Molecular, biochemical and immunological analyses of porcine pancreatic DNase I

Mori, Shinjiro; Yasuda, Toshihiro; Takeshita, Hitoshi; Nakajima, Tamiko; Nakazato, Emiko; Mogi, Kouichi; Kaneko, Yasushi; Kishi, Koichiro

doi:10.1016/s0167-4838(01)00196-0

Cited by 22 publications

(23 citation statements)

References 29 publications

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“…All results evidenced that the enzymatic activity requires Ca 2þ /Mg 2þ , incubation temperature of 378C and neutral pH, moreover, this activity is inhibited by the addition of Zn 2þ . These biochemical characteristics are very similar to those generally reported for the mammalian DNase I (Mori et al 2001).…”

Section: Discussionsupporting

confidence: 88%

DNase I activity in pig MII oocytes: implications in transgenesis

Zannoni

Spinaci²,

Bernardini³

et al. 2006

Reproduction

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Several reliable methods to produce transgenic animals utilize the male genome. After penetration into oocyte, sperm DNA undergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated in the zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of a DNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in rat immature oocytes and the presence of nuclease activities has been shown in avian oocytes. The present study was conducted to verify whether a DNase-I like activity is present in MII mature pig oocytes. To do this, oocyte extracts were assessed for nuclease activity by a plasmid degradation assay and by zymography; these analyses evidenced a 33 kDa, Ca 21 /Mg 21 dependent DNase I-like activity that was inhibited by Zn 21 . A further identification of DNase I was achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activity was confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNA transferred to oocyte by spermatozoa during sperm mediated gene transfer in vitro fertilisation protocol seemed to be protected from DNase I degradation and to persist in the ooplasm till 6 h. These results, together with the high efficiency of sperm based transgenesis methods, suggest that the association with spermatozoa protects exogenous DNA from nuclease activities.

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Section: Discussionsupporting

confidence: 88%

DNase I activity in pig MII oocytes: implications in transgenesis

Zannoni

Spinaci²,

Bernardini³

et al. 2006

Reproduction

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“…For each species of actin, we evaluated the possibility of both physical and functional interactions with PLD1. To determine whether membrane-associated G-actin binds to PLD1, we took advantage of the fact that DNase I binds specifically to G-actin but not F-actin (57,58 Triton X-100, 1% octyl glucoside, and 1% deoxycholate. Lysates were incubated for 16 h at 4°C with DNase I that was covalently linked to Sepharose 4B beads.…”

Section: Addition Of Purified Actinmentioning

confidence: 99%

Regulation of Phospholipase D Activity by Actin

Kusner¹,

Barton²,

Wen

et al. 2002

Journal of Biological Chemistry

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Many critical cellular processes, including proliferation, vesicle trafficking, and secretion, are regulated by both phospholipase D (PLD) and the actin microfilament system. Stimulation of human PLD1 results in its association with the detergent-insoluble actin cytoskeleton, but the molecular mechanisms and functional consequences of PLD-actin interactions remain incompletely defined. Biochemical and pharmacologic modulation of actin polymerization resulted in complex bidirectional effects on PLD activity, both in vitro and in vivo. Highly purified G-actin inhibited basal and stimulated PLD activity, whereas F-actin produced the opposite effects. Actin-induced modulation of PLD activity was independent of the activating stimulus. The efficacy and potency of the effects of actin were isoform-specific but broadly conserved among actin family members. Human ␤␥-actin was only 45% as potent and 40% as efficacious as rabbit skeletal muscle ␣-actin, whereas its inhibitory profile was similar to the single actin species from the yeast, Saccharomyces cerevisiae. Use of actin polymerization-specific reagents indicated that PLD1 binds both monomeric G-actin, as well as actin filaments. These data are consistent with a model in which the physical state of the actin cytoskeleton is a critical determinant of its regulation of PLD activity.

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“…The formation of a stable complex between actin and Dnase1 allowed its crystallization and the determination of its atomic structure [18]. However, actins from more divergent sources often do not inhibit or bind to bovine Dnase1 [19][20][21].…”

Section: Introductionmentioning

confidence: 99%

“…Mutations of one of these three amino acids leads to reduction of the affinity or almost complete loss of binding and inhibition of Dnase1 by G-actin [25]. Such mutations also occur naturally in some mammals, porcine (Sus domesticus) [20] and rat (Rattus norvegicus) [26]; avian (Gallus domesticus) [21]; reptiles like rat snake (Elaphe quadrivirgata) [24]; and amphibians like african clawed frog (Xenopus laevis) [19] Dnase1. Therefore binding and inhibition of Dnase1 by actin is probably a recent achievement of some animals with a particular, so far unknown advantage.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of deoxyribonuclease I by actin is to protect cells from premature cell death

Eulitz

Mannherz

2007

Apoptosis

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Deoxyribonuclease I (Dnase1) is the major extracellular endonuclease. It is secreted by digestive glands into the alimentary tract and into the plasma, lacrimal fluid and urine by hepatocytes, lacrimal glands and renal proximal tubular cells, respectively. In many species the activity of Dnase1 is inhibited by monomeric actin. However, the biological significance of this high affinity interaction is unknown. We generated a Dnase1 mutant with extremely reduced actin binding capacity. EGFP-constructs of wild-type and mutant Dnase1 were transfected into MCF-7 breast cancer cells and apoptosis or necrosis was induced by staurosporine or oxidative stress. During apoptosis faster chromatin fragmentation occurred in cells transfected with mutant Dnase1. When wt (wild-type)- or mutated Dnase1 were added to cells after induction of necrosis, faster chromatin degradation occurred in the presence of mutant Dnase1. Inclusion of actin under these conditions inhibited chromatin degradation by wt- but not by mutated Dnase1. Thus, inhibition of Dnase1 by actin may serve as a self-protection mechanism against premature DNA degradation during cell damage.

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Molecular, biochemical and immunological analyses of porcine pancreatic DNase I

Cited by 22 publications

References 29 publications

DNase I activity in pig MII oocytes: implications in transgenesis

DNase I activity in pig MII oocytes: implications in transgenesis

Regulation of Phospholipase D Activity by Actin

Inhibition of deoxyribonuclease I by actin is to protect cells from premature cell death

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