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

The present study was undertaken to investigate the Ca2+ binding properties of sarcoplasmic reticulum Ca(2+)-ATPase after removal of the cytoplasmic regions by treatment with proteinase K. One of the proteolysis cleavage sites (at the end of M6) was found unexpectedly close to the predicted membrane-water interphase, but otherwise the cleavage pattern was consistent with the presence of 10 transmembrane ATPase segments. C-terminal membranous peptides containing the putative transmembrane segments M7 to M10 accumulated after prolonged proteolysis, as well as large water-soluble fragments containing most of the phosphorylation and ATP-binding domain. Ca2+ binding was intact after cleavage of the polypeptide chain in the N-terminal region, but cuts at other locations disrupted the high affinity binding and sequential dissociation properties characteristic of native sarcoplasmic reticulum, leaving the translocation sites with only weak affinity for Ca2+. High affinity Ca2+ binding could only be maintained when proteolysis and subsequent manipulations took place in the presence of a Ca2+ concentration high enough to ensure permanent occupation of the binding sites with Ca2+. We conclude that in the absence of Ca2+, the complex of membrane-spanning segments in proteolyzed Ca(2+)-ATPase is labile, probably because of relatively free movement or rearrangement of individual segments. Our study, which is discussed in relation to results obtained on Na+,K(+)-ATPase and H+,K(+)-ATPase, emphasizes the importance of the cytosolic segments of the main polypeptide chain in exerting constraints on the intramembranous domain of a P-type ATPase.
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PMID:Do transmembrane segments in proteolyzed sarcoplasmic reticulum Ca(2+)-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K? 765 31

The 53 kDa glycoprotein from sarcoplasmic reticulum was shown to be protected from proteolysis by trypsin, V8 proteinase and proteinase K in intact vesicles yet readily digested in the presence of the non-denaturing detergent C12E8. Competitive ELISAs with a library of seven monoclonal antibodies raised against the 53 kDa glycoprotein showed that the epitopes for these antibodies were only accessible in C12E8 solubilised and not intact sarcoplasmic reticulum. When the monoclonal antibodies against the 53 kDa glycoprotein were assessed for their effect on the uptake of Ca2+ by sarcoplasmic reticulum no effect was detected; neither were these antibodies able to augment the inhibitory influences of anti-(Ca(2+)-Mg2+)-ATPase monoclonal antibodies on Ca2+ uptake. These data indicate that the 53 kDa glycoprotein is located in the lumen of the sarcoplasmic reticulum.
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PMID:Evidence for the lumenal location of the 53 kDa glycoprotein of sarcoplasmic reticulum. 768 Sep 1

Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei.
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PMID:High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei. 775 27

To localize transmembrane segments in the carboxyl-terminal third of the Neurospora plasma membrane H(+)-ATPase, we constructed fusion proteins on the cDNA level. These contained DNA fragments encoding hydrophilic residues of the amino and carboxyl termini of the H(+)-ATPase with a DNA fragment encoding the putative transmembrane segment. To report translocation into microsomes, a DNA fragment encoding three consensus N-linked glycosylation sites was engineered carboxyl-terminal to the putative transmembrane segment. Fusion proteins were synthesized in a Neurospora in vitro translation system supplemented with homologous microsomes. By the criteria of glycosylation of fusion proteins by microsomes, sedimentation of products with microsomes after alkaline extraction, and analysis of protected fragments generated from proteinase K digestion of integrated products, we localized six transmembrane segments in the carboxyl-terminal third of the H(+)-ATPase. These results support a 10-segment model of the Neurospora H(+)-ATPase.
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PMID:The membrane topology of the carboxyl-terminal third of the Neurospora plasma membrane H(+)-ATPase. 789 44

The purified (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum was subjected to extensive proteolysis by using trypsin and proteinase K. This digestion led to the elimination of a considerable portion of the protein, so that the lipid to protein weight ratio was increased from 0.44 in the purified ATPase to 1.20 after extensive proteolysis. After the digestion, the residue was found to be considerably enriched in hydrophobic amino acids. FT-IR spectroscopic studies indicated that the secondary structure of the proteolytic residue was enriched in alpha-helix with 75%, compared with 48% in the intact purified ATPase. FT-IR studies using ATR polarization showed that the alpha-helical part of the residue of proteolytic digestion was considerably more polarized than the purified ATPase, indicating that, on average, the alpha-helices of the residual protein should lie with an orientation closer to the normal to the plane of the membrane. Thermal denaturation studies showed that the residue of proteolysis was considerably more stable than the intact purified ATPase. This would be compatible with the residue being protected from denaturation by its hydrophobic location within the membrane. This study is experimental evidence of the alpha-helical structure of the membrane part of this protein, as suggested by predictions made from its known primary structure (Brandl et al., 1986).
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PMID:Extensive proteolytic digestion of the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum leads to a highly hydrophobic proteinaceous residue with a mainly alpha-helical structure. 803 59

The Neurospora plasma membrane H(+)-ATPase is a polytopic integral membrane protein. To localize transmembrane segments, mutants were constructed that contained the amino and carboxyl termini of the H(+)-ATPase with putative transmembrane segment. A stretch of amino acid residues from yeast invertase that has three consensus N-linked glycosylation sites was placed carboxyl terminal of the putative transmembrane segment. RNA transcripts of these mutants were translated in a Neurospora in vitro system that was supplemented with microsomes from Neurospora. By the criteria of glycosylation of the polypeptide chain, resistance to extraction at pH 11.5, and protection from proteinase K digestion, only one transmembrane segment could be identified within the amino acid residues 272-314 of the primary sequence of the H(+)-ATPase.
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PMID:Topology of the Neurospora plasma membrane H(+)-ATPase. Localization of a transmembrane segment. 810 34

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3'-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A novel method for measuring protein expression at the cell surface. 812 1

Membrane topology of the H+,K+-ATPase has been studied after proteolytic degradation of the protein by proteinase K. Proteinase K had access to either the cytoplasmic part of the protein or to both sides of the membrane. Fourier transform infrared attenuated total reflection spectroscopy indicated that membrane-associated domain of the protein represented about 55% of the native protein, meanwhile the cytoplasmic part represented only 27% of the protein. The secondary structure of the ATPase and of its membrane-associated domains was investigated by infrared spectroscopy. The secondary structure of the membrane-associated structures and of the entire protein was quite similar (alpha-helices, 35%; beta-sheets, 35%; turns, 20%; random, 15%). These data were in agreement with 10 alpha-helical transmembrane segments but suggested a participation of beta-sheet structures in the membrane-associated part of the protein. Polarized infrared spectroscopy indicated that the alpha-helices were oriented nearly perpendicular to the membrane plane. No preferential orientation could be attributed to the beta-sheets. Monitoring the amide hydrogen/deuterium exchange kinetics demonstrated that the membrane associated part of the ATPase molecule is characterized by a relatively high accessibility to the solvent, quite different from that observed for bacteriorhodopsin membrane segments.
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PMID:Fourier transform infrared spectroscopy study of the secondary structure of the gastric H+,K+-ATPase and of its membrane-associated proteolytic peptides. 899 57

Differential scanning calorimetry has been used to characterize the thermal denaturation of gastric (H+,K+)-ATPase. The excess heat capacity function of (H+,K+)-ATPase in highly oriented gastric vesicles displays two peaks at 53.9 degrees C (Tm1) and 61.8 degrees C (Tm2). Its thermal denaturation is an irreversible process that does not exhibit kinetic control and can be resolved in two independent two-state processes. They can be assigned to two cooperative domains located in the cytoplasmic loops of the alpha-subunit, according to the disappearance of the endothermic signal upon removal of these regions by proteinase K digestion. Analysis of the thermal-induced unfolding of the enzyme trapped in different catalytic cycle intermediates has allowed us to get insight into the E1-E2 conformational change. In the E1 forms both transitions are always observed. As Tm1 is shifted to Tm2 by vanadate and ATP interaction, the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdomain interactions. Stabilization of the E2 forms results in the disappearance of the second transition at saturation by K+, Mg2+-ATP, and Mg2+-vanadate as well as in significant changes in Tm2 and DeltaH1. The catalytic domain melts following a process in which intermolecular interactions either in the native or in the unfolded state might be involved. Interestingly, the E2-vanadate-K+ form displays intermediate properties between the E1 and E2 conformational families.
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PMID:Structural domain organization of gastric H+,K+-ATPase and its rearrangement during the catalytic cycle. 899 35

The trivalent cation aluminum can cause chronic cytotoxicity in plants, animals and microorganisms. It has been suggested that Al interaction with cell membranes and enzyme metal binding sites may be involved in Al cytotoxicity. In this study, the binding of Al to microsomes and liposomes was found to be lipid dependent with the signal transduction element phosphatidylinositol-4,5-bisphosphate having the highest affinity for Al with an Al:lipid stoichiometry of 1:1. Al binding was only reduced in the presence of high concentrations of Ca2+ (> 1 mM). Both citrate and, to a lesser extent, malate were capable of preventing Al lipid binding, which is consistent with the involvement of these organic acids in a recently described Al detoxification mechanism in plants. The effects of AICl3, Al-citrate and ZnSO4 on metal-dependent enzyme activities (enolase, pyruvate kinase, H+-ATPase, myosin, Calpain, proteinase K, phospholipase A2 and arginase) was assayed in vitro. While Zn2+ was capable of inhibiting all the enzymes except the H+-ATPase, AlCl3 and Al-citrate had minimal effects except for with phospholipase A2 where an interaction with AlCl3 occurred. However, this could be negated by the addition of citrate. The results indicate that, contrary to current hypotheses, the toxic mode of Al is not through an interaction with enzymatic catalytic metal binding sites but may be through the interaction with specific membrane lipids.
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PMID:Aluminum interaction with plasma membrane lipids and enzyme metal binding sites and its potential role in Al cytotoxicity. 900 May 12


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