scholarly journals Molecular-mass heterogeneity of Griffonia simplicifolia lectin IV subunits. Differences in the oligosaccharide moieties in the N-terminal region

1990 ◽  
Vol 272 (2) ◽  
pp. 343-350 ◽  
Author(s):  
P V Nikrad ◽  
J R Pearlstone ◽  
M R Carpenter ◽  
R U Lemieux ◽  
L B Smillie
Keyword(s):  
Monosaccharide Composition ◽  
Unique Sequence ◽  
Beta Subunit ◽  
The Other ◽  
Alpha Subunit ◽  
Sequence Analyses ◽  
Griffonia Simplicifolia ◽  
Sds Page ◽  
Dimeric Protein ◽  
Covalently Linked

Lectin IV of Griffonia simplicifolia (Mr approximately 56,000), which has a strong affinity for both the Lewis b and Y blood-group determinants, is a dimeric protein of two subunits, alpha (29 kDa) and beta (27 kDa), separable by SDS/PAGE and containing covalently linked oligosaccharide. After digestion with N-glycanase, the protein migrates as a single band with a mobility identical with that of the beta-subunit. After cleavage with hydroxylamine of 3H-labelled, but otherwise intact, lectin, the radioactively labelled oligosaccharide was found to be associated with two blocked N-terminal peptides separable by h.p.l.c. and having identical amino acid compositions. One of these had three or four glucosamine residues per molecule, whereas the other had only one or two. Sequence analyses of these, as well as of a 21 kDa hydroxylamine-cleaved fragment and of the intact lectin pretreated with pyroglutamate aminopeptidase, have provided a unique sequence for residues 1-62 of the two subunits. Evidence is presented for two sites of N-linked oligosaccharide attachment at Asn-5 and Asn-18. Whereas the alpha-subunit has oligosaccharide linked to both sites, the beta-subunit has carbohydrate associated with only one (Asn-18). Sugar analyses of the whole lectin reveal a monosaccharide composition of (Xyl)3(Fuc)2(Man)10(GlcNAc)6, representing 6.4% of the mass of the molecule. Taken together with the susceptibility of the Asn-5 linkage (but not of Asn-18) to N-glycanase digestion, the observations indicate that the structures of the oligosaccharides at residues 5 and 18 are different.

scholarly journals A human leukocyte differentiation antigen family with distinct alpha-subunits and a common beta-subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule.

1983 ◽  
Vol 158 (6) ◽  
pp. 1785-1803 ◽  
Author(s):  
F Sanchez-Madrid ◽  
J A Nagy ◽  
E Robbins ◽  
P Simon ◽  
T A Springer
Keyword(s):  
Human Leukocyte ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Complement Receptor ◽  
Lymphocyte Function ◽  
Subunit Dissociation ◽  
Molecular Weights ◽  
Alpha Subunits ◽  
Sds Page

The human lymphocyte function-associated antigen-1 (LFA-1), the complement receptor-associated OKM1 molecule, and a previously undescribed molecule termed p150,95, have been found to be structurally and antigenically related. Each antigen contains an alpha- and beta-subunit noncovalently associated in an alpha 1 beta 1-structure as shown by cross-linking experiments. LFA-1, OKM1, and p150,95 alpha-subunit designations and their molecular weights are alpha L = 177,000 Mr, alpha M = 165,000 Mr, and alpha X = 150,000 Mr, respectively. The beta-subunits are all = 95,000 Mr. Some MAb precipitated only LFA-1, others only OKM1, and another precipitates all three antigens. The specificity of these MAb for particular subunits was examined after subunit dissociation by high pH. MAb specific for LFA-1 or OKM1 bind to the alpha L- or alpha M-subunits, respectively, while the cross-reactive MAb binds to the beta-subunits. Coprecipitation experiments with intact alpha 1 beta 1-complexes showed anti-alpha and anti-beta MAb can precipitate the same molecules. In two-dimensional (2D) isoelectric focusing-SDS-PAGE, the alpha subunits of the three antigens are distinct, while the beta-subunits are identical. Biosynthesis experiments showed alpha L, alpha M, and alpha X are synthesized from distinct precursors, as is beta. The three antigens differ in expression on lymphocytes, granulocytes, and monocytes. During maturation of the monoblast-like U937 line, alpha M and alpha X are upregulated and alpha L is downregulated. Some MAb to the alpha subunit of OKM1 inhibited the complement receptor type three. LFA-1, OKM1, and p150,95 constitute a novel family of functionally important human leukocyte antigens that share a common beta-subunit.

scholarly journals Evidence for a covalent intermediate in the S-adenosyl-l-methionine-dependent transmethylation reaction catalysed by sirohaem synthase

1996 ◽  
Vol 313 (2) ◽  
pp. 415-421 ◽  
Author(s):  
Sarah C. WOODCOCK ◽  
Martin J. WARREN
Keyword(s):  
Binding Assay ◽  
Protein A ◽  
Tight Binding ◽  
The Other ◽  
Multifunctional Enzyme ◽  
Sds Page ◽  
Covalently Linked ◽  
Covalent Intermediate ◽  
Uroporphyrinogen Iii Methylase

CysG, also known as uroporphyrinogen III methylase and sirohaem synthase (CysG; EC 2.1.1.107), is a multifunctional enzyme that is able to transform uroporphyrinogen III into sirohaem via two S-adenosyl-L-methionine (AdoMet)-dependent transmethylations, an NAD+-dependent dehydrogenation and a ferrochelation. The apparent tight binding of AdoMet to this multifunctional enzyme is investigated. The use of a rapid AdoMet binding assay demonstrates that CysG becomes labelled with both [methyl-3H]AdoMet and [carboxyl-14C]AdoMet. Further experiments show that the CysG-AdoMet complex is subsequently able to methylate uroporphyrinogen III. CysG remains associated with the labelled constituents of the AdoMet even after denaturation with urea and SDS/PAGE, suggesting that the AdoMet has become covalently linked to the protein. A rapid examination of some of the other transmethylases involved in corrin biosynthesis reveals that they bind the AdoMet in a similar fashion. A multistep transmethylation mechanism is proposed to explain the observed results.

The Stratus immunofluorometric assay system evaluated for quantifying human choriogonadotropin in serum.

1986 ◽  
Vol 32 (7) ◽  
pp. 1402-1404 ◽  
Author(s):  
L C Rogers ◽  
S E Kahn ◽  
T H Oeser ◽  
E W Bermes
Keyword(s):  
Monoclonal Antibodies ◽  
Regression Equation ◽  
Assay System ◽  
Beta Subunit ◽  
The Other ◽  
Alpha Subunit ◽  
Human Choriogonadotropin ◽  
Beta Hcg

Abstract We evaluated the Stratus (American Dade, Miami, FL), an automated immunofluorometric assay system, for the quantification of human choriogonadotropin (hCG) in serum or plasma. The assay is based on the "sandwich" (two-site) immunoassay methodology: use of two monoclonal antibodies, one specific for the alpha subunit and the other for the beta subunit, results in an assay that is specific for the intact hCG molecule. Results for the first sample are obtained in 7 min; subsequent additional values are produced at 1-min intervals. Inter-run precision (CV), estimated from replicate determinations of sera, was 4.5% at an hCG concentration of 38 int. units/L, 4.9% at 114, and 6.1% at 194. Intrarun CV was less than 2% at all three concentrations. Correlations of results for 127 specimens analyzed in duplicate with the Stratus (y) and by a radioimmunoassay (x) for beta hCG (Gamma Dab M [cf931125I] beta-hCG, Travenol-Genentech Diagnostics, Cambridge, MA) yielded the following regression equation: y = 0.969x - 6.0 (r = 0.995). The Stratus immunofluorometric system provides a rapid and convenient assay of hCG in serum or plasma.

scholarly journals Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1.

1983 ◽  
Vol 158 (2) ◽  
pp. 586-602 ◽  
Author(s):  
F Sanchez-Madrid ◽  
P Simon ◽  
S Thompson ◽  
T A Springer
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Features ◽  
Cell Interactions ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Complement Receptor ◽  
Receptor Function ◽  
Alpha Subunits ◽  
Sds Page

Mouse Mac-1, a complement receptor-associated surface structure on macrophages, and LFA-1, a function-associated structure on lymphocytes, comprise a novel family of leukocyte differentiation antigens participating in adhesive cell interactions. Mac-1 and LFA-1 contain alpha-subunits of 170,000 and 180,000 Mr, respectively, and beta-subunits of 95,000 Mr noncovalently associated in alpha 1 beta 1 complexes. The structural relation between the alpha- and between the beta-subunits, and the location of functionally important sites on the molecules, have been probed with antibodies. Both non-cross-reactive and cross-reactive monoclonal antibodies (MAb) and antisera prepared to the purified molecules or the LFA-1 alpha-subunits were used. Reactivity with individual subunits was studied by immunoprecipitation after dissociation induced by high pH treatment, or by immunoblotting after SDS-PAGE. Cross-reactive epitopes on Mac-1 and LFA-1 were found to be present on the beta-subunits, which were immunologically identical. Non-cross-reactive epitopes that are distinctive for Mac-1 or LFA-1 were localized to the alpha-subunits. MAb to LFA-1 alpha-subunit epitopes inhibited CTL-mediated killing. Two MAb to Mac-1 alpha-subunit epitopes but not a third MAb to a spatially distinct alpha-epitope inhibited complement receptor function. Neither function was inhibited by a MAb binding to a common beta-subunit epitope. Therefore, sites of Mac-1 and LFA-1 involved in their respective adhesion-related functions, as well as distinctive structural features, have been localized to the alpha-subunits.

scholarly journals A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Thermal Stability ◽  
Pichia Pastoris ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

scholarly journals Amino acid sequence of the human fibronectin receptor.

1987 ◽  
Vol 105 (3) ◽  
pp. 1183-1190 ◽  
Author(s):  
W S Argraves ◽  
S Suzuki ◽  
H Arai ◽  
K Thompson ◽  
M D Pierschbacher ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Calcium Binding ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Receptor Subunit ◽  
Fibronectin Receptor ◽  
Adhesion Receptor ◽  
Receptor Beta

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.

scholarly journals The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 411-421 ◽  
Author(s):  
S J Yeaman ◽  
P Cohen ◽  
D C Watson ◽  
G H Dixon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.

An evaluation of four serum tests for pregnancy.

1983 ◽  
Vol 29 (3) ◽  
pp. 561-563 ◽  
Author(s):  
K W Ryder ◽  
R A Munsick ◽  
T O Oei ◽  
P C Young ◽  
H F Blackford
Keyword(s):  
Positive Result ◽  
Early Pregnancy ◽  
Beta Subunit ◽  
The Other ◽  
Analytical Sensitivity ◽  
Negative Results ◽  
Human Choriogonadotropin ◽  
Beta Hcg

Abstract We evaluated four pregnancy tests (Biocept-G, Beta-CG, Preg/Stat, and HCG-Beta Screen), using sera from 59 nonpregnant subjects and 77 patients with serum human choriogonadotropin beta-subunit (beta-hCG) concentrations ranging from 4 to 100 000 int. units/L. The results obtained for each test were compared with the results predicted on the basis of the sample's beta-hCG concentration and the beta-hCG concentration the manufacturer claimed necessary for a positive result (the test's analytical sensitivity). Biocept-G had the best sensitivity (100%), specificity (98.9%), and accuracy (99.2%). Beta-CG had the poorest sensitivity (86.4%), Preg/Stat the poorest specificity (87.5%), and accuracy (92.6%). We confirmed the manufacturer's claimed analytical sensitivity (200 int. units/L) for the Biocept-G procedure, but our calculated analytical sensitivity for the other tests was significantly different from that claimed by their manufacturers. Best results were obtained with Biocept-G, but with its analytical sensitivity of 200 int. units/L, samples from early pregnancy will give negative results. None of the pregnancy tests evaluated here will establish the presence or absence of early pregnancy with certainty.

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