• Journal of Life Science
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• v.25 no.1
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• pp.1-7
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• 2015

Cyclin D is a member of the cyclin protein family, which plays a critical role as a core member of the mammalian cell cycle machinery. D-type cyclins (D1, D2, and D3) bind to and activate the cyclin-dependent kinases 4 and 6, which can then phosphorylate the retinoblastoma tumor suppressor gene products. This phosphorylation in turn leads to release or derepression of E2F transcription factors that promote progression from the G1 to S phase of the cell cycle. Among the D-type cyclins, cyclin D3 encoded by the CCND3 gene is one of the least well studied. In the present study, we have investigated the biochemistry of the proteolytic mechanism that leads to loss of cyclin D3 protein. Treatment of human prostate carcinoma PC-3-M cells with lovastatin and actinomycin D resulted in a loss of cyclin D3 protein that was completely reversible by the peptide aldehyde calpain inhibitor, LLnL. Additionally, using inhibitors for various proteolytic systems, we show that degradation of cyclin D3 protein involves the $Ca^{2+}$-activated neutral protease calpain. Moreover, the half-life of cyclin D3 protein half-life increased by at least 10-fold in PC-3M cells in response to the calpain inhibitor. We have also demonstrated that the transient expression of the calpain inhibitor calpastatin increased cyclin D3 protein in serum-starved NIH 3T3 cells. These data suggested that the function of cyclin D3 is regulated by $Ca^{2+}$-dependent protease calpain.

• BMB Reports
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• v.28 no.2
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• pp.138-142
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• 1995

An anticoagulant/fibrinolytic protease was purified to homogeneity from the earthworm Lumbricus rubellus. The protein was a single chain glycoprotein of 32 kDa that exhibited strong proteolytic activity on human thrombin and fibrin clots. Proteolytic degradation of these plasma proteins by the purified enzyme occurred at a neutral pH range. Among several human plasma proteins tested as possible substrates for the protease reaction, the 32 kDa enzyme specifically hydrolyzed both thrombin and fibrin polymers without affecting other proteins, such as serum albumin, immunoglobulin, and hemoglobin. Treatment of the purified enzyme at neutral pH with either phenylmethylsulfonylfluoride or soybean trypsin inhibitor resulted in a loss of catalytic activity. The enzyme hydrolyzed the chromogenic substrate H-D-Phe-L-Pipecolyl-L-Arg-p-nitroanilide with a $K_m$ value of 1.1 ${\mu}M$ at a neutral pH. These results suggest that the anticoagulant/fibrinolytic enzyme from Lumbricus rubellus is a member of the serine protease family having a trypsin-like active site, and one of the potential clevage sites for the enzyme is the carbonyl side of arginine residues in polypeptide chains.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.73-79
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• 2010

The Korea Polar Research Institute (KOPRI) has assembled a culture collection of cold-adapted bacterial strains from both the Arctic and Antarctic. To identify excellent protease-producers among the proteolytic bacterial collection (874 strains), 78 strains were selected in advance according to their relative activities and were subsequently re-examined for their extracellular protease activity on $0.1{\times}$ ZoBell plates supplemented with 1% skim milk at various temperatures. This rapid and direct screening method permitted the selection of a small group of 15 cold-adapted bacterial strains, belonging to either the genus Pseudoalteromonas (13 strains) or Flavobacterium (2 strains), that showed proteolytic activities at temperatures ranging between $5-15^{\circ}C$. The cold-active proteases from these strains were classified into four categories (serine protease, aspartic protease, cysteine protease, and metalloprotease) according to the extent of enzymatic inhibition by a class-specific protease inhibitor. Since highly active and/or cold-adapted proteases have the potential for industrial or commercial enzyme development, the protease-producing bacteria selected in this work will be studied as a valuable natural source of new proteases. Our results also highlight the relevance of the Antarctic for the isolation of protease-producing bacteria active at low temperatures.

• Journal of Life Science
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• v.6 no.4
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• pp.241-249
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• 1996

A collagenase was isolated from the culture filtrate of Vibrio mimicus (ATCC 33658). The enzyme was purified to homogeneity by ammonium sulfate precipitation and DEAE-Sephadex A-50 chromatography, which an activity recovery of 22%. The molecular weight of the purified enzyme was estimated to be 42 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration, indication a monomer structure. The optimum pH and temperature od the enzyme for insoluble collagen (Type I) were around 7.75 and 28$\circ$C, respectively. Some chelating agents and serine protease inhibitor inactivated the enzyme, but L-cysteine and histidine did not affect the activity. The amino acid composition indicated that the collagenase contained high amounts of amino acid residues of glycine and alanine. The K$_{m}$ and R$_{cat}$/K$_{m}$ values for the collagenase, using insoluble collagen (type I) as substrate, were 2.86 mg/ml and 972.28 U/mg-protein, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.2
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• pp.119-128
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• 2013

A protease inhibitor was fractionated from fish eggs using methods based on protein solubility. Fractionation efficiency was evaluated with regard to percent recovery and total inhibitory activity (U). The fractionation of protease inhibitor (PI) from egg extracts of skipjack tuna (ST, Katsuwonus pelamis), yellowfin tuna (YT, Thunnus albacores), and Alaska pollock (AP, Theragra chalcogramma) was performed by precipitation with cold acetone or ammonium sulfate (AS). Fractions exhibiting the strongest inhibitory activity contained 20-40% (v/v) cold acetone or 40-60% saturated AS fractions. AS fractionation was more effective in isolating PI than was precipitation with acetone. The total inhibitory activity and percent recovery of fraction obtained with AS 40-60% toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 4,976 U and 24.2% for ST, 3,331 U and 38.1% for YT, and 4,750 U and 43.8% for AP, respectively. In comparisons against six commercial proteases, 40-80% AS fractions, made by combining the 40-60% and 60-80% AS fractions from fish egg extract, exhibited the strongest inhibition of trypsin when using a casein substrate. These results suggest that fish eggs act as serine protease inhibitors and may be useful for protease inhibition in foodstuffs.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.667-667
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enganced by the increase of agitation speed. Two formas of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0 and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both protease were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were 50℃ and 45℃, respectively. About 56% of the original protease BK7-2 activity remained after being treated at 50℃ for 30 min but protease BK7-1 was rapidly inactivated at above 25℃. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Journal of Microbiology
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• v.41 no.3
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• pp.207-211
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• 2003

Extracellular protease, from Aeromonas hydrophila Ni 39, was purified 16.7-fold to electrophoretic homogeneity with an overall yield of 19.9％, through a purification procedure of acetone precipitation, and Q Sepharose and Sephacryl S-200 chromatographies. The isoelectric point of the enzyme was 6.0 and the molecular mass, as determined by Sephacryl S-200 HR chromatography, was found to be about 102 kDa. SDS/PAGE revealed that the enzyme consisted of two subunits, with molecular masses of 65.9 kDa. Under standard assay conditions, the apparent $K_{m}$ value of the enzyme toward casein was 0.32 mg/ml. About 90％ of the proteolytic activity remained after heating at 60$^{\circ}C$ for 30 min. The highest rate of azocasein hydrolysis for the enzyme was reached at 60$^{\circ}C$, and the optimum pH of the enzyme was 9.0. The enzyme was inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), by about 87.9％, but not by E64, EDTA, pepstatin or 1,10-phenanthroline. The enzyme activity was inhibited slightly by Ca$\^$2＋/, Mg$\^$2＋/ and Zn/supb 2＋/ ions.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.677-684
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{\circ}C$ and $45^{\circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{\circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{\circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.49-54
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• 2009

The mushroom Cordyceps militaris has been used for a long time in eastern Asia as a nutraceutical and in traditional Chinese medicine as a treatment for cancer patients. In the present study, a cytotoxic antifungal protease was purified from the dried fruiting bodies of C. militaris using anion-exchange chromatography on a DEAE-Sepharose column. Electrophoretic analyses indicated that this protein, designated C. militaris protein(CMP), has a molecular mass of 12 kDa and a pI of 5.1. The optimum conditions for protease activity were a temperature of $37^{\circ}C$ and pH of $7.0{\sim}9.0$. The enzyme activity was specifically inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. Amino acid composition of intact CMP and amino acid sequences of three major peptides from a tryptic digest of CMP were determined. CMP exerted strong antifungal effect against the growth of the fungus Fusarium oxysporum, and exhibited cytotoxicity against human breast and bladder cancer cells. These results indicate that C. militaris represents a source of a novel protein that might be applied in diverse biological and medicinal applications.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.63-63
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• 2003

$\alpha$$_1$-Antityrpsin is a member of the serine protease inhibitor (SERPIN) family that shares a common tertiary structure. The reactive site loop (RSL) of serpins is exposed at one end of the molecule for protease binding. Upon cleavage by a target protease, the RSL is inserted into the major $\beta$-sheet A, which is a necessary process for formation of a tight inhibitory complex. Various biochemical and structural studies suggest that the rate of the RSL insertion upon binding a target protease is critical for inhibitory activity, and it is thought that helix F region (thFs3A and helix F) located in front of $\beta$-sheet A, should be lifted for the loop insertion during complex formation.