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J Hakimi

Publications and source records attributed to J Hakimi.

At least 55 records · Page 3Linked to original sources
PubMed1988

Human interleukin-2 anti-idiotypes.

We describe the generation of a monoclonal anti-idiotypic (anti-id) antibody directed against affinity purified rabbit antibodies (id) to recombinant human IL-2. This monoclonal antibody, 6A12, has the ability to inhibit the neutralization of IL-2 activity by the id, suggesting that it may bind at or near the IL-2 neutralizing site on the id. 6A12 exhibits IL-2 agonist activity on PHA-activated human T cells. The purified IgG of 6A12 is also shown to bind to a purified soluble recombinant p55 subunit of the IL-2 receptor. Furthermore, purified 6A12 shows inhibition of IL-2 activity in an IL-2 dependent mouse T cell line (CTLL) and this inhibition can be reversed by excess IL-2. These results suggest that although 6A12 may not be an exact 'internal image' of the receptor binding site of IL-2, it may bind to at least the P55 subunit of the ligand binding site on the high affinity IL-2 receptor complex.

Animals↗
PubMed1987

Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction.

The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (approximately 11 micrograms/10(6) cells/48 h) in rodent cells using gene-linked co-amplification. The soluble forms of IL-2R were shown to retain IL-2 affinity shown by cell-surface IL-2R (Kd approximately 18 nM) and were purified to homogeneity using IL-2 affinity chromatography. Purified, recombinant IL-2R and biotinylated IL-2 were used to establish a solid-phase receptor binding assay. Binding of IL-2-biotin was demonstrated to be dose-dependent at concentrations ranging from 10 to 1000 ng/ml, and the specificity of receptor-ligand binding was demonstrated by competition with non-biotinylated IL-2 and with anti-receptor antibodies known to block IL-2 binding in vivo. This immunosorbent receptor assay offers a simple and rapid method for studying the binding of IL-2 to human IL-2R.

Animals↗
PubMed1987

A cDNA presumptively coding for the alpha subunit of the receptor with high affinity for immunoglobulin E.

Rat mast cells and a neoplastic analogue such as rat basophilic leukemia (RBL) cells have receptors that have exceptionally high affinity for immunoglobulin E (IgE). When aggregated, these receptors induce cellular degranulation. The alpha chain of the receptor contains the binding site for IgE; the function(s) of the noncovalently associated beta and gamma chains is (are) still undefined. Using a cDNA library constructed from the mRNA of RBL cells, we have isolated a cDNA clone whose sequence predicts a putative 23-residue signal peptide, followed by a sequence that accurately predicts the amino acid composition, the peptide molecular weight, and six peptide sequences (encompassing 59 residues or 26% of the total number) determined for the alpha chain by direct analysis. These findings provide strong evidence that the cDNA codes for the alpha chain, even though expression has not been unambiguously achieved. The sequence suggests that the alpha chain contains a 180-residue extracellular portion with two homologous domains of approximately 35 residues, a 20-residue transmembrane segment containing an aspartic acid, and a 27-residue cytoplasmic portion containing 9 basic amino acids. The sequence shows no homology with the low-affinity receptor for IgE from lymphocytes but over 30% homology with an Fc gamma receptor.

Amino Acid Sequence↗
PubMed1987

Induction of differentiation of the human leukemia cell line, KU812.

We treated KU812 cells from different passage levels with compounds which induce differentiation of other leukemic cell lines. Early passages contained small subpopulations of basophil and erythrocyte-like cells, but later passages consisted of more uniform cells with a myelomonocytic phenotype. When cultured with 1 nM actinomycin D, the cells became nonproliferative, developed pale cytoplasm and segmented nuclei, and lost nonspecific esterase activity. Thus, KU812 cells can be induced to mature to cells with a myelomonocytic phenotype. However, their expression of previously reported basophil characteristics diminishes with continued passage, and may be a capability of only a subpopulation of the cells.

Cell Differentiation↗
PubMed1986

Biological properties of a recombinant human immunoglobulin epsilon-chain fragment.

A recombinant human immunoglobulin epsilon-chain gene expression product (rFc epsilon) was compared with a human E myeloma protein in the affinity for epsilon-chain Fc fragment receptors (Fc epsilon R) on cultured human basophils. The association-dissociation kinetics of the rFc epsilon-Fc epsilon R interaction are indistinguishable from that of E myeloma protein, indicating that rFc epsilon and IgE have identical affinity for the receptors. The recombinant gene product sensitizes cultured basophils for anti-IgE-induced histamine release. A dose-response curve of histamine release indicates that the gene product is equally efficient in transducing the signal for degranulation as the natural IgE. Both the rFc epsilon and IgE lost the affinity for Fc epsilon R by heating at 56 degrees C. Upon renaturation by passage through a solution of 6 M guanidine hydrochloride, rFc epsilon recovered both the affinity for Fc epsilon R and the original CD spectra. On the other hand, renaturation of heat-denatured IgE largely restored optical activity above 250 nm but restored neither the affinity for Fc epsilon R nor the CD spectrum below 220 nm. The results suggest that either the amino acid sequence or the carbohydrate present in the myeloma protein, but not the rFc epsilon, may interfere with refolding of the receptor-binding structures.

Basophils↗
PubMed1984

Biosynthesis and processing of ribophorins in the endoplasmic reticulum.

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.

Animals↗
PubMed1982

Glycosylation of intracellular Sindbis virus glycoproteins.

Oligosaccharides of purified intracellular Sindbis virus glycoproteins have been examined by high-resolution Bio-Gel chromatography. The array of glycopeptides from cellular E1 and E2 appeared similar to the glycopeptides (S1, S2, S3, and S4) found in mature virus glycoproteins described previously [Hakimi, J., & Atkinson, P. H. (1980) Biochemistry 19, 5619]. However, compared to its viral counterpart, intracellular E1 glycoprotein also contained larger sized mannosyl oligosaccharides. B and PE2 proteins were found to contain an array of primarily large mannosyl oligosaccharides (8-10 hexose units). No sialyl glycopeptides were found on these proteins regardless of labeling time. By contrast, the products of PE2 cleavage (E2 and E3) contained sialyl glycopeptides similar to those found in mature virus (S1, S2, and S3). E2 also contained smaller mannosyl oligosaccharides (8-5 hexose units) similar to its viral counterpart. Current evidence shows that sialyl and galactosyl transferases are in or near the Golgi region. Thus we conclude that cleavage of PE2 with a Man8 oligosaccharide structure occurs in the Golgi region and not in the plasma membrane as suggested by others.

Animals↗
PubMed1982

The purification and characterization of alpha-L-fucosidase from the hepatopancreas of Octopus vulgaris.

We have isolated and purified, by affinity chromatography with Agarose-epsilon-amino-caproyl-fucosamine, an alpha-L-fucosidase [alpha-L-fucoside fucohydrolase EC 3.2.1.51] from the hepatopancreas of Octopus vulgaris. In the purified fraction only fucosidase activity could be detected. However, two protein bands, one major (about 95 per cent) and one minor (about 5 per cent), were evident on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing also revealed two activities, pI 8.1 (major) and pI 7.3 (minor). Under denaturing conditions the molecular weight of the major band was estimated to be 52,000 while that of the minor one was 43,000. Only one major activity peak with an apparent molecular weight of 70,000--75,000 was detected by gel filtration chromatography. The enzyme has two optimal pH values, and the relative activities are temperature-dependent; one optimum is at pH 5.5 +/- 0.2 and the other at pH 3.0 +/- 0.2. We found that the enzyme has a maximum activity at about 70 degrees C, but 50 per cent of the enzyme was inactivated at 70 degrees C after 5 min. The purified enzyme, using p-nitrophenyl-L-fucoside as substrate, has a specific activity of 38.9 units/mg of protein, Km of 3.58 x 10(-4) M and Vmax of 65 mumol/min/mg of protein. alpha-L-Fucose acts as a competitive inhibitor, with a K1 of 1.2 x 10(-3) M. alpha-L-Fucosidase released radioactive fucose from cellular glycopeptides, but no detectable free fucose was released fom 5 natural substrates.

Animals↗
PubMed1981

Application of high-field proton magnetic resonance spectroscopy in the structural determination of membrane-derived Sindbis virus glycopeptides.

Sindbis virus membrane glycopeptides have ben purified in chemical quantities and their oligosaccharide structures analyzed by 1H NMR spectroscopy at 360 MHz. Interpretable spectra could be obtained with approximately 100 micrograms of oligosaccharide. Spectral analysis of the sialyl glycopeptides S1, S2, and S3 at high and low temperatures confirms their structures to be NANA alpha (2,3)Gal beta (1,4)-GlcNAc beta (1,2)Man alpha (1,6)-[NANA alpha (2,3)Gal beta (1,4)-GlcNAc beta (1,2)Man alpha (1,3)]-Man beta (1,4)GlcNAc beta (1,4)-[Fuc alpha-(1,6)]-GlcNAc beta 1-Asn. These are heterogeneous with respect to sialic acid (NANA). Spectra of two endo-beta-N-acetylglucosaminidase products of the S4 glycopeptides are reported. The interpretation of these spectra is consistent with Man5GlcNAc and Man7GlcNAc oligosaccharide structures. Their chemical shifts are essentially identical with those reported for ovalbumin glycopeptides of the same composition, with exception to the perturbations arising from their oligosaccharide nature.

Animals↗
PubMed1981

Determination of the Structure of four glycopeptides from hen ovalbumin using 360-MHz proton magnetic resonance spectroscopy.

Four glycopeptides have been purified by Dowex and Bio-Gel P2 chromatography from Pronase digests of hen ovalbumin. The high-resolution proton magnetic resonance spectra of these glycopeptides and various products of their enzymatic digestion have been obtained at 360 MHz. By use of information derived from the spectra of a number of model compounds, an unambiguous assignment of all C1-H and Man C2-H resonances in the spectra can be made. On this basis structures are proposed for the four glycopeptides which are identical with those structures previously deduced from destructive chemical methods.

Animals↗
PubMed1981

Demonstration of heterogeneity of chick ovalbumin glycopeptides using 360-MHz proton magnetic resonance spectroscopy.

Ovalbumin glycopeptides AC-C and AC-D at various stages of purification were studied by high-field proton magnetic resonance spectroscopy (1H NMR). In a homogeneous substance, the intensity of the various resonances appears in integral amounts, while subintegral intensities usually denote mixtures of structure. We show how 1H NMR can be used to nondestructively assay the purification of major components from mixtures. In glycopeptide AC-C we have spectroscopic evidence for the four different glycopeptide species, three of which have been described [Shepherd, V., & Montgomery, R. (1978) Carbohydr. Res. 61, 147; Tai, T., Yamashita, K., Ito, S., & Kobata, A. (1977) J. Biol. Chem. 252, 6687]. However, we did detect a fourth structure not previously reported. In glycopeptide AC-D, we have spectroscopic evidence for five different compounds, only two of which have been previously reported (Tai et al., 1977; Shepherd & Montgomery, 1978).

Animals↗
PubMed1980

Growth-dependent alterations in oligomannosyl glycopeptides expressed in Sindbis virus glycoproteins.

Sindbis virus was used to examine host cell growth-dependent expression of oligomannosyl glycopeptides within single species of viral glycoproteins. Four mannosyl oligosaccharides ranging from 5 to 8 mannose residues were separated on Bio-Gel P4 column chromatography. Virus derived from rapidly growing chicken embryo fibroblasts displayed greater quantities of larger sized oligomannosyl glycopeptides in intact virus and in purified Sindbis virus glycoproteins E1 and E2 compared with virus from nongrowing cells. The pattern of mannosyl oligosaccharides in the two glycoproteins differed remarkably. E1 and E2 contained predominantly Man5GlcNAc and Man7GlcNAc, respectively; in addition, E1 relative to E2 contained more complex-type glycopeptides than mannosyl-type glycopeptides. These data clearly demonstrate that host-dependent alterations in glycosylation are expressed in Sindbis virus and that differences in specific glycosylation patterns are obscured in oligosaccharides derived from mixtures of glycoproteins. It is apparent that processing of these cotranslated glycoproteins can yield different patterns of oligosaccharide structures.

Animals↗