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H L Cooper

Publications and source records attributed to H L Cooper.

At least 55 records · Page 3Linked to original sources
PubMed1983

Rapid protein phosphorylation induced by phorbol ester in HL-60 cells. Unique alkali-stable phosphorylation of a 17,000-dalton protein detected by two-dimensional gel electrophoresis.

Treatment of the leukemic promyelocytic cell line, HL-60, with phorbol-12-myristate-13-acetate (PMA), has been shown to induce terminal differentiation of the cells into monocytes. We found that this effect involves a rapid increase in phosphorylation of a 17-kDa protein (pp17, pI approximately 5.5) and a 27-kDa protein (pp27, pI approximately 5.5) as detected by two-dimensional gel electrophoresis. Within 15 min there was 5- to 7-fold and 3- to 4-fold increase in the phosphorylation of pp17 and pp27, respectively. The rate of phosphorylation remained accelerated over a period of 60-min incubation with PMA. In contrast to these specific changes, there was no effect of PMA on total 32Pi incorporation into HL-60 cells during the same period. The phosphoester bond of the pp17, but not of pp27, revealed a remarkable stability to alkali treatment of the electrophoretic gels. However, phosphoamino acid analysis showed that both pp17 and pp27 proteins are phosphorylated only at serine residues. These two phosphoproteins were located exclusively in the cytosol and were absent from the crude membrane fraction. We further demonstrated that several inactive derivatives of phorbol ester failed to cause an increase in phosphorylation of pp17 and pp27, suggesting that these specific phosphorylation events are intimately related to the induction of differentiation in HL-60 cells by PMA.

Cell Differentiation↗
PubMed1983

Identification of the hypusine-containing protein hy+ as translation initiation factor eIF-4D.

A single protein of Mr 17,000-19,000 and pI approximately equal to 5.1, found in all animal cells we have studied to date, undergoes post-translational modification in growing cells to form the unusual amino acid hypusine. Because of the association of this modification with the increasing rate of protein synthesis during lymphocyte growth stimulation, its subcellular distribution, and its widespread occurrence and structural conservation among animal cells, we considered the possibility that this protein might be a translation initiation factor. Purified rabbit reticulocyte factors (eukaryote initiation factors) eIF-4C and eIF-4D were chosen for study because of their Mr (17,000-19,000) and acidic pI. The hypusine-containing protein and purified eIF-4D showed identity of electrophoretic mobility in both isoelectric focusing and NaDodSO4/polyacrylamide gel electrophoresis dimensions, while eIF-4C was clearly nonidentical. Purified eIF-4D contained approximately 1 mol of hypusine per mol of protein. Since only one protein has thus far been observed to contain hypusine, we conclude that eIF-4D is the hypusine-containing protein. On the basis of relative synthesis among lymphocyte proteins and detection by Coomassie blue staining, we also conclude that eIF-4D is a major cell protein. It is possible that the activity of this factor is modulated by It is possible that the activity of this factor is modulated by post-translational hypusine formation, which may play a role in regulation of protein synthesis during lymphocyte growth stimulation.

Animals↗
PubMed1983

Rapid turnover of HLA proteins in quiescent lymphocytes: proposed connection with immunologic surveillance.

Current immunologic theory defines a role for HLA-A, -B, -C proteins in cells presenting foreign antigens to cytotoxic T lymphocytes. No clear function, however, is assigned to the prominently represented HLA molecules on the T cells themselves. To investigate this question, the synthesis and turnover of HLA-A, -B, -C molecules in human peripheral lymphocytes was studied. Newly synthesized HLA-A, -B, -C and beta 2-microglobulin molecules were identified among total lymphocyte proteins by two-dimensional gel electrophoresis and specific immunoselection. In resting T lymphocytes, the polymorphic set of HLA-A, -B, -C proteins was among the most prominently synthesized and rapidly turned over of total cell proteins. Its half-life was 6 to 7 hr, which is considerably shorter than that of total cell protein. HLA and beta 2M were the major proteins of the plasma membrane undergoing active synthesis and turnover in resting T lymphocytes. After growth stimulation, the rate of HLA synthesis increased to a lesser degree than total protein synthesis. Survival of newly synthesized HLA was increased, however, indicating reduction in HLA turnover. The result was the accumulation of HLA in the plasma membrane. It is suggested that continuous degradation and replacement reflects the biochemical nature of the participation of HLA molecules in immunologic surveillance by quiescent lymphocytes. Cessation of HLA turnover, with accumulation of surface HLA molecules, is a biochemical adjustment related to T cell activation.

Antigens, Surface↗
PubMed1982

The biosynthesis of protein-bound hypusine (N epsilon -(4-amino-2-hydroxybutyl)lysine). Lysine as the amino acid precursor and the intermediate role of deoxyhypusine (N epsilon -(4-aminobutyl)lysine).

The major labeled constituent produced in cellular protein during the incubation of Chinese hamster ovary (CHO) cells with [3H]putrescine or [terminal methylenes-3H]spermidine was identified as hypusine (N epsilon -(4-amino-2-hydroxybutyl)lysine). This unusual amino acid was found to occur predominantly in one relatively acidic low molecular weight protein. When CHO cells were labeled with [4,5-3H)lysine, a small portion of the radioactivity of the cellular protein fraction, after release by proteolytic digestion or acid hydrolysis, chromatographed at the position of hypusine. Oxidative degradation of this isolated labeled material yielded labeled lysine, thus, providing evidence that lysine is the amino acid precursor of hypusine. Upon incubation of CHO cells with the metal chelator, alpha,alpha-dipyridyl, and either [4,5]3H]lysine or [terminal methylenes-3H]spermidine, label was incorporated into a protein-bound material, the chromatographic properties of which, after release by digestion, were found to be different from those of hypusine. This constituent of cell protein was identified as the unhydroxylated form of hypusine, deoxyhypusine (N epsilon -(4-aminobutyl)lysine). Evidence that the normal biosynthesis of hypusine proceeds through hydroxylation of deoxyhypusine was obtained by demonstration of conversion of protein-bound deoxyhypusine to protein-bound hypusine both in intact cells and in cell-free lysate. In the presence of the metal chelator, alpha,alpha-dipyridyl, deoxyhypusine accumulated in a single protein whose two dimensional electrophoretic properties were indistinguishable from those of the usual hypusine-containing protein. This finding supports the proposed mechanism in which peptide-bound lysine is converted to peptide-bound hypusine through hydroxylation of the transitory intermediate, deoxyhypusine.

Animals↗
PubMed1982

Posttranslational formation of hypusine in a single major protein occurs generally in growing cells and is associated with activation of lymphocyte growth.

Growing lymphocytes perform a novel chemical modification of a single protein (Hy+: approximately 18 kd, pI approximately 5.1), resulting in the formation of the unusual amino acid, hypusine (N epsilon-[4-amino-2-hydroxybutyl]lysine). This posttranslational event occurs only following activation of lymphocyte growth. Hypusine formation increases at a rate parallel to protein synthesis during the first 24 hr of growth stimulation, beginning before 6 hr of growth. At all times, hypusine is restricted primarily to the single protein, Hy+. In resting cells, the unmodified substrate protein, Hy0, is continuously synthesized and maintained in a steady-state pool of significant size. In several other cell lines, hypusine formation was also observed in a single protein of approximately 18 kd, pI approximately 5.1, indistinguishable electrophoretically from the lymphocyte protein. Thus Hy+ is a ubiquitous protein showing significant conservation among divergent species. Maintenance by resting lymphocytes of a pool of unmodified protein and early activation during growth of the hypusine-forming enzyme system suggest that this posttranslational modification may be of importance to lymphocyte activation.

Amino Acids↗
PubMed1982

Effect of interferons on protein synthesis in human lymphocytes: enhanced synthesis of eight specific peptides in T cells and activation-dependent inhibition of overall protein synthesis.

Lymphoblastoid and fibroblast IFN inhibited PHA stimulation of overall protein synthesis in human lymphocytes by ca. 30%. Inhibition occurred within the first 6 hr of PHA treatment and was not progressive. DNA synthesis at 48 hr was inhibited to the same extent. Overall protein synthesis in resting lymphocytes was not detectably inhibited by IFN concentrations up to 1000 U/ml. Thus, inhibition of protein synthesis and subsequent reduction of cell proliferation by IFN require certain early events in mitogen activation. Resting lymphocytes were not unresponsive to IFN treatment, however. Two-dimensional electrophoretic analysis of newly synthesized proteins after IFN treatment showed enhanced synthesis of a specific set of eight peptides (I-peptides), which were shown to be synthesized in T lymphocytes. This enhancement was produced by both IFN-alpha and IFN-beta after 4 to 6 hr of exposure and was identical for all lymphocyte donors studied. After growth stimulation, IFN treatment produced no enhancements of additional peptides, although the original eight I-peptides were enhanced as usual. It is concluded that the biochemical activities of the I-peptides, which remain to be determined, cannot inhibit protein synthesis in resting lymphocytes, but may do so after mitogen activation, when the major physiologic restriction of lymphocyte protein synthesis is released. Alternatively, the I-peptides may be unrelated to regulation of protein synthesis but may be involved in viral protection or enhancement of NK activity.

Blood Proteins↗
PubMed1981

Identification of hypusine, an unusual amino acid, in a protein from human lymphocytes and of spermidine as its biosynthetic precursor.

When normal human peripheral lymphocytes are treated with mitogen and grown in the presence of [3H]putrescine or [terminal methylenes-3H]spermidine, label is incorporated predominantly into one cellular protein. The radioactive constituent of this protein was identified as the unusual amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This was accomplished by isolation of the component from proteolytic digests or acid hydrolysates and comparison with authentic hypusine by chromatography, conversion to the 2,4-dinitrophenyl derivative, and oxidative degradation. The observed relationships among intracellular levels of labeled putrescine, polyamines, and protein bound hypusine after growth of cells with the various labeled amines and with or without an inhibitor of polyamine biosynthesis supply evidence that spermidine is the immediate amine precursor of hypusine and that the 4-amino-2-hydroxybutyl portion of hypusine derives from the butylamine moiety of spermidine.

Blood Proteins↗
PubMed1981

Microtubules: are they involved in the initiation of lymphocyte activation?

Purified human blood lymphocytes were stimulated with concanavalin A or phytohemagglutinin. DNA synthesis was measured with 2-h pulses of [3H]thymidine between 48 h and 73 h after stimulation. Colchicine, at concentrations between 0.1 muM and 10 muM, suppressed consequent DNA synthesis without affecting viability of the cells when added at any time up to 18 h before incorporation of [3H]thymidine was assessed. In concanavalin-A-stimulated lymphocytes, removal of the mitogen by methyl alpha-mannoside only prevented proliferation when added initially, but was without any effect when added after 20 h of stimulation, regardless of when DNA synthesis was measured. Thus, there was a period after 20 h of concanavalin A stimulation, when DNA synthesis was still sensitive to colchicine, but no longer required the presence of the mitogen. Colchicine also suppressed incorporation of [3H]leucine into protein, in resting as well as mitogen-stimulated lymphocytes. Similarly, colchicine decreased amino acid transport, as determined by uptake of alpha-amino-isobutyrate, which appeared to be the rate-limiting step in the incorporation of amino acids into protein in colchicine-treated cells. When the rate of protein synthesis was followed by the relative distribution of ribosomal particles, especially the increase of polysomes in activated lymphocytes, colchicine was without any detectable effect. The early increase in the incorporation of [14C]oleate into phospholipids was identical in the presence or absence of the microtubule-active drug. The data strongly suggest that microtubules are not involved in the initiation of lymphocyte growth or mitogenesis.

Biological Transport↗
PubMed1980

Polyamines as physiological substrates for transglutaminases.

When normal human blood lymphocytes are treated with mitogen in the presence of [3H]putrescine, label is incorporated into a few cellular proteins. Labeled N-(gamma-glutamyl) putrescine, N1-(gamma-glutamyl)spermidine, and N8-(gamma-glutamyl)spermidine were identified in exhaustive proteolytic digests of the cellular protein fraction. The enzyme-mediated clotting of rat seminal plasma to which 14C-labeled spermidine and spermine are added is accompanied by incorporation of the polyamines into a number of seminal plasma proteins. Proteolytic digests of the protein fraction from this clotted seminal plasma contain labeled N1-(gamma-glutamyl)spermidine, N8-(gamma-glutamyl)spermidine, N1,N8-bis(gamma-glutamyl)spermidine, N1-(gamma-glutamyl)spermine, and N1,N12-bis(gamma-glutamyl)spermine. These findings support a proposal that polyamines serve as substrates for transglutaminases both in cells and in an extracellular fluid. They show differences in cellular and extracellular substrate properties of the polyamines and indicate cross-linking through these amines in the extracellular system, but provide no evidence for such cross-linking in the cells.

Humans↗
PubMed1980

Computer-assisted analysis of two-dimensional electrophoreses of human lymphoid cells.

Two-dimensional electrophoresis in polyacrylamide gel provides a highly reproducible display of biosynthetically pulse-labeled cellular polypeptides, whose relative rates of synthesis can be judged from the relative spot densities they produce in fluorographic images of the electrophoreses. When experimental and control cell populations synthesize protein at differing rates, either the sample load per gel or the duration of exposure of gel to film may be adjusted to produce comparable experimental and control images with no alteration in the rank order of relative spot density. However, variation in the duration of pulse-labeling may significantly alter relative spot density. Film density saturation and nonlinear response to radiation may also alter relative spot density for polypeptides with very large or very small amounts of radioactivity. Stimulation of human peripheral blood lymphocytes with phytohemagglutinin alters the relative synthesis rates of certain polypeptides visualized on the gels. These can be quantitated by using an automated series of computer algorithms to analyze the images. Data from this analysis confirm statistically significant alterations in the relative densities produced by certain polypeptides. Various human lymphoblastoid cell lines may also be distinguished from each other by this technique.

Cells, Cultured↗