Physical studies on the rabbit hepatic galactoside-binding protein. Effects of calcium and ligands.

Andersen, Thomas T.; Freytag, J W; Hill, Robert L.

doi:10.1016/s0021-9258(18)34293-5

Cited by 44 publications

(20 citation statements)

References 27 publications

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“…(ii) The reported mass of the native receptor is 264 ± 16 kDa (Anderson et al, 1982). The mass of the three proposed heterohexamers would be about 268, 279, and 288 kDa.…”

Section: Resultsmentioning

confidence: 97%

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Herzig¹,

Weigel²

1989

Biochemistry

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We have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degrees C. Specific binding of these 125I-labeled derivatives was approximately 90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/125I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. Cross-linking proceeded rapidly at 4 degrees C as a first-order process (k = 0.25 min-1, t1/2 = 2.8 min). After being cross-linked with any of the NHS/125I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDS-PAGE and autoradiography. Major bands were observed at Mr congruent to 84K, 93K, and 105K corresponding to the expected size of 1:1 adducts between NHS/ASOR (Mr congruent to 41.3K) and the three subunits of the receptor, Mr congruent to 43K, 50K, and 60K. The three subunits, rat hepatic lectin (RHL) 1, 2, and 3, were labeled in the ratio of about 1.0:1.2:1.0, respectively. After cross-linking, a polyclonal goat antibody to the receptor immunoprecipitated up to 100% of the specifically cross-linked NHS/125I-ASOR. Preimmune IgG immunoprecipitated less than 1% of the radiolabeled ligand. Cell surface receptors were cross-linked to NHS-ASOR, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid-Sepharose, and subjected to Western blot analysis. By use of anti-sera specific for RHL 1 or RHL 2/3 (from K. Drickamer), cross-linked complexes of Mr congruent to 85K or approximately 90-115K, respectively, were detected as were un-cross-linked native subunits. The ratio of free to cross-linked subunits was approximately 10:1 for RHL 1 and approximately 0.5:1 for RHL 2/3. We conclude that all three receptor subunits can cross-link to ligand. We propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.

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“…(ii) The reported mass of the native receptor is 264 ± 16 kDa (Anderson et al, 1982). The mass of the three proposed heterohexamers would be about 268, 279, and 288 kDa.…”

Section: Resultsmentioning

confidence: 97%

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Herzig¹,

Weigel²

1989

Biochemistry

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“…The Gal/GalNAc-specific receptors isolated from mammalian livers are all composed of polypeptide chains of kDa in size. These polypeptide chains are apparently not held together by disulfide bonds but exist in aqueous solution in the presence of nondenaturing, neutral detergents as aggregates ranging from 160 kDa (Bezouska et al, 1985) to 600 kDa (Anderson et al, 1982) in size. While human, pig, and rabbit receptors consist of only one subunit or two subunits in a simple ratio (Baenziger & Maynard, 1980;Bezouska et al, 1985;Kawasaki & Ashwell, 1976), the rat receptor has an unusual composition, having two minor polypeptide species (52 and 60 kDa) and one predominant species (43 kDa) which accounts for 60-75% of the total protein (Harford et al, 1982;Schwartz et al, 1981;Warren & Doyle, 1981).…”

Section: Discussionmentioning

confidence: 99%

Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

Lee¹,

Lee²

1987

Biochemistry

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The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which [43 kilodalton (kDa)] exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an 125I-labeled high-affinity reagent [a triantennary glycopeptide containing an aryl azide group on galactosyl residues; Lee, R. T., & Lee, Y. C. (1986) Biochemistry 25, 6835-6841], the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. We postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work [Hardy, M. R., Townsend, R. R., Parkhurst, S. M., & Lee, Y. C. (1985) Biochemistry 24, 22-28].

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“…In any case, the binding behavior of the receptor suggests that the high-affinity binding units are further arranged in (permanent or transient) clusters on the cell surface (Hardy et al, 1985). The molecular mass of the receptor complex in detergent solution of approximately 260 kDa (Andersen et al, 1982) is consistent with a hexamer, implying that this might be a preferred complex also in the membrane. The possibility that formation of higher complexes is induced by polyvalent ligands could provide a mechanism for the observed ligandinduced increase of internalization rate.…”

Section: A Mammalian Hepatic Lectinmentioning

confidence: 95%

“…Ca2+ can be replaced by Sr2+ and Ba2+ (in the order of efficiency Sr2+ > Ca2+ > Ba2+) but not by Mg2+. Ca2+-binding studies performed with the chicken hepatic lectin and Drickamer, 1988;Andersen et al" 1982).…”

Section: Carbohydrate-binding Domainmentioning

confidence: 99%

The asialoglycoprotein receptor: a model for endocytic transport receptors

1990

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Physical studies on the rabbit hepatic galactoside-binding protein. Effects of calcium and ligands.

Cited by 44 publications

References 27 publications

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

The asialoglycoprotein receptor: a model for endocytic transport receptors

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