Suppressive effect of protease inhibitors on heterokaryons containing chick erythrocyte nuclei

Darżynkiewicz, Zbigniew; Chelmicka-Szorc, Ewa; Arnason, Barry G. W.

doi:10.1016/0014-4827(74)90489-3

Cited by 14 publications

(5 citation statements)

References 32 publications

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“…The first protein peak, eluted with starting buffer, contained neutral proteinases active towards casein at pH 7.5 (fractions 10-15), and acid proteinases active towards hemoglobin at pH 3.5 (fractions [14][15][16][17][18][19][20][21][22][23][24][25]. After the gradient elution started the second protein peak appeared at 0.15 M NaC1, possessing only neutral proteinase activity (casein pH 7.5).…”

Section: Resultsmentioning

confidence: 99%

“…There are now several reports dealing with neutral proteinases and inhibitors of these proteinases, since they are thought to be involved in a number of diseases, involving blood-clotting [15,16], inflammation and allergic states [17], emphysema [18], carcinoma [19], diseases of connective tissue [20] and collagen metabolism [2] and finally in protein synthesis [21]. It has been known that neutral proteinases are located in so-called azurophil granules [22].…”

Section: Discussionmentioning

confidence: 99%

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Intracellular Distribution of Neutral Proteinases and Inhibitors in Pig Leucocytes. Isolation of Two Inhibitors of Neutral Proteinases

Kopitar

Lebez

1975

Eur J Biochem

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Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose.Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAEcellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, a,-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used. The third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, a,-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%.In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-1 00 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80 %) of post-granular supernatant was eluted together with ovalbumin ( M , 43000) and the remainder with cytochrome c (12 300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase.Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate.Inhibitors of post-granular supernatant are stable at pH 6 -8, but unstable in the pH range 2 -5 and are thermolabile.Neutral tissue and cell proteinases have been rather poorly investigated in comparison to acid proteinases. The reason is that it was not possible to prepare stable active samples. A higher neutral proteinase activity was obtained only from granule (lysosomal) extracts [l -31, or from so-called reextracts, where the first cell extract was discarded; i.e. only from samples where the cytoplasm had in any case been removed [4,5].The last investigations in this field have shown that besides neutral proteinases inhibitors of these proteinases are also present in the cells and that for a successful isolation of neutral proteinases these inhibitors must be removed first. There are no data concerning the presence of any well-characterized material from animal tissues capable of inhibiting intracellular acid proteinases, such as cathepsin D [6].Davies and coworkers [7] reported in their study of neutral proteinases of polymorphonuclear cells that cytoplasmic -postgranular supernatant fraction caused the strongest inhibition of neutral proteinases. Because the polymorphonuclear cells were obtained from inflammatory exudates, they proposed a ...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Intracellular Distribution of Neutral Proteinases and Inhibitors in Pig Leucocytes. Isolation of Two Inhibitors of Neutral Proteinases

Kopitar

Lebez

1975

Eur J Biochem

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“…The fact that at least some of the globin synthesized early after transplantation was translated from preexisting mRNA was also supported by data showing that early synthesis was at least partially refractory to actinomycin D. The interpretation of such results is, however, tenuous since the precise effects of the drug on the partially decondensed chromatin present in reactivating nuclei is undetermined. In fact, chicken erythrocyte chromatin is apparently unique in that it does not vary in its ability to bind actinomycin D throughout either condensation occurring during normal development or decondensation after nuclear reactivation (15,31). Furthermore, it has also been shown that actinomycin D can inhibit the rate of initiation of polypeptide synthesis (14) and that low levels of the drug result in an accumulation of globin mRNA in murine erythroleukemia cells (56).…”

Section: Methodsmentioning

confidence: 99%

Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

Bruno¹,

Reich²,

Lucas³

1981

Mol. Cell. Biol.

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“…In addition, TLCK has been shown to inhibit protein synthesis in poliovirusinfected HeLa cells (21) and RNA synthesis in lymphocytes (26) in the concentration range (1 X 10-4 M) previously reported (14) to selectively inhibit the growth of SV40-transformed cells. Thus, TLCK may also cause growth inhibition via inhibition of macromolecular synthesis rather than by inhibiting cellular proteases.…”

mentioning

confidence: 98%

“…The mechanism by which TPCK inhibits protein synthesis in mammalian cells is not known; it may inactivate one of the peptide chain elongation factors in protein synthesis as it does in bacteria (24,25), and (or) it may act by inhibiting cellular RNA synthesis (26) which is required for protein synthesis. Whatever the mechanism, the growth inhibitory effects of TPCK can no longer simply be ascribed to inhibition of proteolytic activity.…”

mentioning

confidence: 99%

Non-Selective Inhibition of Transformed Cell Growth by a Protease Inhibitor

Chou¹,

Black²,

Roblin³

1974

Proc. Natl. Acad. Sci. U.S.A.

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The protease inhibitors N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) and N-tosyl-Llysylchloromethyl ketone (TLCK) have previously been shown to selectively inhibit growth of simian virus 40-transformed cells, suggesting that proteolytic enzymes play a role in loss of cellular growth control following viral transformation. In contrast, this study shows that TPCKmediated growth inhibition is non-selective, since the growth of both simian virus 40-transformed and untransformed 3T3 cells is similarly reduced by TPCK treatment. Under certain conditions, TPCK treatment of simian virus 40-transformed cells yields a reversible "growth plateau" condition which mimics, .but is not equivalent to, contact inhibition of growth. The growth inhibitory effects of TPCK are due to inhibition of protein synthesis, since TPCK treatment resulted in a diminution of protein synthesis and since the "growth plateau" effect was also observed in cultures treated with cycloheximide.Untransformed fibroblasts in tissue culture show a characteristic reduction in the frequency of cell division upon formation of a confluent monolayer. This phenomenon has been termed contact inhibition or density-dependent inhibition of cell growth (1, 2). In contrast, simian virus 40 (SV40)-transformed cells continue division beyond confluency and frequently exhibit multilayered cell growth.Exposure of confluent, untransformed fibroblast monolayers to low concentrations of proteolytic enzymes can cause transient release from density-dependent inhibition of growth (3,4

show abstract

Suppressive effect of protease inhibitors on heterokaryons containing chick erythrocyte nuclei

Cited by 14 publications

References 32 publications

Intracellular Distribution of Neutral Proteinases and Inhibitors in Pig Leucocytes. Isolation of Two Inhibitors of Neutral Proteinases

Intracellular Distribution of Neutral Proteinases and Inhibitors in Pig Leucocytes. Isolation of Two Inhibitors of Neutral Proteinases

Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

Non-Selective Inhibition of Transformed Cell Growth by a Protease Inhibitor

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