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H L Malech

Publications and source records attributed to H L Malech.

At least 127 records · Page 7Linked to original sources
PubMed1986

Characterization of the formyl peptide chemotactic receptor appearing at the phagocytic cell surface after exposure to phorbol myristate acetate.

We examined the biochemistry and subcellular source of new formyl peptide chemotactic receptor appearing at the human neutrophil and differentiated HL-60 (d-HL-60) cell surface after stimulation with phorbol myristate acetate (PMA). Formyl peptide receptor was analyzed by affinity labeling with formyl-norleu-leu-phe-norleu-[125I]iodotyr-lys and ethylene glycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis of autoradiographs. PMA, a specific granule secretagogue, increases affinity labeling of formyl peptide receptors on the neutrophil surface by 100%, and on d-HL-60, which lack specific granule markers, by 20%. Papain treatment markedly reduces surface labeling of formyl peptide receptor in both neutrophils and d-HL-60, and results in the appearance of a lower m.w. membrane-bound receptor fragment. PMA stimulation of papain-treated cells increases uncleaved surface receptor on neutrophils by 400%, and on d-HL-60 by only 45%. This newly appearing receptor is the same apparent m.w. (55,000 to 75,000 for neutrophils; 62,000 to 80,000 for d-HL-60) and yields the same papain cleavage product (Mr, 31,000 for neutrophils; Mr, 29,000 for d-HL-60) as receptor on the surface of unstimulated cells. Formyl peptide receptor detected by affinity labeling in neutrophil specific granule-enriched subcellular fractions is identical to receptor found on the surface of unstimulated cells appearing as equal amounts of two isoelectric forms (isoelectric points, 5.8 and 6.2) at Mr 55,000 to 70,000. There is twice as much receptor present in the specific granule-enriched fraction per cell equivalent compared with plasma membrane. Azurophil granules contain trace amounts of receptor. Similar analysis of neutrophils treated with papain before subcellular fractionation shows that papain cleaved receptor fragment is detectable almost exclusively in the plasma membrane-enriched fraction. Most of the affinity-labeled formyl peptide receptor present in specific granule enriched fraction is present in membranes other than plasma membrane or Golgi membrane, because specific granule-enriched fraction contains only a small amount of plasma membrane marker and an amount of Golgi membrane marker equal to that found in plasma membrane-enriched fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1986

Analysis of the peptide subunits of human neutrophil myeloperoxidase.

Myeloperoxidase is a cationic protein present in the azurophilic granules of human neutrophils and constitutes 2% to 5% of neutrophil protein by weight. Despite extensive characterization of the functional importance of myeloperoxidase in the microbicidal activity of neutrophils, there is no consensus regarding the subunit composition of this enzyme or the presence of genetic polymorphism. Using isoelectric focussing and peptide mapping of chymotryptic digests of myeloperoxidase subunits under reducing and nonreducing conditions, we found two different heavy chain peptides that have overlapping but distinctive chymotryptic digest maps. One of these heavy chains has a reducible intrapeptide disulfide bond not found in the other. These results suggest that the model proposed for the structure of myeloperoxidase as a symmetric molecule with two identical heavy-light protomers may not be correct.

Autoradiography↗
PubMed1986

Identification of a differentiation-specific cell surface antigen on HL60 cells that is associated with proliferation.

We have produced a murine IgM monoclonal antibody (Y201) that recognizes a cell surface antigen present on HL60 cells. Seventy percent of uninduced HL60 cells expressed Y201 antigen, while the remainder did not. There were no morphological differences between HL60 cells that expressed Y201 antigen and cells that did not express Y201 antigen. Cells with the greatest number of antigenic sites were found to have greater proliferative capacity in liquid culture and in soft agar than did HL60 cells deficient in this marker. Expression or lack of expression of the Y201 antigen is not constant over a prolonged period in that both subpopulations ultimately reproduced the original pattern of antigenic expression when grown in liquid culture. The antigen identified by Y201 was lost with terminal differentiation of HL60 cells using a variety of inducers. Loss of Y201 antigen during differentiation was associated with a decrease in proliferative capacity in soft agar. Loss of Y201 antigen by greater than 95% of differentiated HL60 cells was associated with loss of proliferative capacity. These data suggest that HL60 cells are heterogeneous in regard to proliferative capacity and that this heterogeneity is associated with expression of the cell surface antigen identified by Y201.

Antibodies, Monoclonal↗
PubMed1986

A neutrophil membrane marker reveals two groups of chronic myelogenous leukemia and its absence may be a marker of disease progression.

An IgG1 monoclonal antibody, 31D8, that recognizes normal neutrophil (PMN) membranes, was used to study PMN from patients with chronic myelogenous leukemia (CML). Nineteen patients with Philadelphia chromosome positive CML were followed over a ten-month period and compared with 23 normals, six patients with leukemoid reactions, and eight patients with phagocytic cell defects. The percentage of PMN binding of 31D8 among normal subjects was variable about a normal distribution with an average of 95 +/- 2% of cells binding 31D8. In contrast, there were two groups of CML patients: in 14 patients 88 +/- 3% PMN bound 31D8 while in the remaining five patients only 6 +/- 6% PMN bound 31D8. PMN 31D8 binding was normal in the control patient groups. Control antibodies 7C3 (binds to PMN precursors) and OKM1 (binds to the CR3 (iC3b) receptor) bound normally to CML neutrophils. Functionally, CML cells had normal chemotaxis to several stimuli and normal superoxide generation to phorbol myristate acetate. However, superoxide production in response to fmet-leu-phe was significantly less in 31D8 negative CML PMN than both 31D8 positive CML PMN and normal PMN which contained 85% 31D8 positive and 15% 31D8 negative PMN. Clinically, 2 of 14 CML patients with 31D8 positive PMN were in blast crisis (one extramedullary) at the time of study and the other 12 patients remained clinically stable in the chronic phase during the ten months of study. In contrast, one of five patients with 31D8 negative PMN was in blast crisis at the time of study and all four of the remaining patients progressed to either the accelerated phase or blast crisis. Three of these patients died of their disease eight to ten months after their initial study. Thus, failure of CML cells to bind 31D8 may be useful for predicting which patients are likely to progress to the accelerated phase or blast crisis.

Antibodies, Monoclonal↗
PubMed1986

Activation of human neutrophils by monoclonal antibody PMN7C3: cell movement and adhesion can be triggered independently from the respiratory burst.

Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.

Antibodies, Monoclonal↗
PubMed1986

Polymorphonuclear leukocyte heterogeneity in neonates and adults.

We have used a mouse monoclonal antibody (31D8) to determine whether differences in neutrophil (PMN) subpopulations might help explain decreased PMN chemotaxis in neonates compared with that of adults. 31D8 has been shown to bind heterogeneously to adult PMNs. Approximately 80% of the PMNs that strongly bind 31D8 (31D8 "bright") are the same cells that depolarize and migrate chemotactically when stimulated with the chemoattractant N-formyl-methionylleucylphenylalanine, while the 20% that weakly bind 31D8 fail to similarly respond. All neonatal PMNs bound 31D8 heterogeneously. There was a smaller population of 31D8 "bright" cells in neonates at birth (76% +/- 6%, n = 45) compared with that of neonates at three to 15 days of age (82% +/- 5%, n = 10, P less than 0.002) and both were smaller than that of adults (88% +/- 4%, n = 45, P less than 0.001 and P less than 0.001). Neonatal cord PMNs, which traversed a micropore filter in a modified Boyden chemotaxis chamber in the presence of a chemoattractant, had an increased percentage of 31D8 "bright" cells (89% +/- 7%) than did PMNs which remained above the filter (82% +/- 7%, n = 10, P = 0.034). PMN chemotaxis was less in neonates at birth (32.7 +/- 4.5 micron) than at three to six days of age (36.8 +/- 11.3 micron) and both were decreased compared with that of adults (69.1 +/- 12.4 micron, P less than 0.001 and P less than 0.001). These findings indicate that decreased PMN chemotaxis in neonates may be due in part to a smaller PMN subpopulation of highly motile cells.

Adult↗
PubMed1985

A transient rise in intracellular free calcium is not a sufficient stimulus for respiratory burst activation in human polymorphonuclear leukocytes.

Binding of murine monoclonal antibody PMN7C3 to human neutrophils results in a large rapid, dose dependent transient increase in intracellular free calcium as measured by QUIN-2 fluorescence. Unlike other calcium mobilizing agents PMN7C3 does not induce any increase in respiratory burst activity over basal level. The PMN7C3 effect requires multivalent binding. Chelation of extracellular calcium does not significantly decrease the fluorescence transient generated by exposure to PMN7C3. Lowering of basal levels of intracellular free calcium concentration by maintaining QUIN-2-loaded PMN in calcium free medium eliminates the fluorescence transient. The observations demonstrate that a cell surface receptor mediated intracellular free calcium transient may be generated without any associated respiratory burst activation.

Antibodies, Monoclonal↗
PubMed1985

Asparagine-linked oligosaccharides on formyl peptide chemotactic receptors of human phagocytic cells.

Formyl peptide chemotactic receptors affinity-labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (where Nle represents norleucine) and ethylene glycol bis(succinimidyl succinate) consist of two isoelectric forms with cell type differences in both apparent size and charge (neutrophils: 55-70 kDa, pI 5.8, and 6.2.; monocytes: 60-75 kDa, pI 5.6 and 6.0; differentiated HL-60 cells: 62-85 kDa, pI 5.6 and 6.0). Endo-beta-N-acetylglucosaminidase F (endo F) cleavage of N-linked oligosaccharides from formyl peptide receptor generates 40-50- and 33-kDa products that can be affinity-labeled. Whereas both pI forms of this receptor from neutrophils are cleaved by endo F to 33-kDa final products, this cleavage does not eliminate pI differences. Tunicamycin decreases expression of formyl peptide receptor on differentiating HL-60 and causes a dose-dependent decrease in size of the major product seen after affinity labeling (0.5 micrograms/ml: 38-48 kDa; 2 micrograms/ml: 32 kDa). Thus, the formyl peptide receptor polypeptide backbone from all three cell types contains at least two N-linked oligosaccharide side chains which contribute to the cell type differences in Mr and are not required for ligand binding. Papain treatment of intact cells generates a membrane-bound formyl peptide receptor fragment that can be affinity-labeled and is of similar size (29-31 kDa) in all three cell types. Endo F treatment of the affinity-labeled papain fragment of formyl peptide receptor does not alter its size, suggesting that this fragment does not contain the N-linked oligosaccharide cleaved by endo F from intact receptor. The results indicate that at least two N-linked oligosaccharide chains are located on the distal 1-3-kDa portion of the receptor polypeptide backbone.

Acetylglucosaminidase↗
PubMed1985

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

Antibodies, Monoclonal↗
PubMed1985

Functional integrity of cytokineplasts: specific chemotactic and capping responses.

Cytokineplasts (CKP) are motile, membrane-bound, anucleate, granule-poor cytoplasmic fragments that are induced from human blood polymorphonuclear leukocytes (PMN) by the brief application of heat. We examined CKP with respect to specific chemotactic and capping responses, the presence of the N-formyl-peptide chemotactide receptor, and evidence of respiratory burst activity and compared them with CB-cytoplasts, which are fragments created by the centrifugation of cytochalasin B (CB)-treated PMN at high speeds. Under agarose, CKP responded chemotactically to both N-formyl-methionyl-leucyl-phenylalanine (fmlp) and zymosan-activated serum; CB-cytoplasts responded to neither chemoattractant. Despite the functional differences, both fragments retained N-formyl-peptide receptors as measured by affinity labeling with N-formyl-norleu-leu-phe-norleu-125I-tyr-lys and autoradiography of dried SDS-PAGE gels. For studies of capping we used a murine monoclonal antibody, PMN7C3, which binds a specific, widely distributed membrane component of intact PMN, and on warming, promptly induces capping of ligand-receptor complexes. Rhodamine-conjugated PMN7C3 at 4 degrees C labeled the surface of CKP homogeneously. As the CKP warmed to 37 degrees C, label became concentrated in small fluorescent caps at the rear of migrating fragments. Although CB-cytoplasts also bound the fluorochromed antibody homogeneously in the cold, on warming they were unable to concentrate the label normally. With respect to respiratory burst activity, the situation in the two fragments was reversed: CKP did not generate superoxide anion when stimulated either with phorbol myristate acetate or with fmlp after pretreatment with CB; CB-cytoplasts, as noted earlier by other investigators, did. These two types of cytoplasts with markedly different capabilities have complementary roles in the analysis of PMN function.

Chemotactic Factors↗
PubMed1985

Biochemical analysis and subcellular localization of a neutrophil-specific antigen, PMN-7, involved in the respiratory burst.

The adherence of serum-opsonized yeast to neutrophils results in phagocytosis of these particulate stimuli and activation of the respiratory burst. Both events are mediated or modulated in part by the surface receptors for IgG and complement. The link between the binding of complex particulate stimuli to the cell surface, and the triggering of these neutrophil functions, is not completely understood. We have previously described an anti-human neutrophil, murine monoclonal antibody PMN7C3, which specifically inhibits the respiratory burst of neutrophils stimulated with serum-opsonized yeast. In the present study, we show that the antigen recognized by PMN7C3 (PMN7 antigen) is present on a number of neutrophil proteins, including the recently described group of related leukocyte membrane glycoproteins CR3, LFA-1, and p150,95. The PMN-7 antigen differs from other antigens associated with the C3bi receptor complex (MAC 1, MO 1, OKM1, OKM10) in that it is present only on neutrophils among peripheral blood cells. Furthermore, the binding of PMN7C3 to the neutrophil surface inhibits the activation of the respiratory burst by serum opsonized zymosan without affecting phagocytosis of these particulate stimuli. The cross-linking of cell surface PMN7 antigen by multivalent antibody is associated with the capping and internalization of antigen-antibody complexes, and appears to be necessary for the expression of maximum inhibition of opsonized zymosan-triggered respiratory burst activity. PMN7C3 also binds to a group of granule-associated proteins biochemically distinct from CR3, LFA-1, and p150,95. These granule-associated proteins containing PMN7 antigen can be mobilized to the cell surface with secretion. PMN7 antigen-bearing proteins may play a role in modulating the activation of the respiratory burst associated with phagocytosis of serum-opsonize zymosan.

Antibodies, Monoclonal↗
PubMed1985

An antibody binding to human neutrophils demonstrates antigenic heterogeneity detected early in myeloid maturation which correlates with functional heterogeneity of mature neutrophils.

An IgG1 mouse monoclonal antibody (31D8) labels a subpopulation of human peripheral neutrophils. Heterogeneous binding to granulocyte precursors is seen in the bone marrow as early as the myelocyte stage of maturation. 31D8 binding is not affected by the in vitro stimulation or incubation of neutrophils. We demonstrate that the cells which bind 31D8 strongly (31D8-bright) are the same cells which depolarize, reduce nitroblue tetrazolium, and migrate chemotactically when stimulated with the chemoattractant N-formylmethionylleucylphenylalanine. Treatment with cytochalasin B modulates functional activity so that all cells depolarize or reduce nitroblue tetrazolium in response to this chemoattractant, but the heterogeneous binding of 31D8 does not change. The stable binding of 31D8 at 37 degrees C and during activation permitted characterization of the functional capacity of the two populations. These results demonstrate neutrophil heterogeneity early in myeloid maturation and correlate antigenic with functional heterogeneity. 31D8 will make it possible to study the significance of neutrophil heterogeneity in a variety of clinical situations.

Antibody Specificity↗
PubMed1983

Cell motility and microtubules in cultured fibroblasts from patients with Kartagener syndrome.

Patients with Kartagener syndrome (KS) show defects in ciliary and flagellar movement that are usually associated with the partial or total absence of dynein side arms from axonemal microtubules. Dynein is essential for such movements, but its involvement in other cellular (particularly microtubule-related) processes is unknown. It has recently been reported that neutrophils from KS patients show impaired motility including responses to chemotactic stimuli, suggesting that dynein-like proteins may be generally involved in motile processes. In support of this, we have now found that spontaneous motility of cultured skin fibroblasts from KS patients is also markedly impaired. Three cell lines derived from skin explants of KS patients with deficient dynein side arms in nasal cilia and eight cell lines derived from normal volunteers were studied. Fibroblasts were seeded into dishes containing colloidal gold-coated cover glasses [Albrecht-Buehler, 1977], incubated for 24h at 37 degrees C, and the area of cell "phagokinetic" tracks determined. Each cell line studied in this manner reproducibly displayed an amount of spontaneous motility characteristic for that cell line. The mean track area (+/- SE) for all control cells studied was 14.6 +/- 0.5 X 10(3) micron2 whereas for KS fibroblasts was 8.7 +/- 0.4 X 10(3) micron2 (P less than 0.001). Immunofluorescence microscopy using antitubulin and antihuman 210 K MAP antibodies revealed no differences in the staining patterns between control and KS fibroblasts. Pinocytic rates were identical, and the complement of tubulin and major microtubule associated proteins as seen on one-dimensional SDS polyacrylamide gel autoradiographs appeared similar for control and KS cells. Thus, the observed motility defect is probably not the result of alterations in the occurrence or distribution of microtubules or in the occurrence or binding of the major microtubule-associated proteins. This defect in cellular motility may be related to the absence of dynein or may reflect another independent cellular defect.

Adenosine Triphosphatases↗
PubMed1983

Biochemical and immunologic analysis of hereditary myeloperoxidase deficiency.

Myeloperoxidase (MPO), a heme enzyme present in the azurophilic granules of human polymorphonuclear neutrophils (PMN), is important in the oxygen-dependent microbicidal activity of PMN. MPO deficiency, defined as the lack of PMN peroxidative activity, is a common genetic defect of human PMN. The purpose of our study was to characterize the structural basis for this loss of enzymatic activity, using protein biochemical and immunochemical techniques to examine PMN from three subjects with partial MPO deficiency and from five subjects with complete MPO deficiency.We purified MPO from normal PMN and defined its electrophoretic mobility after two-dimensional electrophoretic separation, using nondenaturing acidic polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecyl sulfate (SDS) denaturation and SDS-PAGE separation of MPO subunit peptides. In agreement with previous studies, we found that normal MPO had subunits of 59,000 and 13,500 mol wt when subjected to SDS-PAGE under reducing conditions. Granule protein extracts of normal PMN, partially MPO-deficient PMN, and completely MPO-deficient PMN were analyzed with two-dimensional PAGE. Partially MPO-deficient PMN granules contained electrophoretically normal MPO in less than normal amounts, whereas completely MPO-deficient PMN granules contain no protein with the electrophoretic mobility of normal MPO. Using rabbit antiserum against purified MPO, we used immunoautoradiographic analysis to examine whole PMN for peptides immunochemically related to MPO. PMN from normal, partially MPO-deficient, and completely MPO-deficient subjects were solubilized in SDS and component peptides separated by SDS-PAGE. The peptides were electroblotted onto nitrocellulose paper that was exposed sequentially to rabbit anti-MPO and (125)I-protein A before autoradiography. Radiolabeled bands were identical when partially purified MPO or normal PMN were compared except that whole PMN contained a small amount of an immunologically cross-reactive membrane associated material of 75,000-90,000 mol wt. Using a modification of this immunoautoradiographic analysis, we quantitated the relative amounts of MPO peptides in PMN. PMN from MPO-deficient subjects contain 41.0-52.3% the amount of MPO peptides present in normal PMN. Similar analysis showed that completely MPO-deficient PMN lacked any peptides corresponding to MPO peptides.We conclude that partial MPO deficiency is characterized by the presence of electrophoretically and immunologically normal MPO in amounts approximately one-half that seen in PMN from normal subjects. Completely MPO-deficient PMN lack any normal MPO peptides. No MPO-deficient subject studied had an immunologically cross-reacting variant of MPO. Since this deficiency is associated with the absence of more than one peptide, it is possible that the underlying genetic defect may involve: (a) failure to synthesize a single precursor peptide; (b) abnormal regulation of the synthesis of two separate peptides; or (c) an aberration in postsynthetic processing or packaging into azurophilic granules.

Adolescent↗
PubMed1983

Inhibition of zymosan activation of human neutrophil oxidative metabolism by a mouse monoclonal antibody.

We have studied a neutrophil-specific murine monoclonal antibody, PMN7C3 (IgG3), which specifically alters PMN oxidative metabolism stimulated by serum-opsonized zymosan (STZ) or Candida albicans (STC). Polymorphonuclear cells (PMNs) exposed to PMN7C3 show a significant depression in O2- release (52.8% +/- 2.5% of control), H2O2 release (44.4% +/- 6.0% of control), and O2 consumption (73.9% +/- 2.6% of control) in response to STZ. O2 release in response to phorbol myristate acetate (PMA) was modestly reduced (78.4% +/- 3.7%) by PMN7C3 treatment, but not to the extent seen with STZ or STC. PMN7C3 did not affect O2 release by PMNs stimulated by zymosan opsonized with IgG or by S. aureus, A 23187, or FMLP. PMN7C3 was not cytotoxic, did not trigger oxidative metabolism when used as a stimulus, did not alter STZ-induced degranulation, and did not interfere with binding or uptake of STZ by PMNs. Exposure of PMNs to PMN7C3 decreased PMN rosette formation with erythrocytes coated with C3b (54% of control) or C3bi (63% of control), but had no affect on rosette formation with IgG-coated erythrocytes. PMN7C3 does not bind to monocytes and had no affect on rosette formation by this cell type. Binding of antibody PMN7C3 to the neutrophil surface inhibits the oxidative response to opsonized STZ or STC, possibly in part by altering the function or expression of C3b and C3bi receptors. Monoclonal antibodies such as PMN7C3 provide highly specific probes that may be used to define the molecular features of the stimulus-coupled response of PMN activation.

Antibodies, Monoclonal↗
PubMed1981

Hollow cylinder protein in the cytoplasm of human erythrocytes.

A "Hollow Cylinder Protein' (HCP) similar to the protein originally isolated by Harris from human erythrocyte membranes (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534--537) is present in the cytosol of erythrocytes at a concentration of more than 15 micrograms/ml packed erythrocytes. When negatively stained and examined in the electron microscope, cytosol HCP is similar in morphology to the HCP associated with erythrocyte ghost membranes. Cytosol HCP can be purified by isoelectric precipitation at pH 5.2 followed by repeated sucrose gradient centrifugation at alkaline pH. Negatively stained purified cytosol HCP appears as a hollow cylinder with apparent dimensions of 18.0 nm in length by 11.8 nm in diameter and contains a hollow core. Purified cytosol HCP migrates as a single band by non-denaturing polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis shows that it is composed of five peptides having apparent molecular weights 21 500, 23 500, 26 000, 27 500 and 29 000. Chymotryptic peptide maps of each of these bands indicate that each is a unique polypeptide chain. These results indicate that erythrocyte cytosol HCP is a macromolecular complex composed of multiple copies of five non-identical subunits arranged as a hollow cylinder.

Blood Proteins↗