Placenta
Volume 31, Issue 5 , Pages 460-462, May 2010

AMP-Deaminase from Developing Human Placenta

  • A. Swieca

      Affiliations

    • Department of Biochemistry and Clinical Physiology, Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland
    • ,
  • I. Rybakowska

      Affiliations

    • Department of Biochemistry and Clinical Physiology, Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland
    • Corresponding Author InformationCorresponding author. Tel./fax: +48 58 349 14 65.
    • ,
  • R. Milczarek

      Affiliations

    • Department of Pharmaceutical Biochemistry, Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland
    • ,
  • J. Klimek

      Affiliations

    • Department of Pharmaceutical Biochemistry, Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland
    • ,
  • K. Kaletha

      Affiliations

    • Department of Biochemistry and Clinical Physiology, Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland

Accepted 12 February 2010. published online 08 March 2010.

Article Outline

Abstract 

During pregnancy the activity of AMP-deaminase in developing human placenta gradually decreases, being in homogenates of mature, term placenta (∼40 week of gestation) one fourth to one third of that in homogenates of immature (∼25 week of gestation) organ. The gradual decrease of activity correlates inversely with the increasing presence of the form of enzyme predominating in homogenates of the mature placenta. The discrepancy observed indicate that isozymic pattern of AMP-deaminase in developing human placenta changes.

Keywords: AMP-deaminase, Human placenta, Pregnancy

 

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1. Introduction 

AMP-deaminase (EC 3.5.4.6) which catalyzes an irreversible reaction of deamination of AMP to IMP is a highly regulated enzyme of adenine nucleotides catabolism, playing significant role in energetic metabolism of tissues [1]. The product of reaction, IMP, is in the center of purine metabolism and may be further used for biosynthesis of guanine nucleotides being necessary for cell replication [2]. The adenylates are involved mainly in metabolic processes, whereas the guanylates mainly in cell replication. In early stages of pregnancy cell replication in developing placenta is especially intense.

Tissue- and stage-specific isoforms of AMP-deaminase have been identified in different mammalian tissues [3], [4], [5]. In human tissues, three main AMP-deaminase isoforms [M (muscle), L (liver) and E (erythrocyte)] encoded respectively by AMPD1, AMPD2 and AMPD3 genes are expressed [6].

In human term placenta specific deamination of adenylic acid (AMP) has been shown first by Makarewicz in 1971 [7]. In next years specific AMP-deaminase from this tissue was isolated and physico-chemical properties of the purified enzyme were characterized [8], [9]. It has been shown that placental AMP-deaminase is an oligomeric enzyme, regulated allosterically by adenine nucleotides (ATP, ADP) and orthophosphate [8], reacting specifically with antibodies directed against human AMPD2 gene product [9]. The experimental results published recently [10] confirmed the dominating expression of AMPD2 gene in human mature placenta and showed its product [isozyme L] as the prevailing form of AMP-deaminase in this tissue [10]. This same, however, must not be a true in case of the immature placenta, the metabolic profile of which adapts to metabolic requirements of the developing foetus.

In this paper the activity and immunological mark of human placental AMP-deaminase at different stages of pregnancy were compared.

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2. Materials and methods 

2.1. Materials 

Fresh samples of human placentas (obtained in result of natural delivery or spontaneous abortion from Clinic of Obstetric and Gynaecology) were used for preparation of homogenates. The scraped tissue was preliminary washed with physiological saline and then threefold with extraction buffer (0.089 M phosphate buffer, pH 6.5, containing 0.18 M KCl, 1 mM thioethanol, 1 mM phenylmethylsulfonyl fluoride and 1 mg/mL of trypsin inhibitor). After washing, the scraped tissue was ground, weighed and then homogenized with 3 volumes (v/w) of extraction buffer in glass Potter-type homogenizer. The homogenate obtained was filtered through a double layer of gauze and centrifuged first at 10,000×g for 10 min and then at 100,000×g for 1 h. The cytoplasmic fraction obtained was used for enzyme activity determinations.

2.2. Methods 

Activity of AMP-deaminase was determined colorimetrically, by phenol-hypochlorite method of Chaney and Marbach [11]. The incubation medium, in final volume of 0.5 mL, contained 0.1 M potassium-succinate buffer, pH 6.5 and 5 mM substrate (AMP). After equilibration of the incubation medium at 30 °C, 25 μg of homogenate supernatant was added into the medium to start the 15 min reaction. All incubations were carried out in three parallel tubes.

Protein concentration was determined according to the method of Bradford [12].

SDS-PAG electrophoresis and Western blot analysis with the use of polyclonal rabbit antibodies [13] (against AMP-deaminase purified from human mature, term placenta) were performed as described previously [9].

Data concerning enzyme activity and protein level are reported as mean ± standard deviation (±S.D) where (n) represents number of placentas studied. Data were analyzed by One-Way Analysis of Variance (ANOVA). Tukey's post-hoc test was used for further determination of the significance of differences. Statistical significance was considered significant at p < 0.05.

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3. Results and discussion 

Table 1 presents activities of AMP-deaminase in homogenates of human placenta measured at three different stages of pregnancy. As it may be seen from this Table, the activity of enzyme studied decreases with the progress of pregnancy, reaching in homogenate of the mature, term placenta a value being three, four times lower from that measured in homogenate of placenta at 25 week of pregnancy.

Table 1. Activity of AMP-deaminase in homogenates of developing human placenta. Activity of the enzyme was measured in 0.1 M succinate buffer, pH 6.5, at 5 mM substrate (AMP) concentration. Statistical significance of differences between values: (#) p < 0.005 vs. term placenta (40th week-old); (*) p < 0.05 vs. term placenta (40th week-old).
Time of gestationSpecific activity [μmoles/min/mg protein]
25th week-old placenta (n = 3)0.037 ± 0.006#
33rd week-old placenta (n = 3)0.025 ± 0.005*
40th week-old placenta (n = 4)0.010 ± 0.007

Fig. 1a presents results of Western blot analysis performed with the placental homogenates prepared. As it may be seen from this Figure, the 68 kDa protein fragment revealed during SDS-PAG electrophoresis of AMP-deaminase purified from the mature term placenta, representing proteolytically modified subunit of this enzyme [9] was the sole protein reacting with rabbit anti-placental AMP-deaminase antibodies in homogenates tested. A distinct and well visible stain of immunological reaction observed in homogenate of mature, term placenta became weaker and less visible in homogenates of immature placentas. A densitometric analysis performed estimated the relative intensity of immunological staining (Fig. 1b) in homogenates of immature placenta as 90% (homogenate of 33 week-old placenta) and 70% (homogenate of 25 week-old placenta) of that observed in homogenate of mature, term placenta.

  • View full-size image.
  • Fig. 1 

    AMP-deaminase from developing human placenta. a) Western blot analysis of homogenates. 15 μg of placental homogenate protein was applied onto the gel during SDS-PAG electrophoresis. Rabbit antibodies against AMP-deaminase purified from human term (∼40 week-old) placenta were used for immunological staining of the blot. Lane 1 – colour protein standards-Protein Ladder, 11–170 kDa, lane 2 – homogenate from 25 week-old placenta, lane 3 – homogenate from 33 week-old placenta, lane 4 - homogenate from term placenta (∼40 week-old); b) Densitometric analysis of Western blot results. The intensity of immunological was evaluated by using of Quantity One (Biorad) program. Statistical significance of differences between values: (**) p < 0.001, (*) p < 0.005.

Placenta enables the foetus to receive the necessary foodstuffs from its maternal organism. During progress of pregnancy many metabolic functions of placenta are gradually taken over by developing organs of the foetus, allowing it finally to discard placenta at the period of delivery.

The graduate decrease of AMP-deaminase activity in developing human placenta is not very surprising. Similar changes in activity of this enzyme were already observed in the rat [14]. Here the high activity of AMP-deaminase observed in early stages of rat pregnancy has been related with a possible role which this ammonia-producing reaction may fulfil in buffering acidic products of the mostly anaerobic metabolism of the foetus to maintain its acid/basic homeostasis. In later stages of rat pregnancy, when foetal systems for safe ammonia disposal and utilization are already developed, the ammonia-producing capabilities of the rat placenta, including the ammonia-producing activity of placental AMP-deaminase are significantly reduced. In similar way one may to interpret AMP-deaminase activity changes observed in developing human placenta.

Among three AMPD genes only these producing isozymes L and E of AMP-deaminase are expressed in the human term placenta [10]. Expression of AMPD2 gene is here however dominating and this is why AMP-deaminase isolated from this source recalls so much the isozyme isolated from human liver [15], [16]. Strongly sigmoid kinetics and relatively poor catalytic efficiency of AMPD2 gene product are characteristic feature of this isozyme. Experimental results presented on Table 1 and Fig. 1 indicate that expression of AMPD2 gene product seems not to be the dominating form of AMP-deaminase in developing, immature human placentas. The inverse correlation between decreasing activity of placental AMP-deaminase and increasing presence of liver-type enzyme indicate that isoform(s) of AMP-deaminase immunologically different from liver-type isozyme must independently operate in immature human placenta. The preliminary result of RT-PCR analysis (unpublished data) showing in the immature placenta the increased expression of AMPD3 gene indicates that during pregnancy the isozymic pattern of human placental AMP-deaminase really may change.

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Acknowledgements 

This work was supported financially by the Medical University of Gdansk (Grants: ST-40, ST-534) and by Ministry of Science and Information as a research project (3 P05E 144 25).

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References 

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PII: S0143-4004(10)00088-3

doi:10.1016/j.placenta.2010.02.009

Placenta
Volume 31, Issue 5 , Pages 460-462, May 2010

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