Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

Kihn, L; Rutkowski, Dorothy; Stinson, Robert A.

doi:10.1139/o90-166

Cited by 23 publications

(26 citation statements)

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“…Treatment of the enzyme reconstituted into liposome system with phospholipase C confirmed that TNAP interacts with the liposome membrane through the GPI-anchor (Camolezi et al 2002). Similar results were obtained by Kihn et al (1990) for placenta and liver alkaline phosphatase. Solubilization of TNAP with protease (bromelain or tripsin) results in a soluble enzyme without the GPI-anchor.…”

Section: Applications For the Study Of Lipid-protein Interactionssupporting

confidence: 76%

Proteoliposomes in nanobiotechnology

et al. 2012

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Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid-protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid-protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multiprotein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.

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Section: Applications For the Study Of Lipid-protein Interactionssupporting

confidence: 76%

Proteoliposomes in nanobiotechnology

et al. 2012

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“…If the protein products of the ALP gene are iden tical for these three sources of the enzyme then the Mr differences would reflect varia tion in the extent and/or nature of the posttranslational glycosylation. We believe that anomalous migration of the amphiphilic forms of these enzymes is the reason they migrate with an apparent Mr approximately twice that of their hydrophilic equivalents Like the ALP in human liver plasma mem branes and osteosarcoma cells [8,13], the enzyme in the cultured L428 cells was re leased to near 100% by PI-PLC. It is likely, therefore, that the enzyme is anchored in the plasma membrane of the lymphoma cells by the same phosphatidyl-inositol-glycan moiety that anchors the liver and osteosarcoma en zymes.…”

Section: Discussionmentioning

confidence: 96%

“…Triton X-100 was then added back to the recovered aqueous layer to 1 % (v/v) and the samples subjected to standard pore polyacrylamide gel electrophoresis (PAGE) using ALKPHOR gels ac cording to manufacturer's instructions. Gradient gel electrophoresis of samples and molecular weight mark er proteins was performed as described [13] and the apparent molecular weights of the samples calculated using a graph of log Mr versus Rf value [12]. …”

Section: Electrophoresismentioning

confidence: 99%

Characterization of the Alkaline Phosphatase Expressed on the Surface of a Hodgkin's Lymphoma Cell Line

Belland

Visser²,

Poppema³

et al. 1993

Enzyme Protein

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Alkaline phosphatase solubilized from a human Hodgkin’s lymphoma cell line (L428) was compared with purified amphiphilic and hydrophilic forms of the enzyme from human liver, and with the enzyme solubilized from a cultured osteosarcoma cell line (Saos-2). Purified hydrophilic alkaline phosphatases from human placenta and intestine were also compared in some experiments. Alkaline phosphatase was released from the plasma membrane of intact lymphocytes by phosphatidylinositol phospholipase C and thus is anchored to the outside of the plasma membrane by covalently attached phosphatidylinositol. Enzyme released in this way was hydrophilic and that solubilized with Triton X-100 was amphiphilic, as assessed by adsorption to octyl-Sepharose. Lymphocyte alkaline phosphatase, when released from the membrane by phosphatidylinositol phospholipase C or solubilized by Triton X-100, had apparent M(r) values on gradient gel electrophoresis of 227 and 494 kDa, respectively. These values were consistently higher than equivalent ones obtained with enzymes purified from human liver, but were similar to those of cultured osteosarcoma cells. Isoenzyme-specific inhibitors of alkaline phosphatase showed similar patterns of inhibition between the enzyme from L428 cells and the tissue-nonspecific (liver/kidney/bone) isoenzyme from human liver. Heat stabilities were similar for the enzymes from L428 and Saos-2 (bone isoform) cell lines, but differed significantly from those of liver, intestine and placenta. We conclude that the alkaline phosphatase expressed in this lymphoma cell line (L428) has properties that most closely resemble those of the tissue-nonspecific isoenzyme found normally in osteoblasts of bone (bone isoform).

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“…Like other GPI proteins including acetylcholinesterase, GPI-murine B7-1 and B7-2 [76], variant surface glycoprotein (VSG) from Trypanosoma brucei, GPI-human decay accelerating factor [19,70], and GPI-green fluorescent protein (GFP-GPI) [59], PLAP added to the medium spontaneously inserts into biomembranes [54]. Osseous plate AP-GPI and PLAP can also spontaneously insert into preformed liposomes [15,54].…”

Section: Afm Detection Of Ap-gpi In Model Membranesmentioning

confidence: 99%

“…Osseous plate AP-GPI and PLAP can also spontaneously insert into preformed liposomes [15,54]. Compared with reconstitution procedures, where the protein is added before or during the bilayer formation, this process presents the important advantage to insure an exclusive right side outside orientation of the AP-GPI at the external leaflet of the membrane.…”

Section: Afm Detection Of Ap-gpi In Model Membranesmentioning

confidence: 99%

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM

Giocondi

Seantier

Dosset

et al. 2007

Pflugers Arch - Eur J Physiol

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In plasma membranes, most glycosylphosphatidylinositol-anchored proteins (GPI proteins) would be associated with ordered microdomains enriched in sphingolipids and cholesterol. Debates on the composition and the nano-or mesoscales organization of these membrane domains are still opened. This complexity of biomembranes explains the use, in the recent years, of both model systems and atomic force microscopy (AFM) approaches to better characterize GPI proteins/membranes interactions. So far, the studies have mainly been focused on alkaline phosphatases of intestinal (BIAP) or placental (PLAP) origins reconstituted in model systems. The data show that GPI-anchored alkaline phosphatases (AP-GPI) molecules inserted in supported membranes can be easily imaged by AFM, in physiological buffer. They are generally observed in the most ordered domains of model membranes under phase separation, i.e. presenting both fluid and ordered domains. This direct access to the membrane structure at a mesoscopic scale allows establishing the GPI protein induced changes in microdomains size. It provides direct evidence for the temperature-dependent distribution of a GPI protein between fluid and ordered membrane domains. Origins of reported differences in the behavior of BIAP and PLAP are discussed. Finally, advantages and limits of AFM in the study of GPI proteins/membrane domains interactions are presented in this review.

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Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

Cited by 23 publications

References 0 publications

Proteoliposomes in nanobiotechnology

Proteoliposomes in nanobiotechnology

Characterization of the Alkaline Phosphatase Expressed on the Surface of a Hodgkin's Lymphoma Cell Line

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM

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