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)

Affinity chromatography on cyclic AMP columns allowed a two-step isolation of the cyclic-AMP-binding proteins from bovine kidney cytosol. An AMP-binding protein (apparent molecular weight approximately 60 000) and large amounts of a low affinity binding protein ('P35'; apparent subunit size approximately 35 000) were obtained in practically pure form besides the high affinity binding proteins of the R type. Among the R proteins the dimer R2 of the regulatory subunit of protein kinase II (apparent subunit size approximately 54 000) represented the bulk material. Small amounts of monomer, of higher aggregates, and of a protein 'P49' (subunit size approximately 49 000) presumably identical with the regulatory subunit of protein kinase I were also detected. The R protein fraction of kidney also contained a high affinity binding protein of smaller size (designated as R'; molecular weight approximately 37 000) which appeared to be derived from protein R2 of protein kinase II by limited proteolysis. At all stages of purification, R protein and its aggregates could be quantitatively transformed into R' protein (or a closely related polypeptide) by several proteases including the relatively unspecific proteinase K. The degradation product exhibited unchanged cyclic-AMP-binding capacities but had largely lost the ability to inhibit the catalytic subunit C of protein kinase, to be phosphorylated by C, and to form a dimer. Preliminary experiments indicate that protein R' may be a natural component of kidney tissue.
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PMID:Adenosine-3':5'-monophosphate-binding proteins from bovine kidney. Isolation by affinity chromatography and limited proteolysis of the regulatory subunit of protein kinase II. 20 61

Among 21 different polysaccharides tested, 5 greatly enhanced the spontaneous and cyclic AMP-induced formation of exolipase: glycogen, hyaluronate, laminarin, pectin B, and gum arabic. These polysaccharides have in common the tendency to form highly ordered networks because of the branching or helical arrangement, or both, of their molecules. None of the polysaccharides could be utilized by the cells as the sole carbon source. Strong lipid extraction of four different polysaccharides did not reduce their exolipase-enhancing efficacy. At a constant cell density the stimulation of exolipase formation by various concentrations of glycogen followed saturation kinetics, suggesting a limited number of "sites" for the glycogen to act. The active principle present in a solution of pectin was destroyed by degradation (beta-elimination) of the polymer. Hyaluronate lost its exolipase-enhancing activity by exhaustive hydrolysis with hyaluronidase but was resistant to proteinase K. Exopolysaccharide, isolated from growth medium of Serratia marcescens SM-6, enhanced the exolipase formation as efficiently as hyaluronate. The results of this work are discussed mainly in terms of the "detachment hypothesis."
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PMID:Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. 22 24

The proton motive force (delta mu H+) plays an important role, although it is not absolutely essential, in the in vitro translocation of secretory proteins, such as OmpA, across the cytoplasmic membrane of Escherichia coli (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The transient accumulation in membrane vesicles of a possible translocation intermediate of OmpA was observed in the absence of delta mu H+. The intermediate was detected on a polyacrylamide gel as a proteinase K-resistant band corresponding to a molecular weight of 26,000. The intermediate did not possess the signal peptide. The appearance of this band was inhibited in the absence of ATP or the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) and enhanced upon the addition of SecA. Upon the addition of NADH that energizes the membrane, the intermediate was converted to the translocated form of OmpA, even in the presence of AMP-PNP. These results suggest different requirements of ATP and delta mu H+ for the early and late stages of the translocation reaction. The SecA requirement for the early stage of the translocation has also been suggested. In addition to this band, two other bands were observed at higher positions on the gel, when the translocation reaction was performed in the absence of delta mu H+. Although these two bands also represented the mature form of OmpA, which was partly protected from the proteinase K treatment by the membrane vesicles, the accumulation was not transient. These bands did not appear when the translocation reaction was performed in the presence of dithiothreitol. Together with other evidence, the above observations suggest that OmpA, which has an intramolecular disulfide bridge, cannot undergo the translocation unless delta mu H+ is imposed.
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PMID:In vitro analysis of the process of translocation of OmpA across the Escherichia coli cytoplasmic membrane. A translocation intermediate accumulates transiently in the absence of the proton motive force. 255 15

The homopolymer of ADP-ribose, poly(ADP-ribose), was synthesized in vitro by liver nuclei from NAD. The protein-poly(ADP-ribose) adducts were isolated and, after base hydrolysis or proteolysis by proteinase K, the free polymers were separated from NAD, ADP-ribose, AMP and adenosine, and quantitatively determined by reversed-phase chromatography on an Ultrasphere ODS 5-micron column. Oxidation of the polymer by sodium periodate and labeling with 3H by borotritiation maintained the polymeric structure, but its modification was detectable by the chromatographic system employed.
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PMID:Separation of poly(ADP-ribose) by high-performance liquid chromatography. 609 Apr 88

An endonuclease activity associated with purified proteinase K-treated intracisternal A-particles was identified and characterized. The activity required divalent cations, preferring Mn2+ to Mg2+. Salt concentrations above 50 mM inhibited the activity. The endonuclease was greatly stimulated by ATP, ADP, and dATP, whereas AMP appeared to produce a slight inhibition. GTP had no apparent effect on the activity. The enzyme introduced single-stranded nicks into DNA and nicked preferentially supercoiled DNA duplexes in the presence of ATP, although linear duplexes also functioned as substrates. Single-stranded DNA was not nicked to any great extent. The molecular weight of the enzyme was estimated to be about 40,000. The characteristics of this enzyme are very similar to those of the endonuclease found associated with Friend murine leukemia virus.
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PMID:Properties of an intracisternal A-particle-associated endonuclease activity which is stimulated by ATP. 627 25

Mitochondrial gene expression in kinetoplastid organisms such as Trypanosoma, Leishmania and Crithidia requires a posttranscriptional RNA processing event known as kRNA editing. During editing, uridine nucleotides get inserted and deleted into pre-mRNAs directed by small, metabolically stable RNAs, termed guide RNAs. Although the precise mechanism of the reaction is not understood, the accepted working model describes the formation of extended anti-parallel RNA helices between gRNA molecules with pre- and partially edited mRNAs as intermediates. These duplex structures must be separated to ensure the sequential action of multiple gRNAs in a 3' to 5' polarity on the mRNA molecule. In spite of this fact, no unwinding activity has heretofore been identified in kinetoplastid mitochondria. We report the characterisation of a RNA helicase activity within Trypanosoma brucei mitochondrial extracts. The activity unwinds 25- and 48 bp, tailed RNA duplex structures but fails to separate DNA strands. It can be destroyed by heat denaturation as well as by proteinase K treatment. The activity requires magnesium cations and acts in a NTP/dNTP dependent manner. Hydrolysis of a nucleoside triphosphate is required rather than mere NTP binding as deduced from a comparison of unwinding in the presence of ATP and AMP-PCP. RNA duplexes mimicking presumed kRNA editing intermediates are substrates of the unwinding activity and therefore, we address the possible involvement of a RNA helicase activity during kRNA editing.
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PMID:Trypanosoma brucei mitochondria contain RNA helicase activity. 752 33

In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.
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PMID:A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates. 754 14

The gene for a new outer membrane-associated protease, designated OmpP, of Escherichia coli has been cloned and sequenced. The gene encodes a 315-residue precursor protein possessing a 23-residue signal sequence. Including conservative substitutions and omitting the signal peptides, OmpP is 87% identical to the outer membrane protease OmpT. OmpP possessed the same enzymatic activity as OmpT. Immuno-electron microscopy demonstrated the exposure of the protein at the cell surface. Digestion of intact cells with proteinase K removed 155 N-terminal residues of OmpP, while the C-terminal half remained protected. It is possible that much of this N-terminal part is cell surface exposed and carries the enzymatic activity. Synthesis of OmpP was found to be thermoregulated, as is the expression of ompT (i.e., there is a low rate of synthesis at low temperatures) and, in addition, was found to be controlled by the cyclic AMP system.
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PMID:New outer membrane-associated protease of Escherichia coli K-12. 828 30

The ADPglucose pyrophosphorylase (EC 2.7.7.27) from Escherichia coli is allosterically activated by fructose 1,6-bisphosphate and inhibited by AMP. Proteolysis of the enzyme with proteinase K causes loss of activity and generates two peptides, 21 and 28 kDa, from the 49.7-kDa subunit. The presence of ADPglucose, Mg2+, and fructose 1, 6-bisphosphate during the incubation with proteinase K protected the enzyme activity and prevented cleavage at sites Met181-Ala182 and Phe192-Val193. Proteolysis of the protected enzyme removed 10 to 13 amino acids from the N-terminal and 2 amino acids from the C-terminal. The resulting enzyme was almost independent of the need for fructose 1,6-bisphosphate for maximal activity and insensitive to inhibition by AMP. The apparent affinity for the substrates was similar to that of the fully-activated wild-type enzyme. These data suggest that amino acid residues in the N-terminal portion and possibly the C-terminal portion of ADPglucose pyrophosphorylase are part of the regulatory domain of the enzyme, critical for allosteric regulation of the enzyme.
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PMID:The N-terminal region is important for the allosteric activation and inhibition of the Escherichia coli ADP-glucose pyrophosphorylase. 975 Jan 79

Protection of the Ca2+ATPase (SERCA) from proteinase K digestion has been observed following the addition of Ca2+, Mg2+, and nucleotide and interpreted as a substrate-dependent conformational change (1). The protected digestion site is located on the loop connecting the A domain and the M3 transmembrane helix. We studied by mutational analysis the protective effect of AMP-PCP, an ATP analog that is not utilized for enzyme phosphorylation. We found that the nucleotide protective effect is interfered with by single mutations of Arg-560 and Glu-439 in the N domain and Lys-352, Lys-684, Thr-353, Asp-703, and Asp-707 in the P domain. This is consistent with a transition from the open to the compact configuration of the ATPase headpiece and approximation of the N and P domains by interactions with the nucleotide adenosine and phosphate moieties, respectively. The A domain-M3 loop is consequently involved. Protection by nucleotide substrate increased following the mutations of Asp-351 (the residue undergoing phosphorylation by ATP) and neighboring Asn-706 to Ala, underlying the importance of side chain specificity in positioning the nucleotide terminal phosphate and limiting the stability of the substrate-enzyme complex. Protection is not observed when AMP-PCP is added in the absence of Ca2+ or following mutations (E771Q or N796A) that interfere with Ca2+ binding. Therefore, nucleotide binds to the Ca2+-activated enzyme in the open headpiece conformation and the consequent approximation of the N and P domains occurs while the transmembrane domain is still in the Ca2+-bound conformation. Mg2+ is not required for the protective effect of nucleotide, even though it is specifically required for the subsequent catalytic reactions.
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PMID:Substrate-induced conformational fit and headpiece closure in the Ca2+ATPase (SERCA). 1275 Mar 73


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