Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 2.4-kilobase cDNA clone for human steroid-sulfatase (STS) was isolated and sequenced, which encoded an enzymatically active protein. The deduced amino acid sequence comprises 583 amino acids with an N-terminal signal peptide of 21 or 23 residues and four potential N-glycosylation sites. Two of the N-glycosylation sites are utilized and were localized to the asparagine residues 47 and 259. STS has the solubility properties of an integral membrane protein. The resistance of STS toward proteinase K after translocation into microsomes suggests that most, if not all, sequences of STS are exposed at the luminal side of microsomes. The deduced amino acid sequence predicts two membrane-spanning domains (amino acids 185-211 and 213-237) separated by a helix-breaking proline residue. We propose for STS a three-domain model. Two glycosylated luminally oriented domains of 161 and 346 residues are separated by a hydrophobic domain spanning the membrane twice in opposite directions. STS expressed in BHK-21 cells is located predominantly in the endoplasmic reticulum; smaller fractions are found in the Golgi, at the cell surface, multivesicular endosomes, as well as in lysosomes. The stability of STS in lysosomes may be related to the high homology of the two luminal domains of STS with the lysosomal sulfatases, arylsulfatase A, and arylsulfatase B. In spite of its similarity with these two lysosomal sulfatases, STS does not contain mannose 6-phosphate residues and is transported to lysosomes by a mannose 6-phosphate receptor-independent mechanism.
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PMID:Cloning and expression of human steroid-sulfatase. Membrane topology, glycosylation, and subcellular distribution in BHK-21 cells. 266 75

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

The E1-glycoprotein (Mr = 26,014; 228 amino acids) of mouse hepatitis virus A59 is a class III membrane glycoprotein which has been used in this study as a model system in the study of membrane integration and protein transport. The protein lacks an NH2-terminal cleavable signal sequence and spans the viral membrane three times. Hydrophobic domains I and III could serve as signal sequences for cotranslational membrane integration. Domain I alone was sufficient to translocate the hydrophilic NH2 terminus of E1 across the membranes as evidenced by glycosylation of a newly introduced N-glycosylation site. The COOH-terminal part of E1 involving amino acids Leu124 to Thr228 was found to associate tightly with membranes at the post-translational level, although this part of the molecule lacks pronounced hydrophobic sequences. Membrane protection assays with proteinase K showed that a 2-kDa hydrophilic fragment was removed from the COOH terminus of E1 indicating that the protein is largely embedded into the membrane. Microinjection of in vitro transcribed capped and polyadenylated mRNA into CV-1 cells or into secretory AtT20 pituitary tumor cells showed that the E1-protein accumulated in the Golgi but was not detectable at the plasma membrane or in secretory granules. The 28 NH2-terminal hydrophilic amino acid residues play no role in membrane assembly or in intracellular targeting. Various NH2-terminal portions of E1 were fused to Ile145 of the cytoplasmic N-protein of mouse hepatitis virus. The resulting hybrid proteins were shown to assemble into membranes in vitro and were detected either in the rough endoplasmic reticulum or transient vesicles of microinjected cells.
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PMID:Membrane integration and intracellular transport of the coronavirus glycoprotein E1, a class III membrane glycoprotein. 284 93

We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.
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PMID:A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. 330 20

Mouse liver beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the beta-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within the vesicle lumen or only weakly bound to the inside of the vesicle membrane. This conclusion was derived from release studies using low concentrations of Triton X-100 or controlled sonication. Both the intact complex and free egasyn were released in parallel with lumenal proteins, not with intrinsic membrane components. Also, beta-glucuronidase was protected from digestion by proteinase K by the membrane of microsomal vesicles. The hydrophilic nature of both the complex and free egasyn was confirmed by phase separation experiments with the detergent Triton X-114. Egasyn is one of an unusual group of esterases that, despite being located within the lumen or only weakly bound to the lumenal surface of the endoplasmic reticulum, do not enter the secretory pathway.
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PMID:Lumenal location of the microsomal beta-glucuronidase-egasyn complex. 366 91

Monoacylglycerol acyltransferase activity from suckling rat liver was localized to the microsomal subcellular fraction by differential centrifugation and comparison with the partitioning of selected marker enzymes. Chymotrypsin, pronase, and proteinase K inactivated the monoacylglycerol acyltransferase activity in detergent-disrupted microsomes, but not in intact microsomes, falsely suggesting a lumenal location for the enzyme. The impermeant inhibitors mercury-dextran and 4,4'-diisothiocyano,-2,2'-disulfonic acid stilbene inhibited monoacylglycerol acyltransferase in intact microsomes. These data, as well as the lack of latency and the inability of the substrate palmitoyl-CoA to readily permeate hepatic microsomes from suckling rats, strongly suggest that the enzyme's active site faces the cytosolic surface of the endoplasmic reticulum.
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PMID:Subcellular location and topography of rat hepatic monoacylglycerol acyltransferase activity. 388 75

Children with autoimmune chronic active hepatitis may have high titers of antibodies detected by immunofluorescence staining of hepatocytes and tubular cells in rat liver and kidney sections, respectively. These antibodies are directed against antigens contained in microsomal fractions prepared from these two organs. We have found that sera from these patients recognized a 50,000 mol wt protein present in higher concentration in smooth microsome subfractions compared with rough microsome subfractions. This protein is an integral membrane protein and is not glycosylated. It is exposed on the cytoplasmic face of the endoplasmic reticulum and is rather resistant to proteolysis with proteinase K. Since patients with liver disease of different etiology and similar severity of cell lysis do not give rise to liver-kidney microsome antibody (LKMA), lysis of hepatocytes is apparently not a sufficient condition for their development.
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PMID:Anti-liver-kidney microsome antibody recognizes a 50,000 molecular weight protein of the endoplasmic reticulum. 398 71

Transfer of a glycosylphosphatidylinositol (GPI) anchor to proteins carrying a C-terminal GPI-directing signal sequence occurs after protein translocation across the endoplasmic reticulum (ER). We describe the translocation and GPI modification of a model protein, preprominiPLAP, in ER microsomes depleted of lumenal content by high pH washing. In untreated microsomes preprominiPLAP was processed to prominiPLAP and GPI-anchored miniPLAP. Both products were fully translocated, since they resisted proteinase K treatment of the microsomes, and both behaved as membrane proteins by the carbonate extraction criterion. Microsomes depleted of lumenal content were able to translocate and process preprominiPLAP to give protease-protected prominiPLAP, but were unable to convert prominiPLAP to miniPLAP. Loss of GPI anchoring capacity occurred with a wash of pH > 9.5. If the alkaline wash was performed after formation of prominiPLAP conversion to miniPLAP was relatively unimpaired. The results indicate that constituents of the ER lumen, possibly chaperones interacting with the proprotein and/or the GPI anchor precursor, are required in the initial steps of GPI anchoring.
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PMID:Soluble constituents of the ER lumen are required for GPI anchoring of a model protein. 758 98

The type I membrane protein calnexin functions as a molecular chaperone for secretory glycoproteins in the endoplasmic reticulum with ATP and Ca2+ as two of the cofactors involved in substrate binding. Protease protection experiments with intact canine rough microsomes showed that amino acid residues 1-462 of calnexin are located within the lumen of the endoplasmic reticulum. Expression using the baculovirus Sf9 insect cell system of a recombinant truncated calnexin corresponding to residues 1-462 (calnexin delta TMC) revealed an association in vivo with a coexpressed secretory glycoprotein substrate, human immunodeficiency virus type I gp120. For the in vitro characterization of calnexin delta TMC, we purified this secreted form to homogeneity from the medium of Sf9 cells. We demonstrate that the properties of the purified calnexin delta TMC correspond to those of full-length calnexin in canine microsomes with at least one intramolecular disulfide bond and binding to 45Ca2+. Calnexin delta TMC underwent a marked and reversible conformational change following Ca2+ binding as measured by its resistance to proteinase K digestion of a 60-kDa fragment and also by the change from an oligomeric form of calnexin delta TMC to a monomeric form. We also found that calnexin bound Mg-ATP leading to a conformational change from a monomeric to an oligomeric form that coincided as with markedly increased proteinase sensitivity. Our results identify the luminal domain of calnexin as responsible for binding substrates, Ca2+, and Mg-ATP. Because Ca2+ and ATP are required in vivo for the maintenance of calnexin-substrate interactions, conformational changes in the luminal domain of calnexin induced by Ca2+ and Mg-ATP are relevant to the in vivo function of calnexin as a molecular chaperone.
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PMID:Conformational changes induced in the endoplasmic reticulum luminal domain of calnexin by Mg-ATP and Ca2+. 762 14

Mouse vas deferens protein (MVDP) is a major androgen-dependent protein of deferential fluid. It is specifically expressed in the epithelium of the mouse vas deferens. Its amino acid sequence as deduced from the nucleotidic sequence of its cDNA does not possess a signal sequence characteristic of secretory proteins. In vitro, transcription of MVDP cDNA followed by translation of mRNA in the rabbit reticulocyte system, in the absence or the presence of microsomes, demonstrated that there was no internalization of MVDP into microsomes that could protect it from degradation by proteinase K; this confirmed the absence of signal sequence. Moreover, MVDP has its NH2-terminus blocked. To understand how MVDP can be exported, its ultrastructural distribution and secretion process were analyzed by means of electron microscopy. Immunolocalization of MVDP revealed that it was distributed in the whole cytoplasm; it was never detected in the lumen of endoplasmic reticulum, Golgi apparatus, or vesicles but was abundant in apical protrusions and in the fluid, where it was associated with cellular material undergoing degradation. These data clearly demonstrated that exportation of MVDP into the luminal fluid does not occur in the classical manner for secretory proteins but rather involves an apocrine secretion process.
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PMID:Exportation of mouse vas deferens protein, a protein without a signal peptide, from mouse vas deferens epithelium: a model of apocrine secretion. 771 Nov 83


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