Affinity isolation and characterization of immunoglobulin G Fc fragment-binding glycoprotein from human blood platelets.

Immune complexes bind to several eukaryotic cell types including human blood platelets through the Fc fragment of immunoglobulin (Ig) G. Utilizing immobilized Fc fragment of IgG enabled us to isolate from human blood platelets a glycoprotein of an apparent Mr = 255,000 which, upon reduction, dissociated into sub-units of an apparent Mr = 50,000. This Fc fragment-binding glycoprotein has an isoelectric point between pH 6.3 and 6.9 and is composed of 34% hydrophobic, 25% acidic, and 14% basic amino acids. The Fc fragment-binding glycoprotein was also isolated from human platelet membrane preparations and was unaffected by prior treatment of platelets with thrombin. Isolated Fc fragment-binding glycoprotein formed an in vitro complex with aggregated immunoglobulin G. These results suggest that the isolated Fc fragment-binding component may prove useful in studies concerning the functional role of glycoproteins as cellular receptors for the Fc fragment of IgG.     

Structure and expression of Fc gamma receptors on mouse suppressor T cell hybridomas

Antigen-specific and idiotype-specific mouse suppressor T cell hybridomas were analyzed for the presence and specificity of Fc gamma receptors (Fc gamma R) by EA rosetting and by flow microfluorometry with the use of monoclonal antibodies. We found that four hybridomas expressed Fc gamma R specific for IgG1 and IgG2b, one of which became Fc gamma R- during prolonged culture. Four other hybridomas and the fusion parent, BW5147, consistently lacked Fc gamma R. The 125I-labeled Fc gamma R were isolated from surface radioiodinated hybridoma cells solubilized with 1% Nonidet P-40, were purified by using single or repetitive chromatography on mouse IgG-Sepharose columns, and were analyzed by SDS-PAGE. An 125I-labeled 56,000 to 61,000 Mr macromolecule was isolated from each of the Fc gamma R+ hybridomas, but from none of the Fc gamma R- hybridomas nor from BW5147 cells. This macromolecule rebound to insolubilized mouse IgG1, IgG2b, and human Fc fragments, but not to insolubilized mouse IgG2a, IgG3, or IgA or human F(ab')2 fragments, consistent with the specificity observed for Fc gamma R on intact hybridoma cells. The mouse suppressor T cell Fc gamma R differs in size and specificity from mouse B cell Fc gamma R. A 70,000 Mr protein expressed on all hybridomas and on BW5147 cells was radiolabeled and, despite preclearing with ovalbumin-Sepharose, bound to the mouse IgG-Sepharose columns, presumably due to mouse antibodies to gp-70. This macromolecule was completely and specifically removed by using goat antiserum to gp-70.     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Fractionation of polyclonal antibody by isoelectric focusing--differences in cross-reactivity and affinity of rabbit clonotype anti-human thyrotropin antibody

Immunoglobulin G (IgG) fractions prepared from three different batches of rabbit antihuman thyrotropin (hTSH) antisera were fractionated by agarose isoelectric focusing (IEF) in the pH ranges 3 to 10 and 5 to 8. Staining of protein in agarose gel after IEF showed that polyclonal IgG separated into more than 20 protein bands with isoelectric points (pIs) ranging from 6 to 9. The clonotype antibodies to hTSH were recovered from the fractions and subjected to radioimmunoassay for determination of the binding-affinity for hTSH and the cross-reactivity with human chorionic gonadotropin (hCG). The affinity constants of the antibodies recovered ranged from 6.4 X 10(9) M-1 to 3.1 X 10(10) M-1, and the cross-reactivities of the clonotype antibodies differed greatly. A good correlation was observed between the pIs of antibody molecules and their cross-reactivities: antibodies with higher pIs bound hCG more strongly than those with lower pIs. The correlation coefficients between the pIs and cross-reactivities were 0.83, 0.84, and 0.87 in three batches of antibody.     

Rheumatoid factors from patients with rheumatoid arthritis react with beta 2-microglobulin.

IgM rheumatoid factors (RF) from 18 sera of rheumatoid arthritis (RA) patients isolated from monomeric IgG affinity columns showed strongly positive ELISA reactions with human beta 2-microglobulin (beta 2m), as well as with recombinant beta 2m. When the same RA sera were adsorbed to beta 2m-Sepharose affinity columns, eluted material showed predominant IgM anti-Fc of IgG and anti-beta 2m reactivity. Inhibition reactions with "RF" obtained from IgG affinity columns showed slightly higher reactivity of RF for Fc over beta 2m; however, when RF from the same RA serum had been adsorbed to and eluted from beta 2m affinity columns, beta 2m showed greater inhibition than Fc for RF reacting with either beta 2m or Fc on ELISA plates. Thus two overlapping populations of RF were identified in RA sera showing reactivity with both beta 2m and Fc of IgG. When RF were isolated from IgG columns, affinity was slightly higher for Fc than beta 2m. Conversely, RF eluted from beta 2m Sepharose reacted slightly more with beta 2 m than Fc. Trypsin digests of a polyclonal RA IgM RF showed no beta 2m reactivity in Fc mu 5 fragments. Fab mu RF retained slight anti-Fc IgG but no residual anti-beta 2m activity. Monoclonal human IgM, IgG, or IgA RF either from mixed cryoglobulins or EBV-stimulated RA lymphoid cell lines showed negative or occasional weakly positive anti-beta 2m activity. Overlapping 7-mer peptide ELISA analysis of the entire 99-amino acid sequence of beta 2m showed a major RF-reactive linear hydrophilic sequence at positions 56-60 which included a 3-amino acid exact homology to positions 401, 403, and 404 of the C gamma 3 domain. A peptide encompassing this sequence produced 90% inhibition of RF binding to whole beta 2m. Substitution of neutral glycines for each amino acid throughout the reactive epitope at positions 56-66 indicated that lysine at position 58 aspartic acid at 59, and tryptophane at 60 represented major portions of the RF-reactive epitope. These findings indicate that human RF derived from patients with RA react with other epitopes besides those present on IgG Fc, including epitopes on human beta 2m. For many years serum RF3 found in patients with RA have been regarded as premier examples of autoantibodies to autologous IgG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies of the mechanism of the human platelet release reaction induced by immunologic stimuli. III. Relationship between the binding of soluble IgG aggregates to the Fc receptor and cell response in the presence and absence of plasma

Aggregated IgG coupled covalently with bis-diazobenzidine (BDB-IgG) and labeled with 3H-diazobenzene (3H-BDB-IgG) has been used to study the binding of soluble IgG aggregates to human platelets in relationship to the release of the contents of intracellular granules (e.g., serotonin). In washed cell suspensions a minimum of 0.14 to 0.2 mug 3H-BDB-IgG per 5 X 10(8) platelets (40 to 70 aggregates per cell) was required for the triggering of the release reaction and cell aggregation. Binding was independent of divalent cations. The Arrhenius plot gave a straight line between 0 to 37 degrees C and a Q10 of 1.6. Neither inhibitors of the release reaction nor energy metabolism, nor formaldehyde fixation of the platelets affected binding. Bound 3H-BDB-IgG was not significantly eluted by IgG, bovine albumin (BSA), buffer, or plasma. Binding to washed platelets was more strongly inhibited by human IgG than by F(ab')2, bovine IgG, human albumin (HSA), or BSA. Plasma was an even more effective inhibitor of both binding and release. Plasma deficient in IgG or depleted of complement retained its inhibitory capacity. In the presence of plasma, at physiologic ratios of plasma and platelets, no release of serotonin was observed. Binding, although inhibited in rate, nevertheless occurred. It was enhanced by divalent cation chelation and had a Q10 of 2.5. The release reaction of washed platelets to which 3H-BDB-IgG had been bound in the presence of HSA or BSA was also inhibited by the subsequent addition of plasma or plasma proteins (human IgG being more effective than bovine IgG, F(ab')2, HSA, or BSA). 3H-BDB-IgG bound in the presence of either plasma or human IgG did not induce release when the platelets were subsequently suspended in media lacking these proteins. Thus, it appears that the platelet Fc receptor binds 3H-BDB-IgG by a process which is effectively inhibited by plasma, or by free IgG with an intact Fc, and to some extent by high concentrations of other proteins. The effects of bound IgG aggregates are dependent on the other proteins present both during binding and subsequently added. The mechanism of such receptor modulation and its implications in vivo are discussed.     

Human neutrophils and eosinophils have structurally distinct Fc gamma receptors

Human Fc gamma receptors were isolated from surface radioiodinated granulocytes and eosinophils by using repetitive affinity chromatography on human IgG-Sepharose columns. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated that cell preparations containing eosinophils possessed a 43,000 Mr Fc gamma-binding macromolecule. Nylon wool-filtered cells from patients with eosinophilia and cell cultures derived from normal donors provided highly purified eosinophil preparations that expressed only the 43,000 Mr Fc gamma receptor. Granulocyte populations yielded the 52,000 to 68,000 Mr Fc gamma receptor characteristic of neutrophils as well as the Fc gamma-binding macromolecules apparently derived from eosinophils. The 43,000 Mr Fc gamma receptor of the eosinophil and the 31,000 and 34,000 Mr fragments that appear to be derived from it were able to rebind selectively to human IgG1-Sepharose, Fc gamma 1-Sepharose, IgG3-Sepharose, and Fc gamma 3-Sepharose. In contrast, the 52,000 to 68,000 Mr Fc gamma receptor from neutrophils could rebind only to IgG1-Sepharose and Fc gamma 1-Sepharose. The results demonstrate that the Fc gamma receptor of human eosinophils is distinct in structure from the neutrophil Fc gamma receptor and that these Fc gamma receptors, at least in their solubilized states, differ in specificity for human IgG3.     

Isolation of alpha 6 beta 1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment

Ligand affinity chromatography was used to identify receptors on platelets and two adherent cell lines, OV-CAR-4 and HBL-100, for the E8 fragment of murine laminin. A complex of two polypeptides (140 and 110 kDa nonreduced) was bound by the E8 affinity columns from all three cell types and was eluted with EDTA. This heterodimeric complex was identified as the alpha 6 beta 1 integrin by immunoprecipitation with specific antibodies against either the alpha 6 or the beta 1 subunit. The alpha 6 beta 1 integrin did not bind to an affinity column containing fragment P1 originating from a different part of murine laminin which, however, bound the alpha IIb beta 3 integrin from platelets. Furthermore, in immunofluorescence staining, the alpha 6 beta 1 integrin localizes in focal contacts of OVCAR-4 cells attached to laminin and E8 but not to fibronectin substrates. These results, combined with previous antibody inhibition studies, unequivocally identify the alpha 6 beta 1 integrin as a specific receptor for fragment E8. Affinity chromatography of OVCAR-4 and HBL-100 cells on a large pepsin fragment of laminin from human placenta yielded integrin alpha 3 beta 1. When alpha 3 beta 1 was removed from lysates of OVCAR-4 cells by preclearing with an alpha 3-specific monoclonal antibody, alpha 6 beta 1 was able to bind to human laminin as well. Integrin alpha 6 beta 1 on platelets which do not express alpha 3 beta 1 binds directly to human laminin. These results indicate that both alpha 3 beta 1 and alpha 6 beta 1 can act as receptors for human laminin and may interfere by steric hindrance. The alpha 6 beta 4 complex, which is strongly expressed on HBL-100 cells, did not bind to either mouse laminin fragment E8 or human laminin affinity columns.     

Characterization of the Fc receptors of the murine leukemia L1210

A glycoprotein extract prepared from the plasma membranes of L1210 cells was passed over columns of Sepharose 4B to which either heat-aggregated human IgG or F(ab′)₂ fragments had been coupled. The intact IgG column bound 35.7% of the applied counts, whereas the F(ab′)₂ columns bound 2.8%. The bound glycoproteins were eluted with citrate buffer (pH 3.2) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three peaks with apparent molecular weights of 65,000, 45,000, and 28,000 daltons were identified and purified by electroelution from polyacrylamide gels. The isolated proteins were able to bind to the same subclasses of mouse IgG myeloma proteins as the intact L1210 cells, indicating that these molecules are related to L1210 surface Fc receptors. Amino acid analyses of the 3 proteins were markedly similar suggesting that the observed molecular heterogeneity might be due to carbohydrate differences. Neuraminidase digestion of the isolated proteins resulted in mobility shifts on polyacrylamide gel electrophoresis which were consistent with the interpretation that either the isolated proteins have considerably different sialic acid contents, or that removal of the sialic acid results in disaggregation of an Fc receptor molecule.     

Purification and properties of a human plasma endogenous modulator for the platelet tricyclic binding/serotonin transport complex

An endogenous modulator for the site labeled by [3H]imipramine which is putatively coupled to the serotonin transporter in human platelets was isolated and purified from plasma. Procedures included sequential chromatography on Cibacron blue-Sepharose 4B, concanavalin A-Sepharose 4B, Mono Q HR 10/10 anion exchange, DuPont GF-250 gel permeation and Mono S HR 5/5 cation exchange columns. The purified modulator is a protein of Mr 45,000 with a very acidic pK (less than 3) and sensitive to various proteinases but heat- and acid-stable. This protein inhibited [3H]imipramine binding to platelet membranes competitively (IC50 approximately 6 microM) and enhanced serotonin uptake in fresh human platelets (EC50 approximately 7 microM). Various physicochemical properties, including chromatographic, electrophoretic and immunological as well as amino acid composition analysis revealed that the isolated protein is most probably the human alpha 1-acid glycoprotein.     

Selective separation of human peripheral platelets,granulocytes and lymphocytes by surface affinity chromatography

Selective separation of human peripheral platelets, granulocytes and lymphocytes was investigated by column liquid chromatography using methoxyethoxymethyl (MEM) bonded-phase columns (25 × 0.9 cm I.D.). Isotonic solutions containing mono- and disaccharides, methyl-α-d-pyranosides and a physiological saline at pH 7.4 were used as the mobile phase. Granulocytes and lymphocytes were separated on the MEM-Cellulofine GH-25 column by elution with 0.3 M d-mannose solution. The isolation of platelets and lymphocytes from human leukocyte-rich plasma was performed with a MEM-Sephadex G25 column and elution with 0.27 M sucrose solution. On the same column platelets could also be collected selectively by elution with 0.31 M methyl-α-d-mannoside at the high recovery of 100%. The isolated cells were viable for more than 90%.     

Activation of oxidative metabolism of granulocytes and monocytes by IGG-coated platelets from patients with thrombocytopenia

We have previously shown that monoclonal anti-T cell antibodies bound to their specific targets can trigger the activation of monocyte/macrophage oxidative metabolism through an Fc receptor-mediated interaction. The present study demonstrates that IgG coated platelets from patients with thrombocytopenia-associated diseases can induce a similar respiratory burst activation in polymorphonuclear and mononuclear phagocytes from normal individuals. The intensity of the oxidative reaction as measured by luminol-dependent chemiluminescence is in close correlation with the level of surface-bound IgG molecules as determined by a radioactive anti-immunoglobulin assay. This new methodology to evaluating IgG fixed on human platelets by their capacity to trigger the generation of highly reactive oxygen species by granulocytes and monocytes has also suggested a new mechanism in the genesis of thrombocytopenia associated with autoimmune diseases.     

Characterization and isolation of a C-reactive protein receptor from the human monocytic cell line U-937

Human C-reactive protein (CRP) is an acute phase reactant that is opsonic and an activator of macrophage tumoricidal function. CRP also activates the classical C cascade. These activities suggest that CRP might interact with monocytes/macrophages via specific receptors in a manner analogous to the interaction of IgG with FcR. With the use of radio-labeled human CRP, we have observed specific binding of CRP to human blood monocytes and the human monocytic cell line U-937. Binding was saturable at a pathophysiologic concentration of CRP, with an estimated KD of 9.5 x 10(-8) M and 3.6 x 10(5) binding sites/cell. Specific binding was inhibited by polyclonal human IgG as well as an IgG1 myeloma. In the converse experiment, CRP failed to inhibit specific [125I]IgG binding. The mAb IV.3, which inhibits binding of IgG immune complexes to FcRII, did not inhibit CRP binding. A 100-fold excess of phosphorylcholine or the phosphorylcholine binding peptide of CRP (residues 47-63) failed to inhibit binding. Although human rIFN-gamma and PMA increased FcRI expression, these reagents had no affect on CRP receptor expression. A single membrane protein of 38 to 41 kDa from U-937 cells was chemically cross-linked to [125I]CRP; the cross-linking was inhibited by human IgG1 but not the IV.3 mAb. Furthermore, two membrane proteins with a Mr of 38 to 40 kDa and 58 to 60 kDa were isolated by CRP ligand-affinity chromatography. These proteins were of a distinct size from those isolated for FcRI from an IgG ligand matrix. These studies demonstrate specific binding of human CRP to a human monocytic cell line via receptors that are distinct from the IgG FcR and implicate CRP in nonspecific, preimmune host defense reaction mediated by cells of the monocytic lineage.     

Structural polymorphism of the human platelet Fc gamma receptor

A variable T lymphocyte proliferative response to murine IgG1 anti-T3 monoclonal antibodies, in which most North American Caucasians respond whereas a minority do not, is well established. This is most likely the result of a genetic polymorphism manifested by 1) the inability of the monocyte 40-kDa IgG FcR of some individuals to bind murine IgG1, and 2) a distinctive trimorphic pattern on IEF of the monocyte 40-kDa FcR, one form being seen in all individuals who do not respond and another form (or a combination of both forms) being seen in those who do respond. We have evaluated the IEF patterns of the platelet 40-kDa FcR and find that in every individual tested the pattern for platelet FcR correlates with that seen for the monocyte 40-kDa FcR pattern. Furthermore, the platelets of those individuals whose "nonresponder" monocyte 40-kDa FcR did not mediate a murine IgG1 anti-T3 response did not respond with an aggregation reaction to murine IgG1 immune complexes (opsonized E). In contrast, platelets from donors possessing "responder" monocytes displayed positive "aggregation" responses to E coated with murine IgG1 antibody. However, the platelet FcR structural polymorphism described earlier did not correlate with the donor-specific variability in capacity of platelets to respond functionally to aggregated human IgG described in an earlier paper. Rather, the variation in capacity of platelets from individual donors to respond functionally to aggregated human IgG was related to the quantitative expression of platelet FcR. These data indicate that the molecular mechanisms responsible for the platelet 40-kDa FcR structural polymorphism are quite different from the mechanisms governing the variation in quantitative expression of the receptor.     

抗人IgG Fc片段及Fab段单抗的鉴定及应用

本实验采用木瓜酶水解,SPA柱亲合层析等手段得到人IgGFc段及Fab段,以Sigma抗人IgGfFc段和抗人IgG Fab段单抗为标准品,鉴定了细胞库中抗人IgG系列的部分细胞株,得到特异性分泌抗人IgG Fc段和抗人IgG Fab段单抗的细胞各一株。 在上述实验基础上,用抗人IgG Fc及抗人IgG Fab单抗分别制备了Sepharose4B亲合层析柱,提纯了酶解人IgG Fc、Fab片段,经ELISA法鉴定,相互之间无交叉反应。同时用此方法制备了人抗HBe Fab片段,并将该片段进行了过氧化物酶标记,用来配制HBe ELISA诊断盒,证明其生物活性未受影响,而且消除了类风湿因子引起的HBe Ag假阳性现象。因抗HBe单抗来源困难,如采用HBe多抗制备ELISA试剂,本法将是提高质量的一个好方法。     

Purification of antibody and antibody-fragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent

To obtain a more efficient purification process for antibody fragments from an Escherichia coli homogenate, the precipitant, Ethodin (6,9-diamino-2-ethoxyacridine lactate) was introduced to the homogenate. By adding the precipitant a drastic reduction of host cell protein was obtained. The majority of the proteins were recovered in a precipitate with the cell debris, while the antibody or antibody-fragment was recovered in the clarified supernatant. In addition, DNA was also efficiently precipitated when using Ethodin as a precipitation agent. The improved purity of the clarified extract obtained by using the precipitant allows for the use of smaller chromatography columns and may reduce the number of chromatographic steps required in the recovery process. The effect of Ethodin concentration, pH, temperature, and conductivity were investigated. The investigation was performed on two different antibody-fragments, e.g., F(ab')(2) molecules and a full-length antibody produced in E. coli. The two F(ab')(2) proteins were F(ab')(2)A and F(ab')(2)B, which have a similar molecular mass (100 kDa) but different isoelectric points (pIs), i.e., 8.9 and 7.5, respectively. The full-length antibody, Ab (the full IgG form of F(ab')(2)B) has a pI of 7.8 and molecular mass of 150 kDa. The investigation showed that the highest purification factors were obtained at neutral pH, low conductivity, and Ethodin concentrations of 0.6%.     

Sequencing of full-length cDNA encoding the alpha and beta subunits of human casein kinase II from human platelets and megakaryocytic cells. Expression of the casein kinase IIalpha intronless gene in a megakaryocytic cell line

Casein kinase II (CKII) is a ubiquitous protein kinase composed of two subunits, alpha and beta, that can use both ATP and GTP as phosphoryl donors. Two genes located on two separate chromosomes were identified for CKIIalpha: one on chromosome 20 band 13 with an approximate size of 20 kb and a second on chromosome 11 band 15.5-p15.4 that is the same size as the cDNA of locus 20 kb (1.2 kb) and does not contain any introns. The two genes differ in four amino acids. Recently, it has been demonstrated that a membrane-associated platelet-derived CKII phosphorylates coagulation factor Va. The mRNA encoding the platelet CKII was isolated from fresh human platelets, and the corresponding cDNAs encoding the alpha and beta subunits of human platelet CKII were produced and sequenced. The cDNA for platelet CKIIalpha was found to be 99.7% homologous to the CKIIalpha intronless gene, having the same characteristic amino acid residues at positions 128, 256, 287, and 351. However, the cDNA of platelet CKIIalpha has a different amino acid at position 236 (Arg --> His), which is not found in the intronless gene. The cDNA of the CKIIbeta subunit was completely identical with the sequence of the CKIIbeta subunit isolated from other tissues. Since platelets arise from megakaryocytes, mRNA was isolated from the megakaryocytic cell line MEG-01 and the cDNA for CKIIalpha was cloned and sequenced. The cDNA was found to be identical to the intronless gene found in platelets. We have also investigated the expression of the intronless gene in several other cell lines. Expression of the intronless gene was only found in cell line MEG-01. Our data demonstrate expression of the CKIIalpha intronless gene in megakaryocytes and platelets.     

Comparative studies of canine colipase and lipases from bovine, porcine, canine, human and rat pancreases.

1. Colipase was purified from canine pancreatic juice and found to have certain specificity in its reaction with various pancreatic lipases. 2. This colipase will stimulate the lipolytic activities of lipases isolated from canine, bovine and porcine pancreas but not lipases from a fungus, or from human and rat pancreases. 3. Characterization of these lipases showed (a) the molecular dimension of rat lipase is very different from the other lipases; (b) the pIs of canine, porcine and bovine lipases are almost identical but different from the pIs of rat, human and Candida (a fungus) lipases; and (c) the antiserum prepared against canine lipase will also react with lipases from human, hog and cow pancreases but not with rat and Candida lipases. 4. These physical differences can explain partly the difference in reaction between the various lipases and the canine colipase.     

A comparative study of the effect of modification of the surface of human platelets on the receptors for aggregated immunoglobulins and for ristocetin-von Willebrand factor

The receptors for aggregated immunoglobulin G (IgG) (an Fc receptor) and for ristocetin-von Willebrand factor on human platelets were studied by means of various modifications of the platelet surface. The expression of these receptors was measured by the agglutination of platelets to ristocetin in the presence of von Willebrand factor, which is part of the factor VIII complex, and by the binding of aggregated IgG coupled to ³H-labelled diazobenzen. Treatment of platelets with chymotrypsin, trypsin, papain and pronase which removed protein and glycoprotein from the platelet under conditions where the release reaction was inhibited caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Induction of membrane changes with ADP and of the release reaction with the ionophore A23187 abolished agglutination to ristocetin-von Willebrand factor but did not alter the receptor for aggregated IgG. Possible contributions of unspecific membrane changes, produced by protease treatment of platelets, to the modification of receptor expression were eliminated by the use of formaldehyde-treated platelets. Trypsin, papain and pronase destroyed the ability of these platelets to agglutinate to ristocetin-von Willebrand factor but produced no change in the binding of aggregated IgG. Therefore, the receptor for ristocetin-von Willebrand factor is truly sensitive to proteolysis while the Fc receptor is not, but is partially masked by protease-sensitive material.     

Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells

We have studied the distribution of myosin molecules in human cells using myosin-specific antibody coupled with fluorescent dyes. Rabbits were immunized with platelet myosin or myosin rod. They produced antisera which precipitated only myosin among all the components in crude platelet extracts. From these antisera we isolated immunoglobulin-G (IgG) and conjugated it with tetramethylrhodamine or fluorescein. We separated IgG with 2-5 fluorochromes per molecule from both under- and over-conjugated IgG by ion exchange chromatography and used it to stain acetone-treated cells. The following controls established the specificity of the staining patterns: (a) staining with labeled preimmune IgG; (b) staining with labeled immune IgG adsorbed with purified myosin; (c) staining with labeled immune IgG mixed with either unlabeled preimmune or immune serum; and (d) staining with labeled antibody purified by affinity chromatography. In blood smears, only the cytoplasm of platelets and leukocytes stained. In spread Enson and HeLa cells, stress fibers stained strongly in closely spaced 0.5 mum spots. The cytoplasm stained uniformly in those cells presumed to be motile before acetone treatment. In dividing HeLa cells there was a high concentration of myosin-specific staining in the vicinity of the contractole ring and in the mitotic spindle, especially the region between the chromosomes and the poles. We detected no staining of erythrocytes, or nuclei of leukocytes and cultured cells, or the surface of platelets and cultured cells.