggests that a 10K mol wt hydrophilic, carboxy-terminal fragment (from amino acid number 86 to amino acid number 175) of this glycoprotein is exposed on the cytoplasmic side of the ER membrane. A model of how NS28 folds in the ER membrane is proposed.
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PMID:Topography of the simian rotavirus nonstructural glycoprotein (NS28) in the endoplasmic reticulum membrane. 283 61

No functional role could yet be established for the glycosylated beta-subunit of the Na,K-ATPase. In this study, we describe the intracellular processing of the beta-subunit as a glycoprotein in toad bladder cells and the consequences of its structural perturbation with glycosylation inhibitors on the cellular expression of the alpha- and beta-subunits and on the structural and functional maturation of the enzyme. Controlled trypsinolysis of homogenates from pulse-labeled cells reveals that the beta-subunit is subjected to glycosylation-dependent structural rearrangements during its intracellular routing. Inhibition of correct terminal glycosylation of the beta-subunit with deoxynojirimycin or swainsonine has no effect on the trypsin sensitivity of the alpha-subunit, its ability to perform cation-dependent conformation changes or the cellular Na,K-ATPase activity. Acquisition of core-sugars is sufficient for the enzyme to assume its catalytic functions. On the other hand, complete inhibition of glycosylation with tunicamycin leads to a destabilization of both the beta- and the alpha-subunits as judged by their higher trypsin sensitivity. In addition, tunicamycin treatment results in a decrease of the amount of newly synthesized beta- and alpha-subunit indicating that a glycoprotein, possibly the beta-subunit itself, plays a role in the efficient accumulation of the alpha-subunit in the endoplasmic reticulum.
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PMID:Role of the Na,K-ATPase beta-subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors. 284 51

Extracted human deciduous teeth undergoing physiological root resorption were fixed with a mixture of formaldehyde and glutaraldehyde and processed for scanning (SEM) and analytical transmission electron microscopy, as well as acid trimetaphosphatase cytochemistry. The granulated tissues, which are rich in multinucleated odontoclasts and capillary vessels, formed various resorption lacunae on the resorbing dentin surfaces. SEM observations of dentin surfaces treated with sodium hypochlorite revealed two types of resorption lacunae: deep, round lacunae in which the peritubular matrix of dentinal tubules was strongly dissolved; and shallow, irregular lacunae with intact peritubular matrix. In trypsin-treated materials, the resorption surfaces were characterized by the presence of numerous collagen fibers in both the peritubular and intertubular matrices, suggesting demineralization of the surface dentin. Odontoclasts were characterized by the presence of abundant mitochondria, perinuclear stacks of Golgi membranes, various lysosomes, numerous endocytotic vacuoles, and a well-developed ruffled border against the resorption lacunae. Most endocytotic vacuoles were distributed in the cytoplasm between the ruffled border and the nuclei. In undemineralized ultrathin sections, the surface dentin of resorption lacunae consisted of collagen fibers and apatite crystals and had a lower packing density than those in unresorbed, deeper dentin. Many apatite crystals were demonstrated to be present in the extracellular channels of the ruffled border and in adjacent endocytotic vacuoles derived from it. Lysosomes located in the perinuclear cytoplasm of odontoclasts contained amorphous dense material and/or a small amount of crystals. An energy-dispersive x-ray microanalysis of apatite crystals in undemineralized sections indicated that the energy spectrum peaks of Ca and P detected from crystals in resorbing dentin were much lower than those in unresorbed dentin. Similarly, lower spectrum peaks of Ca and P were obtained from crystals found in the ruffled border and endocytotic vacuoles of odontoclasts. A slight trace Ca peak also was detected in the amorphous dense material in lysosomes of odontoclasts. The enzyme cytochemistry of lysosomal acid trimetaphosphatase indicated that odontoclasts had intense enzymatic activity in the Golgi membranes, endoplasmic reticulum cisternae, lysosomes, and endocytotic vacuoles. Dense reaction precipitates of enzymatic activity also were found along the dentin surfaces of resorption lacunae occupied by odontoclast ruffled borders.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Dentin resorption mediated by odontoclasts in physiological root resorption of human deciduous teeth. 285 Dec 63

We report the first isolation of purified coated vesicles (CVs) from thyroid gland. Bovine thyroid CVs were isolated by differential centrifugation, including a step through sucrose-D2O, using a modification of the method described by Nandi et al. (1) for bovine brain CVs. The CVs were characterized by electron microscopy, sedimentation properties, and SDS-PAGE of the protein components. Thyroglobulin (Tg) was found to be associated with the purified CVs. When the thyroid CVs were exposed to conditions known to remove the protein coat from brain CVs, such as low ionic strength at pH 8.5, most of the Tg dissociated from the vesicles along with the coat proteins. Moreover, the Tg remaining with the uncoated vesicles (UVs) was trypsin sensitive, and therefore judged to be associated with the external surface of the vesicle. Since ligand-receptor complexes are normally located within CVs and not on their outer surface, no evidence was found for Tg-receptor complexes within thyroid CVs. Thyroid slices were incubated in the presence of [35S] methionine with subsequent isolation of labeled CVs in order to study the incorporation of newly-synthesized proteins into these structures. At 0.5 and 2 hours of incubation, the 180K MW subunit of clathrin, as well as other proteins, but not Tg, had become labeled in the purified CVs. Extracellular 19S-[35S] thyroglobulin was isolated from the incubation medium, however, demonstrating release of newly-synthesized Tg (presumably into cut follicles). It is concluded that thyroid CVs do not seem to be involved in the secretion of newly-synthesized Tg from the rough endoplasmic reticulum into the follicular lumen. While a possible role of thyroid CVs in the reabsorption of small quantities Tg by micropinocytosis cannot be completely excluded, the present data do not support a primary role for thyroid CVs in either endocytosis or exocytosis of Tg.
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PMID:Coated vesicles from the thyroid gland: isolation, characterization, and a search for a possible role in thyroglobulin transport. 286 11

Hexachloro-1,3-butadiene (HCBD) is a substrate for the hepatic microsomal glutathione transferases and is metabolised at higher rates by these enzymes than their cytosolic counterparts. Conjugation reactions catalysed by the microsomal and cytosolic transferases have been studied and characterized using this substrate and 1-chloro-2,4-dinitrobenzene (CDNB). In rat liver microsomes the Km values for HCBD and CDNB were 0.91 and 0.012 mM and in cytosol 0.51 and 0.10 mM respectively. Vmax values for HCBD were 1.39 and 0.35 nmol conjugate formed/min/mg protein for microsomes and cytosol respectively. In microsomal systems HCBD was a potent competitive inhibitor of the metabolism of CDNB with a Ki value of approximately 10 microM. However, CDNB did not inhibit HCBD metabolism significantly. These data suggest that more than one microsomal enzyme is involved in HCBD metabolism. The microsomal membrane could be solubilized without significant inhibition of HCBD activity; however, some detergents did inhibit the conjugation reaction. Activity was also lost on treatment of microsomal membranes with trypsin indicating the enzyme is localized on the cytoplasmic surface of the endoplasmic reticulum. Pretreatment of the rats with Aroclor 1254, 3-methylcholanthrene or phenobarbital did not change the microsomal conjugation of HCBD or CDNB with glutathione. Of seven species investigated, a human liver sample showed the highest ratio of microsomal to cytosolic glutathione transferase activity for HCBD (in microsomes 40-fold higher specific activity than in cytosol). Glutathione conjugation appears to play a critical role in the toxicity and carcinogenicity of some halogenated hydrocarbons. These data substantiate the potentially important role for the microsomal glutathione transferase in catalysing these reactions.
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PMID:Properties of the microsomal and cytosolic glutathione transferases involved in hexachloro-1:3-butadiene conjugation. 291 21

Human liver microsomal fractions exhibit ATP-supported Ca2+ uptake which is half-maximal at 7 X 10(-7) M free Ca2+ in the presence of oxalate. Ca2+ uptake is coupled to a Ca2+-stimulated ATPase activity, which is half-maximal at 4 X 10(-7) M free Ca2+. Catalysis involves formation of an Mr = 116,000 phosphoprotein with stability characteristics of an acylphosphate compound suggested to represent a phosphoryl protein intermediate of the Ca2+-ATPase. Phosphorylation is half-maximal at about 10(-6) M free Ca2+. The Mr = 116,000 protein is highly susceptible to proteolysis with trypsin. The phosphorylated active site was localized in an Mr = 58,000 primary tryptic fragment and in an Mr = 34,000 subfragment. Analyses on the mechanism of the Ca2+-ATPase suggest the following reaction sequence: formation of an ADP-reactive phosphoenzyme (Mr = 116,000) with bound Ca2+, which can transphosphorylate its Pi to ADP, giving rise to synthesis of ATP; reversible transformation of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, which cannot further react with ADP; hydrolytical cleavage, probably catalyzed by Mg2+, of the ADP-unreactive phosphoenzyme with liberation of Pi. Comparison with the Ca2+-transport ATPase in sarcoplasmic reticulum of skeletal muscle led us to suggest that the Mr = 116,000 Ca2+-ATPase belongs to the class of E1P . E2P-ATPases and might be operative as a Ca2+-transport ATPase at the level of the endoplasmic reticulum in human liver.
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PMID:Ca2+-activated ATPase in microsomes from human liver. 295 25

Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers we constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here we report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is approximately equal to 9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. Containing only two cysteine residues, the protein does not have consensus sequences for asparagine-linked glycosylation, amphipathic alpha-helix, or the N-terminal leader signal sequences for entry into endoplasmic reticulum, although hydrophobic segments for potential membrane associations exist. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant (no lysine or arginine) and highly negatively charged (13 aspartic plus glutamic residues). Within this segment are five repeats of (Asp/Glu)-Trp-(Ser/Thr); two of these are nearly tandem repeats of Thr-Glu-Asp-Trp-Ser-Ala-Xaa-Pro.
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PMID:Increased mRNA expression of a laminin-binding protein in human colon carcinoma: complete sequence of a full-length cDNA encoding the protein. 297 Jun 39

Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparently located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.
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PMID:Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae. 300 42


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