Evidence that neutral protease from calf thymus chromatin is a serine type enzyme

Kurecki, T; Kowalska-Loth, Barbara; Toczko, Kazimierz; Chmielewska, Izabela

doi:10.1016/0014-5793(75)80044-5

Cited by 17 publications

(12 citation statements)

References 5 publications

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“…The procedure for the isolation of these proteases usually involves extracting purified nuclei with 24 mM EDTA [41] and then separating the protease from the chromatin by extraction with high salt [42]. In general these proteases are not Cazf-activated [42-471, degrade specific histones (H2A [42], lysine-rich histones [45], H1, H2A, H2B and H4 [44]) and are inhibited by soybean trypsin inhibitor [46]. In summary, the protease described here has characteristics different from those described above; in particular the protease can be separated from the nucleus with 3 mM EDTA in buffers of low ionic strength.…”

Section: Discussionmentioning

confidence: 99%

Properties of a Ca²⁺‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells

Nelson

Traub

1981

European Journal of Biochemistry

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A Ca2 +-activated neutral protease is described which, when tested against various native proteins, appears to be specific for vimentin, the 58000-M, subunit protein of intermediate-sized (7-11 nm) filaments in Ehrlichascites-tumour cells. The protein subunits of other classes of intermediate-sized filaments have been tested ; neurofilament protein and glial fibrillary acidic protein are not degraded, however skeletin, the subunit protein of intermediate-sized filaments in smooth muscle, is degraded. The protease is found associated with the detergentresistant cytoskeleton of Ehrlich-ascites-tumour cells; proteins, other than vimentin, present in this structure are not degraded. The protease is activated by Ca2+ and Sr2+ but not by other divalent cations tested: the CaZf concentration required for activation is 10 pM. The pH optimum is between pH 7.5 and 8.0, and the KCI concentration required for optimal activity is 100 mM. The protease is inhibited by 1 -chloro-3-tosylamido-7-amino-L-2-heptanone hydrochloride and L-1-tosylamido-2-phenylethyl chloromethyl ketone but not by soybean trypsin inhibitor; inhibition by phenylmethylsulphonyl fluoride is moderate. The high substrate specificity of the protease suggests it may play a role in vimentin intermediate-sized filament protein turnover in Ehrlich-ascitestumour cells

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Section: Discussionmentioning

confidence: 99%

Properties of a Ca²⁺‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells

Nelson

Traub

1981

European Journal of Biochemistry

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“…Many kinds of cellular serine proteinase have been reported. Some enzymes of nuclear origin are thought to degrade histones, non-histone proteins, and ribosomal proteins (26)(27)(28)(29)(30). However, our enzyme did not hydrolyze histones.…”

Section: Discussionmentioning

confidence: 77%

A Serine Proteinase from Cow Snout Epidermis Degrades High Molecular Weight Keratins

Toku

Inoue

Nakada

1986

The Journal of Dermatology

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This study describes a serine proteinase which degrades high molecular weight polypeptides of keratins. The enzyme was coextracted with 8M urea together with keratins and separated from the keratins by gel filtration chromatography. Molecular weight of the enzyme was estimated at 25,000 daltons by gel filtration. Revealed characteristics of the enzyme were as follows; 1) maximally active at pH 8.5-9.0, 2) requires 0.04% SDS for maximum activity, 3) heat stable in the absence of SDS up to 65°C for 2 min, 4) sensitive against serine protease inhibitor, phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, 5) highly specific for natural protein substrates, such as keratin polypeptides.

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“…Our thiol protease was stimulated 3 -4-fold by addition of a low concentration of DNA (2.5 pg/ 1.0 ml) as well as RNA (data not shown). Some other pro-teases belong to the class of serine proteases [40,411, and two belong to the class of thiol proteases [44,46]. The optimal pH of one of the thiol proteases (29 kDa) was pH 4.5 and was shifted to pH 5.5 by the addition of DNA.…”

Section: Discussionmentioning

confidence: 99%

“…Although nothing difinite is known about their physiological functions and their regulation, it has been suggested that nuclear proteases may play a role in the derepression of genes [5]. Such proteases have been purified from calf thymus (15.4 kDa; [40]) and rat liver (200 kDa; [41]), (25 kDa; [42]), (103 kDa; [43]), (40 kDa; [44]), (27 kDa; [45]) or (29 kDa; [46]). A serine protease which is activated by addition of DNA has been purified from rat liver chromatin and has a molecular mass of 25 kDa, but the effective concentration of DNA was observed to be rather high, namely 500 pg/l.O ml [47].…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a protease from Xenopus embryos

Miyata¹,

Yoshida²,

Kihara³

1989

European Journal of Biochemistry

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A proteolytic enzyme was purified from Xenopus embryos. The purification procedure consisted of fractionation of an extract of embryos with acetone, gel filtration of Sephadex G-75 and chromatography on carboxymethyl-cellulose and hydroxylapatite. The preparation of enzyme appeared to be homogeneous as judged by electrophoresis in polyacrylamide gels. This protease had a molecular mass of 43 -44 kDa and was composed of two subunits with molecular masses of 30 kDa and 13 kDa. The optimal pH of the reaction catalysed by the protease was approximately 4.0. This proteolytic activity was inhibited by antipain, leupeptin and iodoacetic acid; it was not affected by phenylmethylsulfonyl fluoride and pepstatin; and it was enhanced by dithiothreitol. In the presence of RNA, the optimal pH was shifted from pH 4.0 to pH 4.5. The protease was activated by addition of total RNA from Xenopus embryos, by poly(rU) or poly(rG). In contrast, after addition of tRNA or poly(rC), no activation of the protease was observed.Nearly all proteins in animal cells are continuously degraded to their constituent amino acids and this process contributes in an important way to the control of the levels of many enzymes and functional factors [l -31. Despite the importance of this process, the exact identification of the proteolytic enzymes that catalyze the degradation of proteins in vivo remains obscure. Animal cells contain many different proteases and there are both lysosomal and non-lysosomal pathways of protein degradation [4-61. Two of the major types of pathways for the degradation of proteins are clearly distinguishable, but many features of both pathways are not well understood. In general, the lysosomal proteases are small (20 -40 kDa) and they are maximally active under acidic conditions.Recent studies have indicated that the degradation of many intracellular proteins also occurs in the cytoplasm [7, 81. Although the mechanism of selection of substrates for degradation in the cytoplasm have not yet been established, many non-lysosomal proteases have been purified and characterized. For example, ATP-dependent proteases, multicatalytic proteases, and Ca2 +-dependent proteases have been found in animal cells as non-lysosomal proteases [4 -61.It has been reported that proteases play an important role in embryonic development [9 -121. We reported previously that a protease inhibitor, antipain, inhibits the incorporation of [3H]uridine into RNA in Xenopus embryos [13] and that the level of the proteolytic activity which is sensitive to antipain increased by a factor of several thousand during development up to the tail-bud stage [14]. In this report, we describe the purification of the antipain-sensitive enzyme and some of its properties. MATERIALS AND METHODS Chemicals Preparation ojeggs and embryosXenopus embryos obtained after mating were dejellied and allowed to develop to the tail-bud stage at room temperature. The embryos were immersed in cold acetone and homogenized in a glass homogenizer. The homogenate was centrifuged at 3000 r...

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Evidence that neutral protease from calf thymus chromatin is a serine type enzyme

Cited by 17 publications

References 5 publications

Properties of a Ca²⁺‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells

Properties of a Ca²⁺‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells

A Serine Proteinase from Cow Snout Epidermis Degrades High Molecular Weight Keratins

Purification and characterization of a protease from Xenopus embryos

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Evidence that neutral protease from calf thymus chromatin is a serine type enzyme

Cited by 17 publications

References 5 publications

Properties of a Ca2+‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells

Properties of a Ca2+‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells

A Serine Proteinase from Cow Snout Epidermis Degrades High Molecular Weight Keratins

Purification and characterization of a protease from Xenopus embryos

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Properties of a Ca²⁺‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells

Properties of a Ca²⁺‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells