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)

Membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. These microvilli are covered by a sialomucin complex, composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the associated concanavalin A (Con A)-binding glycoprotein ASGP-2. Limited proteolysis of the microvilli releases large, highly glycosylated fragments of ASGP-1 from the microvilli and increases the association of ASGP-2 with the Triton-insoluble microvillar microfilament core (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988). To analyze the topography of ASGP-2 in the membrane and its association with the microfilament core, microvilli were treated with proteinase K for timed intervals and centrifuged. The pelleted microvilli were extracted with Triton X-100 for the preparation of microfilament cores and Triton-soluble proteins or with 0.1 M carbonate, pH 11, for the preparation of microvillar membranes depleted of peripheral membrane proteins. These microvilli fractions were analyzed by dodecyl sulfate gel electrophoresis, lectin blotting with Con A and L-phytohemagglutinin, and immunoblotting with anti-ASGP-2. The earliest major proteolysis product from this procedure was a 70 kDa membrane-bound fragment. At longer times a 60 kDa released fragment, 30-40 kDa Triton-soluble fragments, and 25-30 kDa membrane- and microfilament-associated fragments were observed. Phalloidin shift analysis of microfilament-associated proteins on velocity sedimentation gradients indicated that the 25-30 kDa fragments were strongly associated with the microfilament core. From these studies we propose that ASGP-2 has a site for indirect association with the microfilament core near the membrane on a 15-20 kDa segment.
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PMID:Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli. 267 61

Smooth, rough, and neutral forms of lipopolysaccharide (LPS) from Pseudomonas aeruginosa were used to assess the appropriate conditions for effective enzyme-linked immunosorbent assay (ELISA) of LPS. Each of these forms of well-defined LPS was tested for the efficiency of antigen coating by various methods as well as to identify an appropriate type of microtiter plate to use. For smooth LPS, the standard carbonate-bicarbonate buffer method was as efficient as the other sensitivity-enhancing plate-coating methods compared. The rough LPS, which has an overall hydrophobic characteristic, was shown to adhere effectively, regardless of the coating method used, to only one type of microtiter plate, CovaLink. This type of plate has secondary amine groups attached on its polystyrene surface by carbon chain spacers, which likely favors hydrophobic interactions between the rough LPS and the well surfaces. Dehydration methods were effective for coating microtiter plates with the neutral LPS examined, which is composed predominantly of a D-rhamnan. For the two dehydration procedures, LPS suspended in water or the organic solvent chloroform-ethanol was added directly to the wells, and the solvent was allowed to dehydrate or evaporate overnight. Precoating of plates with either polymyxin or poly-L-lysine did not give any major improvement in coating with the various forms of LPS. The possibility of using proteinase K- and sodium dodecyl sulfate-treated LPS preparations for ELISAs was also investigated. Smooth LPS prepared by this method was as effective in ELISA as LPS prepared by the hot water-phenol method, while the rough and neutral LPSs prepared this way were not satisfactory for ELISA.
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PMID:Appropriate coating methods and other conditions for enzyme-linked immunosorbent assay of smooth, rough, and neutral lipopolysaccharides of Pseudomonas aeruginosa. 749 23

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

Sm23, a surface protein of the human parasite Schistosoma mansoni, belongs to the family of "cysteine-rich, hydrophobic proteins," which are expressed on mammalian hematopoietic cells or tumor cells. Sm23 shares the highly conserved hydrophobicity profile of these proteins, which predicts four transmembrane segments, but is in addition linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor. Our results suggest that Sm23 uses both the potential transmembrane domains and the GPI anchor for membrane insertion: (a) Sm23 was not released from the surface after cleavage with phosphatidylinositol-specific phospholipase C (PIPLC). (b) In a Triton X-114 phase-separation system, native [3H]ethanolamine- or [35S]methionine-labeled Sm23 partitioned into the detergent phase. Upon removal of the GPI anchor by PIPLC, the majority of the molecules stayed in the detergent-phase as expected of a transmembrane protein. (c) When full-length recombinant Sm23 was transcribed and translated in vitro, the polypeptide chain was inserted into microsomal membranes: Sm23 stayed associated with the membranes when they were incubated with carbonate buffer at pH 11.5, and membrane bound Sm23 was protected from digestion with proteinase K. (d) Recombinant Sm23, when expressed in the baculovirus expression system, was transported to the surface of infected insect cells, and similarly to the native protein it was not released from these cells after cleavage with PIPLC.
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PMID:Schistosoma mansoni: Sm23 is a transmembrane protein that also contains a glycosylphosphatidylinositol anchor. 816 Nov 93

ORF 1a of lactate dehydrogenase-elevating virus, strain P (LDV-P), encodes a protein of 2206 amino acids. Eisenberg hydrophobic moment analysis of the protein predicted the presence of eleven transmembrane segments in the C-terminal half of the molecule (amino acids 980-1852) that flank the serine protease domain. cDNAs encoding ORF 1a protein segments encompassing transmembrane segments 5 to 11 and its amphipathic C-terminal end as well as the N-terminal 80 amino acids of the downstream ORF 1b protein were transcribed and the transcripts in vitro translated in the absence and presence of microsomal membranes. The synthesis of the protein products with putative transmembrane segments was enhanced by the presence of the microsomal membranes and the proteins became membrane associated. When synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the membrane pellet when synthesized in the presence of membranes. Furthermore, the latter proteins were not released from the membranes by disruption of the membrane vesicles in carbonate buffer, pH 11.5, and large portions of the proteins were resistant to digestion by trypsin, chymotrypsin and proteinase K. No N-glycosylation was observed and only little, if any, processing of the protein by the putative serine protease. The results indicate that the C-terminal half of the ORF 1a protein represents a non-glycosylated integral membrane protein. Potential modes of synthesis and function of the protein are discussed. In addition, the results showed that the synthesis of the ORF 1a protein was generally terminated at its termination codon, but that read-through into the ORF 1b gene occurred with low frequency.
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PMID:Membrane association of the C-terminal half of the open reading frame 1a protein of lactate dehydrogenase-elevating virus. 877 92

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein G alpha i3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]methionine, two pools of GAIP--a soluble and a membrane-anchored pool--were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the G alpha i subfamily, G alpha i1, G alpha i2, G alpha i3, G alpha z, and G alpha o, but not with members of other G alpha subfamilies, G alpha s, G alpha q, and G alpha 12/13. The C terminus of G alpha i3 is important for binding because a 10-aa C-terminal truncation and a point mutant of G alpha i3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of G alpha i3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP's possible involvement in membrane trafficking.
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PMID:GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of G alpha i subunits. 898 88

The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for expression of the mitochondrial COX1 gene, which encodes subunit I of cytochrome c oxidase. In a pet309 null mutant, there is a defect both in accumulation of COX1 pre-RNA, if it contains introns, and in translation of COX1 RNAs [Manthey, G. M. & McEwen, J. E. (1995) EMBO J. 14, 4031-4043]. To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p was tagged at the carboxy terminus with an epitope from the human c-myc protein. A monoclonal antibody against the c-myc epitope detected a 98-kDa protein in mitochondria of yeast cells that expressed the PET309-c-myc fusion protein from a high copy number plasmid. This protein was not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector. Additional analyses of mitochondrial subfractions demonstrated that the PET309-c-myc fusion protein is localized specifically in the inner mitochondrial membrane. It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondria with a disrupted outer membrane). These results indicate that Pet309p spans the inner membrane, with domain(s) exposed to the intermembrane space side of the membrane. How Pet309p may function in concert with other gene products necessary for COX1 RNA translation or accumulation, such as Mss51p or Nam1p, respectively, is discussed.
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PMID:The Saccharomyces cerevisiae Pet309 protein is embedded in the mitochondrial inner membrane. 969 14

Poly(lactide-co-trimethylene carbonate)s were prepared by the lipase-catalyzed ring-opening copolymerization of lactide and trimethylene carbonate having carbonate content from 0 to 100%. Their thermal properties and enzymatic degradability were measured. The L,L-, D,D- and D,L-lactides were copolymerized with trimethylene carbonate by porcine pancreatic lipase to produce random copolymers having molecular weights of up to 21000. The glass transition temperature (Tg of the copolymer was dependent on the carbonate content and the Tg values linearly decreased from 35 degrees C (polylactide) to -8 degrees C [poly(trimethylene carbonate)]. Among the lipases tested, the porcine pancreatic lipase and proteinase K showed biodegradability towards poly(ester-carbonate)s.
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PMID:Novel lipase-catalyzed ring-opening copolymerization of lactide and trimethylene carbonate forming poly(ester carbonate)s. 1041 63

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an autosomal recessive neurodegenerative disorder caused by mutations in SURF1. Although SURF1 is ubiquitously expressed, its expression is lower in brain than in other highly aerobic tissues. All reported SURF1 mutations are loss of function, predicting a truncated protein (hSurf1) product. Western blot analysis with anti-hSurf1 antibodies demonstrated a specific 30 kDa protein in control fibroblasts, but no protein in LS patient cells. Steady-state levels of both nuclear- and mitochondrial-encoded COX subunits were also markedly reduced in patient cells, consistent with a failure to assemble or maintain a normal amount of the enzyme complex. An epitope (FLAG)-tagged hSurf1 was targeted to mitochondria in COS7 cells and a mitochondrial import assay showed that the hSurf1 precursor protein (35 kDa) was imported and processed to its mature form (30 kDa) in a membrane potential-dependent fashion. The protein was resistant to alkaline carbonate extraction and susceptible to proteinase K digestion in mitoplasts. Mutant proteins in which the N-terminal transmembrane domain or central loop were deleted, or the C-terminal transmembrane domain disrupted, did not accumulate and could not rescue COX activity in patient cells. Co-expression of the N- and C-terminal transmembrane domains as independent entities also failed to rescue the enzyme deficiency. These data demonstrate that hSurf1 is an integral inner membrane protein with an essential role in the assembly or maintenance of the COX complex and that insertion of both transmembrane domains in the intact protein is necessary for function.
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PMID:Expression and functional analysis of SURF1 in Leigh syndrome patients with cytochrome c oxidase deficiency. 1055 3

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.
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PMID:CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells. 1077 13


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