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)

The orientation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by proteolytic degradation of purple membrane sheets, reconstituted vesicles, and whole cells, with the following results: (i) Bacteriorhodopsin in purple membrane sheets is cleaved at a single site by Pronase or trypsin; a polypeptide segment of about 15 amino acids is lost from the carboxyl end. Carboxypeptidase A sequentially releases amino acids from the carboxyl end; the tetrapeptide sequence -Ala-Ala-Thr-Ser(COOH) was tentatively deduced for this terminus. (ii) The apomembrane, which lacks retinal, undergoes a second cleavage with trypsin releasing a fragment of approximately 6300 molecular weight from the amino terminus. (iii) Vesicles reconstituted from the purple membrane sheets and synthetic lecithins, in which the direction of proton pumping is opposite to that in the whole cells, have the carboxyl terminus of bacteriorhodopsin accessible to proteolysis. (iv) In envelope vesicles, which largely pump protons in the same direction as the whole cells, the carboxyl terminus is largely protected against proteolysis. (v) Treatment of whole cells with proteinase K hydrolyzes the cell wall proteins but has no effect on acteriorhodopsin. However, the same treatment after lysis of the cells results in degradation of the hydrophilic region at the carboxyl terminus. The results show that the carboxyl terminus as well as the additional cleavage site near the amino terminus observed in apomembrane are on the cytoplasmic side of the purple membrane.
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PMID:Orientation of bacteriorhodopsin in Halobacterium halobium as studied by selective proteolysis. 27 65

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.
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PMID:Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel. 128 5

A homogeneous serine proteinase was isolated from cultural filtrates of the extreme halophilic bacteria Halobacterium mediterranei 1538 using affinity chromatography on bacitracin-Sepharose, ultrafiltration and gel filtration on Sephadex G-75, with a 48% yield and 260-fold purification. The enzyme was completely inactivated by specific inhibitors of serine proteinases, PMSF and DFP, as well as by Hg2+ and PCMB. The enzyme activity was strongly dependent of NaCl concentration, the enzyme being inactivated below 0.75 M NaCl. Inactivation of the enzyme was also seen in the presence of 2-7% organic solvents. The pH optimum for Glp-Ala-Ala-Leu-pNA hydrolysis is 8.0-8.5; Km is 0.14 mM, kcat is 36.9 s-1. The stability optimum lies at pH 5.5-8.0, temperature optimum is at 55 degrees C. The enzyme molecular weight is 41,000 Da; pI is 7.5. The substrate specificity of the enzyme is comparable to that of secretory subtilisins; the extent of protein substrate hydrolysis is similar to that of proteinase K. The N-terminal sequence of Halobacterium mediterranei serine proteinase, Asp-Thr-Ala-Asn-Asp-Pro-Lys-Tyr-Gly-Ser-Gln-Tyr-Ala-Pro-Gln-Lys-Val-Asn- Ala- Asp-, reveals a 50% homology with the aminoterminal sequence of Thermoactinomyces vulgaris serine proteinase. Hence, the serine proteinase secreted by halophilic bacteria may be considered as a structural and functional analog of eubacterial enzymes.
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PMID:[Serine proteinase from the archaebacterium Halobacterium mediterranei--an analog of eubacterium subtilisin]. 139 Dec 25

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.
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PMID:Binding of heparan sulfate to Staphylococcus aureus. 154 63

An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.
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PMID:Purification and characterization of an enterotoxin from Bacteroides fragilis. 154 60

A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins).
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PMID:A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins. 163 13

The codon of the catalytic serine in the active site of the vacuolar serine proteinase yscB (PrB) was changed to alanine, yielding the mutant gene prb1-Ala519. Following replacement of the wild-type PRB1 allele with prb1-Ala519, only a 73-kDa molecule was detected by immunoprecipitation with PrB-specific antiserum. The size of the mutant molecule corresponds to the unprocessed cytoplasmic precursor (pre-super-pro-PrB), as detected in sec61 mutants, when translocation into the endoplasmic reticulum is blocked. However, the mutant molecule is completely translocated into the secretory pathway, as indicated by protection from proteinase K digestion in spheroplast lysates in the absence of detergent. When N-glycosylation was inhibited in prb1-Ala519 mutant cells by tunicamycin, a smaller molecule of about 71 kDa appeared consistent with single N-glycosylation and signal-sequence cleavage of the translocated mutant PrB molecule in the endoplasmic reticulum. Thus, the active-site mutation prevents the wild-type processing of the N-glycosylated 73-kDa precursor of PrB to the 41.5 kDa pro-PrB in the endoplasmic reticulum. In order to characterize the processing of wild-type super-pro-PrB in more detail, we generated antibodies against the non-enzymatic superpeptide domain of the 73-kDa precursor expressed in Escherichia coli. We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDS/PAGE is generated in the endoplasmic reticulum. Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wild-type cells. Correspondingly, the superpeptide was virtually undetectable by immunoblotting wild-type cell extracts. In contrast, no degradation of radioactively labeled 41-kDa superpeptide was observed within 60 min in mutant strains deficient in the vacuolar proteinase yscA (PrA), in which maturation of vacuolar pro-PrB to active PrB is blocked. Accordingly, superpeptide antigenic material was readily detected by immunoblotting cell extracts and enriched in vacuolar preparations of PrA deficient mutant cells. These results indicate that the superpeptide and pro-PrB travel to the vacuole, where the superpeptide is rapidly degraded upon pro-PrB activation to PrB. Using purified vacuoles, rapid degradation of the superpeptide was reconstituted in vitro by addition of either mature PrA or mature PrB. However, the PrA-triggered in vitro degradation of the superpeptide required PrB activity, as this process was inhibited in the presence of the PrB inhibitor chymostatin.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biogenesis of the yeast vacuole (lysosome). Mutation in the active site of the vacuolar serine proteinase yscB abolishes proteolytic maturation of its 73-kDa precursor to the 41.5-kDa pro-enzyme and a newly detected 41-kDa peptide. 173 47

The crystal structure of the transition state analog complex formed covalently between proteinase K and methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethyl ketone was determined by x-ray diffraction methods at a resolution of 2.2 A and refined by constrained least squares to an R factor of 19.8% for the 11864 structure amplitudes greater than 1 sigma F. The chloromethyl ketone group is covalently linked with the active site functional groups His69(N epsilon) and Ser224(O gamma). The former has substituted for chlorine and the latter has attacked the carbon of the ketone group, thereby forming the tetrahedral carbon atom of the transition state analog. The peptide part of the inhibitor is in an extended conformation and fills subsites S1 to S5 of the substrate recognition site. Its backbone hydrogens bond with strands 100-104 and 132-136 of the substrate recognition site as the central strand of a three-stranded antiparallel beta-pleated sheet. This sheet formation is associated with a movement by approximately 1 A of strand 100-104 which is probably associated with the insertion of the bulky proline side chain. The methoxysuccinyl group is stacked on the phenolic side chain of Tyr104 that is a part of the bottom of the recognition site. Biochemical studies show that shorter inhibitors of this type are less effective than the longer one, because there are fewer hydrogen bonding and van der Waals/stacking interactions.
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PMID:Inhibition of proteinase K by methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethyl ketone. An x-ray study at 2.2-A resolution. 189 49

Co-translational protein translocation across the endoplasmic reticulum membrane is interrupted by particular amino acid sequences which are called stop-transfer sequences. Since the stop-transfer process should reflect the character of the protein translocation machinery, systematic examination on the structural requirements for stop-transfer sequences should give information about the translocation process. By the manipulation of the cDNA of interleukin 2, a typical secretory protein, the middle portion of the molecule was replaced with systematically constructed hydrophobic stretches, and two positively or negatively charged amino acid residues were introduced just behind the hydrophobic stretches. The modified proteins were synthesized with an in vitro transcription-translation system in the presence of dog pancreas rough microsomes, and their topologies in the membrane were examined with proteinase K digestion. The efficiency of stop-translocation depended on the hydrophobicity and the length of the inserted stretch. The segments followed by positively charged residues interrupted the translocation more efficiently than those with negatively charged residues. We observed that more than 19 alanine residues were required for efficient stop-translocation, whereas only 9 leucine residues were sufficient. We suggest that the positively charged residues following the hydrophobic stretches promote stop-translocation of the peptides through the channel.
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PMID:Systematic analysis of stop-transfer sequence for microsomal membrane. 202 23

Co-translational translocation of proteins across the membrane of rough endoplasmic reticulum (ER) is interrupted by particular amino acid sequences, which are functionally termed "stop-transfer sequence." We analyzed the structural requirements for the interruption of the peptide translocation. By the manipulation of the cDNA of interleukin 2 (IL2), which passes through ER membrane co-translationally, the middle portion of the IL2 molecule was replaced with systematically altered hydrophobic segments, leucine, alanine, or leucine/alanine mixed clusters. Furthermore, charged amino acid residues were introduced just downstream of the hydrophobic segments. These modified IL2 peptides were synthesized with wheat germ cell-free system in the presence of rough microsomes and the topology of the peptides in the microsomes was assessed by post-translational digestion with proteinase K. We obtained the following results. (i) Each modified protein was processed to the mature form but the extent of stop-translocation varied widely. The ratio of the stopped to the translocated products increased as the length and hydrophobicity of the inserted segment increased. (ii) Shorter hydrophobic segments than naturally occurring native transmembrane segment promoted stop-translocation. (iii) Proteins with hydrophobic segments followed by positive charges were more efficiently stop-translocated than those having negative charges. (iv) If the hydrophobicity of the segment was sufficiently high, the positive charges after the segment were not essential for stop-translocation. We also suggest that the stop-transfer process includes protein-protein interaction between the hydrophobic segment and translocation channel.
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PMID:Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane. 208 36


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