Characterization of soluble vs membrane-bound human placental 5′-nucleotidase

Klemens, Mary R.; Sherman, William R.; Holmberg, Nels; Ruedi, J M; Low, Martin G.; Thompson, Linda F.

doi:10.1016/0006-291x(90)91601-n

Cited by 44 publications

(35 citation statements)

References 21 publications

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“…A soluble form of 5NT with enzymatic and immunological characteristics identical to the GPI-anchored form is found in blood (Klemens et al, 1990;discussed in Zimmerman, 1992). Our data raise the possibility that some of this pool is cleaved by a phospholipase C or D at the sinusoid and never reaches the apical domain.…”

Section: Discussionmentioning

confidence: 99%

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5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Schell¹,

Maurice²,

Stieger³

et al. 1992

The Journal of Cell Biology

115

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Abstract. In hepatocytes, all newly synthesized plasma membrane (PM) proteins so far studied arrive first at the basolateral domain; apicaUy destined proteins are subsequently endocytosed and sorted to the apical domain via transcytosis. A mechanism for the sorting of newly synthesized glycophosphatidylinositol (GPI)-linked proteins has been proposed whereby they associate in lipid microdomains in the trans-Golgi network and then arrive at the apical domain directly. Such a mechanism poses a potential exception to the hepatocyte rule. We have used pulse-chase techniques in conjunction with subcellular fractionation to compare the trafficking of 5' nucleotidase (5NT), an endogenous GPI-anchored protein of hepatocytes, with two transmembrane proteins. Using a one-step fractionation technique to separate a highly enriched fraction of Golgi-derived membranes from ER and PM, we find that both 5NT and the polymeric IgA receptor (plgAR) traverse the ER and Golgi apparatus with high efficiency. Using a method that resolves PM vesicles derived from the apical and basolateral domains, we lind that 5NT first appears at the basolateral domain as early as 30 rain of chase. However the subsequent redistribution to the apical domain requires >3.5 h of chase to reach steady state. This rate of transcytosis is much slower than that observed for dipeptidylpeptidase IV, an apical protein anchored via a single transmembrane domain. We propose that the slow rate of transcytosis is related to the fact that GPI-linked proteins are excluded from clathrin-coated pits/vesicles, and instead must be endocytosed via a slower nonclathrin pathway.

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Section: Discussionmentioning

confidence: 99%

“…These observations have led to the notion that a portion of 5NT is anchored by a transmembrane domain (Klemens et al, 1990). However, when this possibility has been addressed using other biochemical or molecular biological tests (Bailyes et al, 1990;Ogata et al, 1991), all evidence supports an exclusive GPI anchorage for 5NT.…”

Section: Discussionmentioning

confidence: 99%

5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Schell¹,

Maurice²,

Stieger³

et al. 1992

The Journal of Cell Biology

115

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“…1984; Thompson et al, 1987;Klemens et al, 1990;Misumi et al, 1990;Thompson et al, 1990). To determine the subcellular localization of 5'-NT, homogenates of the embryo-decidual unit on day 7 were fractionated by centrifugation ( Table 2).…”

Section: Subcellular Localization Of 5'-nt Is Consistent With Expressmentioning

confidence: 99%

Adenosine levels in the postimplantation mouse uterus: Quantitation by HPLC‐fluorometric detection and spatiotemporal regulation by 5′‐nucleotidase and adenosine deaminase

Blackburn

Gao

Airhart

et al. 1992

Developmental Dynamics

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Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryodecidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmollmg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmoVmg protein) and day 5 (0.59 nmoYmg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmollmg protein) and day 7 (0.10 nmollmg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual :zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast ((days 7-13) and the metrial gland (days 11-13).Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation.The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6 1 l), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities. Purine nucleoside phosphorylase and xanthine oxidase, which are two catabolic enzymes acting subsequent to 5'-NT and ADA in the sequential degradation of AMP to xanthine, remained low and constant in the tissues examined suggesting that the catabolic pathway is geared toward regulation of adenosine le...

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Section: Introductionmentioning

confidence: 99%

“…However, a 'soluble' form of ecto-5 -NT has also been described in synovial fluid [6] and human placenta [7]. This species seemed to be generated from the hydrolysis of the GPI anchor, catalysed by phosphatidylinositol-specific phospholipase C (PI-PLC), as shown by similarities in the N-terminal sequence, apparent molecular mass, low K m values towards AMP and enzymespecific inhibition by nucleoside di-and triphosphates [7]. From a comparative study of the catalytic properties of three forms of the enzyme, namely, native ecto-5 -NT linked to the plasma membrane of pig lymphocytes, the solubilized form obtained Abbreviations used: GPI, glycosylphospatidylinositol; IEF, isoelectric focusing; MALDI, matrix-assisted laser-desorption ionization; ecto-5 -NT, ecto-5 -nucleotidase; 5 -NT, 5 -nucleotidase; PI-PLC, phosphatidylinositol-specific phospholipase C; RP, reverse phase.…”

Section: Introductionmentioning

confidence: 99%

Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma

Fini

Talamo

Cherri

et al. 2003

Biochemical Journal

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Ecto-5 -nucleotidase (ecto-5 -NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5 -NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln 547 and/or Phe 548 . Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn 403 . Significant differences in substrate specificity were observed between isoforms and, on the basis of molecularmodelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5 -NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.

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Characterization of soluble vs membrane-bound human placental 5′-nucleotidase

Cited by 44 publications

References 21 publications

5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Adenosine levels in the postimplantation mouse uterus: Quantitation by HPLC‐fluorometric detection and spatiotemporal regulation by 5′‐nucleotidase and adenosine deaminase

Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma

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