Purification and properties of bovine liver plasma membrane 5′ nucleotidase

Harb, Jean; Méflah, Khaled; Duflos, Yves; Bernard, Serge

doi:10.1111/j.1432-1033.1983.tb07806.x

Cited by 52 publications

(29 citation statements)

References 41 publications

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“…Under dissociating conditions (for instance in the presence of 2 M urea) its AMPase activity is rapidly and irreversibly reduced. Thus the smallest functional form of chicken gizzard 5'-nucleotidase appears to be the homodimer, which is in agreement with results deduced from chemically cross-linking the enzyme isolated from bovine liver [27]. However, the possibility can not be excluded that in vivo 5'-nucleotidase will form larger molecular mass aggregates as it tends to do when solubilized in 0.1% Triton X-100.…”

Section: Discussionsupporting

confidence: 79%

An improved procedure for purifying 5′‐nucleotidase from various sources

Dieckhoff

Knebel

Heidemann

et al. 1985

European Journal of Biochemistry

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5′‐Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5′‐nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E. M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J. P. (1984) Biochem. J. 221, 369–377). Gel filtration, using non‐denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5′‐nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5′‐nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5′‐nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F‐actin than the enzyme isolated from rat liver.

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Section: Discussionsupporting

confidence: 79%

An improved procedure for purifying 5′‐nucleotidase from various sources

Dieckhoff

Knebel

Heidemann

et al. 1985

European Journal of Biochemistry

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“…The hybrid glycosylation persisted at the 2-h chase point and at steady state. Similar results have been found for 5NT in bovine liver (Harb et al, 1983) and in the I-LS hepatoma cell line (van den Bosch et al, 1986), although 5NT has been reported to be completely endo H resistant in primary cultures of hepatocytes (Baron and Luzio, 1987). We also find a hybrid glycosylation of 5NT in our enriched PM fraction; the endo H sensitivity appears similar in frac- b and e of the one-step Golgi flotation.…”

Section: Appearance Of Complex Glycosylated Formssupporting

confidence: 76%

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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“…The purification factor obtained in the present study lies among the highest values reported for 5'-nucleotidase [11,[15][16][17][18]. To our knowledge our preparation shows the highest specific activity reported so far.…”

Section: Purification Of 5'-nucleotidasesupporting

confidence: 53%

“…Concanavalin A inhibits 5'-nucleotidase from various sources [16,18,22,24]. The inhibition of the rat renal enzyme is shown in figure 4.…”

Section: Effect Of Concanavalin a (Assay 2)mentioning

confidence: 99%

Purification and Properties of a 5'-Nucleotidase from Rat Renal Membranes

Hir¹,

Gandhi²,

Dubach³

1989

Enzyme

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5'-Nucleotidase activity was solubilized from a particulate fraction of rat renal homogenates by Sulphobetaine 14. An 11,430-fold purification was achieved by a two-step chromatographic procedure using concanavalin-A Sepharose and ADP-agarose. SDS-PAGE of the purified material revealed a single polypeptide band with a M(r) of 69,000. The enyzme exhibited absolute specificity for 5'-mononucleotides. Among 7 tested substrates, adenosine monophosphate (AMP) showed the highest value of V/K(m). The K(m) for 5'-AMP is 5.1 μmol/1 and V is 632 pmol/min/mg. The plot of activity versus pH shows a broad plateau between pH 6.8 and 8.0. The hydrolysis of 5'-AMP was competitively inhibited by adenosine 5'-triphosphate (ATP; K; = 1.2 μmol/1), adenosine 5'-diphosphate (ADP; K(i) = 0.032 μmol/1) and a,ß-methyleneadenosine 5'-diphosphate (AOPCP; K(i) = 0.005 μmol/1). All of the 5 detergents tested activated the enzyme. Sulphobetaine 14 was the most potent and resulted in a 4-fold stimulation by increasing V without change of Km. Addition of exogenous divalent cations was not required for activity. However, the enzyme was inhibited by EDTA. This inhibition was overcome by the addition of Co^2+, M^n2+ and to a lesser extent of Mg^2+. Hg^2+, Zn^2+, Cu^2+ and Pb2+ inhibited in the low micromolar range. The properties of this enzyme from the rat kidney are similar to those reported in the literature for ecto 5'-nucleotidases from other

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Purification and properties of bovine liver plasma membrane 5′ nucleotidase

Cited by 52 publications

References 41 publications

An improved procedure for purifying 5′‐nucleotidase from various sources

An improved procedure for purifying 5′‐nucleotidase from various sources

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]

Purification and Properties of a 5'-Nucleotidase from Rat Renal Membranes

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