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)

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography pand gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000-24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.
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PMID:Isolation of specific protease inhibitors from Neurospora crassa. 13 53

The objective of this research was to formulate a mixture of commercial proteases that would mimic the rate and extent of protein degradation obtained using strained ruminal fluid. The proteolytic activity of strained ruminal fluid and several commercial proteases was characterized using 13 L-amino acid p-nitroanilides as artificial substrates. A mixture of Streptomyces griseus protease, chymotrypsin, and proteinase K at .042, 2.5, and .5 enzyme units/mL, respectively, was similar to the activity of strained ruminal fluid against the same artificial substrates. However, degradative activities were different in incubations with feed proteins as substrates. The rates of degradation of expeller soybean meal, solvent soybean meal, and casein were .08, .05, and .08/h, respectively, using the enzyme mixture and .03, .15, and .24/h using strained ruminal fluid. A second experiment compared degradative activity of S. griseus protease at .066 enzyme units/mL, ficin at .5 enzyme units/mL, and a mixture of trypsin, carboxypeptidase B, chymotrypsin, and carboxypeptidase A at 116.6, .5, 2.5, and .5 enzyme units/mL, respectively. Protein degradation rates obtained with strained ruminal fluid were two to six times faster than those obtained with the enzyme mixtures. A third experiment compared the degradability of 15 feed proteins with the mixture of trypsin, carboxypeptidase B, chymotrypsin and carboxypeptidase A to that with strained ruminal fluid. Degradation rates obtained using strained ruminal fluid ranged from .007 to .217/h; degradation rates using the enzyme mixture ranged from .010 to .079/h and were lower (P = .004) than with strained ruminal fluid. Overall, the experiments indicated that the commercial enzymes tested did not mimic the protein degradative activity of strained ruminal fluid.
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PMID:Characterization of the proteolytic activity of commercial proteases and strained ruminal fluid. 870 28

Bacterial surface hydrophobicity (SH) plays a role in adhesion of bacteria to host surfaces and ingestion by phagocytic cells. Streptococcus dysgalactiae (n = 60) isolated from bovine intramammary infections were examined for expression of SH after growth in Todd-Hewitt broth (THB) and THB supplemented with skim milk, whey, lactose, and casein. Strains were significantly more hydrophobic after growth in THB and THB plus whey and more hydrophilic after growth in THB plus skim milk. Both trypsin and proteinase K abolished SH in three strains tested. Mild pepsin treatment had little effect on SH, while heat treatment at 70 degrees or 80 degrees C abolished SH in two strains tested. A hydrophilic strain of S. dysgalactiae did not adhere as well to bovine mammary epithelial cells as a hydrophobic strain. Trypsin treatment significantly reduced adherence of a hydrophobic strain of S. dysgalactiae to epithelial cells while adherence of a hydrophilic strain remained unaltered. A hydrophilic strain of S. dysgalactiae was significantly more resistant to phagocytosis by bovine mammary gland macrophages than a hydrophobic strain. Differences in expression of SH may play an important role in determining the ability of S. dysgalactiae to establish successfully within the mammary gland.
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PMID:Influence of Streptococcus dysgalactiae surface hydrophobicity on adherence to mammary epithelial cells and phagocytosis by mammary macrophages. 877 7

BODIPY-alpha-casein is a new fluorescent protein substrate designed for fluorescence polarization studies to measure proteolytic activity at any pH over the range from pH 2 to 11. Kinetic protease assays in real-time were performed in 1 to 5 min using an FPM-1 fluorescence polarization instrument. A purified enzyme or bacterial culture was mixed with the BODIPY-alpha-casein in a buffer of an appropriate pH and the decrease in fluorescence polarization was automatically recorded at 0.5-min intervals. The initial decrease in fluorescence polarization with time was dependent on protease concentration. In 3-min assays at 37 degrees C, the sensitivity of detection was 8 mU for pepsin at pH 2.0, 1 mU for papain at pH 6.0, 0.6 mU for proteinase K at pH 7.4, and 2 mU for Streptomyces griseus alkaline protease at pH 11. Only 1-10 microliters of a growing culture was necessary to assay the protease activity of Porphyromonas gingivalis or Treponema denticola, oral bacteria that possess certain proteases on their surfaces. These assays have clinical applications, since certain pathogens use proteolytic activity as a virulence mechanism and differ from their nonpathogenic counterparts in this characteristic. Fluorescence polarization assays are simple, rapid, and reproducible.
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PMID:BODIPY-alpha-casein, a pH-independent protein substrate for protease assays using fluorescence polarization. 895 19

Mammalian embryos are known to exhibit delayed development and have lower hatching rates in vitro than in vivo because of inadequate culture condition. These discrepancies may be due to a deficiency of the paracrine factors and proteolytic enzymes which exist in the oviduct and uterus. In order to evaluate the effects of proteases on embryonic development and hatching, 2-cell mouse embryos were cultured for 72 h with or without proteases. The addition of 1.0 microg/ml pronase (PE) and/or 0.1 microg/ml proteinase K (PK) did not affect embryonic development up to the blastocyst stage (94.1% versus 88.2%; 92.2% versus 90.2%, respectively) but significantly increased the hatching rate (60.4% versus 39.2%, 71.8% versus 35.3%, respectively). However, the addition of alpha-chymotrypsin (Chymo) was detrimental to embryonic development and hatching. Changes in the structure of the zona pellucida (ZP) structure of embryos which had been cultured in human tubal fluid (HTF) medium with PE and PK were assessed by fluorescein isothiocyanate-conjugated (FITC)-casein. Embryos cultured in HTF-PE and PK were not stained with FITC-casein. When these embryos were cultured within oviducts, their perivitelline space (PVS) became strongly stained with FITC-casein which was easily removed by phosphate-buffered saline washing. This suggests that PE and PK altered the structure of the ZP. We suggest that the addition of PE and PK to culture media may accelerate the hatching of embryo, by structurally altering the ZP and PVS. This may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.
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PMID:The supplementation of culture medium with protease improves the hatching rate of mouse embryos. 943 92

Proteolytic activity of polyclonal IgG antibodies (Abs) from the blood of AIDS patients was analyzed for the first time. These Abs were shown to display higher activity in hydrolysis of beta-casein than in hydrolysis of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT) or human serum albumin (HSA). Several abzymatic criteria were applied and it was shown that RT, HSA, and beta-casein hydrolyzing activities are an intrinsic property of polyclonal Abs from AIDS patients. Casein-hydrolyzing Abs were detected in the blood serum for 95% of AIDS patients, and it was shown that they possess serine protease-like catalytic activity. The substrate specificities of polyclonal Ab proteases and typical human proteases are different. Depending on the patient, the IgGs exhibit various pH optima of proteolytic activity. The products of casein hydrolysis by Ab proteases were different from those in the case of trypsin, chymotrypsin, and proteinase K.
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PMID:Proteolytic activity of IgG antibodies from blood of acquired immunodeficiency syndrome patients. 1654 61

We report the initial characterization and expression of sfp2, a gene encoding a keratinolytic serine protease from Streptomyces fradiae var. k11. Recombinant SFP2 was expressed in and secreted from the yeast Pichia pastoris with a final yield of 78 mg/L (136.2 U/mL caseinolytic activity) after 25 h of induction. The recombinant enzyme was purified using by ammonium sulfate precipitation and gel filtration chromatography to electrophoretic homogeneity, which was appropriately glycosylated and had a molecular mass of 26.0 kDa. The purified recombinant SFP2 was characterized. The optimal pHs and temperatures of SFP2 for proteolysis of casein and keratin azure were pH 10.0, 60 degrees C, and pH 9.0, 55 degrees C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co(2+) and Cr(3+) and enhanced by Ni(2+) and Cu(2+). The K(m) of 0.45 mmol/L and V(max) of 19.84 mmol/min mg were calculated using N-succinyl-Ala-Ala-Pro-Phe-pNA as a substrate. We tested the activity of SFP2 with soluble and insoluble substrates; SFP2 was more specific for keratinous substrates compared with proteinase K and other commercial proteases.
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PMID:Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris. 1740 67

Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi- phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.
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PMID:Improved detection of multi-phosphorylated peptides by LC-MS/MS without phosphopeptide enrichment. 1764 8

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.
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PMID:Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum. 1765 25

This article describes a fluorescence polarization (FP)-based protease assay on a microfluidic device that is compatible with fast and reproducible analyses of protease activities. The optical systems were arranged for simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes, and the binding of tetramethylrhodamine (TMR) labeled-biotin with streptavidin was utilized for optimizing FP detection in continuously flowing solutions within 74-microm wide, 12-microm deep microchannels of a glass chip. In developing off-chip FP-based assays for proteinase K, trypsin, papain and elastase, TMR conjugated-casein protein (TMR-alpha-casein) was employed as a universal substrate. After optimization of the hydrodynamic flow control to allow complete mixing of TMR-alpha-casein and short proteolysis time as possible, and of buffer composition to minimize protein sticking problems, the developed assay was transferred to the microfluidic chip by monitoring FP changes of TMR-alpha-casein in the main microchannel. The results indicate that the proposed device would serve as an integrated microfluidic platform with automated injection of reacting species, diffusion-controlled mixing, reaction and detection for protease activities without the need to separate the products.
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PMID:A microfluidic protease activity assay based on the detection of fluorescence polarization. 1772 68


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