Degradation of the human erythrocyte membrane band 3 studied with monoclonal antibody directed against an epitope on the cytoplasmic fragment of band 3

The mouse hybridoma monoclonal antibody BIII.136 of the IgG2a class is specific for human erythrocyte band-3 protein. It was shown by means of immunoblotting and immunoprecipitation assays that the antibody recognized an epitope located in the cytoplasmic pole of the band-3 molecule within approximately 20 kDa from the N-terminal end. The N-terminal fragments of band-3 protein, migrating in SDS/polyacrylamide gel electrophoresis in the 60-kDa, 40-kDa and 20-kDa regions, were detected with the antibody in untreated red-cell membranes as products of autolysis of band-3 protein. A correlation was found between the amount of these fragments and erythrocyte age, which suggests that partial degradation of band 3 proceeds in vivo during senescence of erythrocytes. The further degradation of band-3 protein in vitro was not observed in intact erythrocytes stored at 4 degrees C, but progressed distinctly after hemolysis of red cells, during washing and storing the membranes.     

Preparation and Characterization of Monoclonal Antibodies to Serotonin Binding Protein

Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 X 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44-10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44-10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44-10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with MAB 44-10 were located immunocytochemically in sections of rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of antisperm antibodies on early cleavage of fertilized ova.

Early cleavage of zygotes was abnormal after oocytes were fertilized with sperm cells from one of 64 infertile couples studied in our human in vitro fertilization and embryo development procedure. The abnormal cleavage, which did not affect fertilization, was not due to polyspermy or parthenogenetic activation of oocytes, but to antisperm antibodies present on the sperm cells. These antibodies did not react with zona-intact or zona-free human oocytes, although they reacted with the acrosomal (strongly) and tail (weakly) regions of human sperm. The antibodies recognized a double band comprising two glycoproteins of 14 +/- 3 and 18 +/- 3 kDa and a protein band of 22 +/- 3 kDa on the Western blot of lithium diiodosalicylate-solubilized human sperm extract. Antisera were raised in rabbits against the double band (14 +/- 3- and 18 +/- 3-kDa antigens) and the 22 +/- 3-kDa protein. Passive transfer of affinity-purified human or rabbit immunoglobulins directed against the double band (14 +/- 3- and 18 +/- 3-kDa antigens), but not those directed against the 22 +/- 3-kDa protein, caused a significant inhibition of early cleavage of oocytes without affecting pronuclear formation in mice. These results suggest that the sperm surface antigens of 14 +/- 3 and 18 +/- 3 kDa may provide an extranuclear signal to oocytes to divide in mice and humans. These antigens may also find clinical applications in the management of immunoinfertility and in the development of an antisperm contraceptive vaccine for humans.     

The 165-kDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel

The dihydropyridine receptor purified from rabbit skeletal muscle yields in the presence of dithiothreitol and sodium dodecyl sulfate on polyacrylamide gels bands of apparent molecular mass 165 +/- 5, 130 +/- 5, 55 +/- 3, 32 +/- 2 and 28 +/- 1 kDa (chi +/- SEM, n = 12). Under nonreducing conditions, the 130 kDa and 28-kDa peptides migrate as a single peptide of 165 kDa. These peptides were separated on a HPLC size-exclusion column. The specific absorption coefficients of the isolated peptides were determined. From these a stoichiometry of 1:1.7 +/- 0.2:1.4 +/- 0.3 (chi +/- SEM of 12 experiments with three different preparations) was calculated for the 165-kDa, 55-kDa and 32-kDa peptides. The relative amount of the 130/28-kDa peptide varied with different preparations. Tryptic, chymotryptic and V-8 protease peptides of the isolated proteins suggested that the 130/28-kDa peptide was not related to the 165-kDa peptide. The dihydropyridine photoaffinity analog (+/-)-azidopine was specifically incorporated only into the 165-kDa peptide with an efficiency of about 2.4%. The azido analog of desmethoxyverapamil, LU 49888, was specifically incorporated into the same peptide with an efficiency of 1.5%. These results suggest that only the 165-kDa peptide contains the regulatory sites detected so far in the voltage-operated L-type calcium channel. They suggest further that the 130/28-kDa peptide, which migrates as a 165-kDa peptide under nonreducing conditions, does not contain high-affinity binding sites for the calcium channel blockers.     

A new trophoblast-derived growth factor from human placenta: purification and receptor identification

This paper describes the identification and characterization of a new peptide growth factor. The peptide was isolated from trophoblastic brush border membranes of human placenta. The purified preparation was homogeneous and consisted of a single polypeptide of Mr 34 000 with a pI of about 6.0. This peptide stimulated DNA replication in cultured fibroblasts. The following association was seen between activity and protein: During DEAE-cellulose chromatography, both the 34-kilodalton (kDa) protein and the mitogenic activity displayed identical binding and salt dependence of elution. Nondenaturing electrophoresis at pH 8.3 revealed a comigration of the 34-kDa protein and the DNA replication stimulatory activity. Identical electrophoretic mobilities were displayed for both activity and protein at pH 7.0. These results demonstrate that the preparation is homogeneous and show that growth factor activity is intrinsic to the 34-kDa polypeptide. Binding of the 125I-labeled 34-kDa mitogen to target fibroblastic cells was specific; i.e., nanomolar concentrations of the unlabeled 34-kDa protein competed effectively with the labeled protein, whereas a variety of well-characterized growth factors and hormones were unable to compete even at micromolar levels. Thus the 34-kDa protein interacts with target cells through highly specific surface receptors. Chemical cross-linking techniques were used to investigate the identity of the receptor for the 34-kDa mitogen. Cross-linking of fibroblastic cells containing bound 125I-labeled 34-kDa protein generated a radiolabeled complex of 86 kDa in all four cell types examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cellular proteins which can associate specifically with polyomavirus middle T antigen in human 293 cells include the major human 70-kilodalton heat shock proteins.

We compared the proteins which associate with middle T antigen (MT) of polyomavirus in human cells infected with Ad5(pymT), a recombinant adenovirus which directs the overexpression of MT, with the MT-associated proteins (MTAPs) previously identified in murine fibroblasts expressing MT. MTAPs of 27, 29, 36, and 63 kilodaltons (kDa) appeared to be fairly well conserved between the two species, as judged by comigration on two-dimensional gels. Several 61-kDa MTAP species detected in MT immunoprecipitates from both cell sources also comigrated on these gels. However, no protein comigrating precisely with the murine 85-kDa MTAP could be detected in the human cells. Furthermore, two proteins of 72 and 74 kDa associated with wild-type MT in the infected human cells but not in murine fibroblasts expressing MT. It had been previously reported for murine cells that the 70-kDa heat shock protein associates with a particular mutant MT but not with wild-type MT (G. Walter, A. Carbone, and W.J. Welch, J. Virol. 61:405-410, 1987). By the criteria of comigration on two-dimensional gels, tryptic peptide mapping, and immunoblotting, we showed that the 72- and 74-kDa proteins that associate with wild-type MT in human cells are the major human 70-kDa heat shock proteins.     

Kinetic independence between red cell anion exchange and urea transport

Urea equilibrium exchange fluxes were measured in human red cells under conditions which recruit the anion transporter into an outward-facing or an inward-facing state (with respect to the anion transport site). Regardless of these conditions, urea transport always occurred at the same rate: 41 +/- 2 mol.(kg cell solids.min)-1 with 1.5 M urea at 0 degrees C. These data suggest that the pathway on the band-3 protein which mediates anion transport is kinetically uncoupled from urea transport and is probably not involved in the transport of urea across the red cell membrane.     

Processing and localization of the african swine fever virus CD2v transmembrane protein

The African swine fever virus (ASFV)-encoded CD2v transmembrane protein is required for the hemadsorption of red blood cells around infected cells and is also required for the inhibition of bystander lymphocyte proliferation in response to mitogens. We studied the expression of CD2v by expressing the gene with a V5 tag downstream from the signal peptide near the N terminus and a hemagglutinin (HA) tag at the C terminus. In ASFV-infected cells, a full-length glycosylated form of the CD2v protein, which migrated mainly as a 89-kDa product, was detected, as well as an N-terminal glycosylated fragment of 63 kDa and a C-terminal nonglycosylated fragment of 26 kDa. All of these forms of the protein were localized in the membrane fraction of cells. The 26-kDa C-terminal fragment was also produced in infected cells treated with brefeldin A. These data indicate that the CD2v protein is cleaved within the luminal domain and that this occurs in the endoplasmic reticulum or Golgi compartments. Confocal microscopy showed that most of the expressed CD2v protein was localized within cells rather than at the cell surface. Comparison of the localization of full-length CD2v with that of a deletion mutant lacking all of the cytoplasmic tail apart from the 12 membrane-proximal amino acids indicated that signals within the cytoplasmic tail are responsible for the predominant localization of the full-length and C-terminal 26-kDa fragment within membranes around the virus factories, which contain markers for the Golgi compartment. Processing of the CD2v protein was not observed in uninfected cells, indicating that it is induced by ASFV infection.     

Involvement of protein serine and threonine phosphorylation in human sperm capacitation.

The involvement of serine and threonine phosphorylation in human sperm capacitation was investigated. Anti-phosphoserine monoclonal antibody (mAb) recognized six protein bands in the 43-55-kDa, 94 +/- 2-kDa, 110-kDa, and 190-kDa molecular regions, in addition to a faint band each in the 18-kDa and 35-kDa regions. Anti-phosphothreonine mAb recognized protein bands in six similar regions, except that the 18-kDa, 35-kDa, and 94 +/- 2-kDa protein bands were sharper and thicker, and an additional band was observed in the 110-kDa molecular region. In the 43-55-kDa molecular region, there was a well-characterized glycoprotein, designated fertilization antigen, that showed a further increase in serine/threonine phosphorylation after exposure to solubilized human zona pellucida. In a cell-free in vitro kinase assay carried out on beads or in solution, four to eight proteins belonging to similar molecular regions, namely 20 +/- 2 kDa, 43-55 kDa, 94 +/- 2 kDa, and 110 +/- 10 kDa, as well as in 80 +/- 4 and 210 +/- 10 kDa regions, were phosphorylated at dual residues (serine/tyrosine and threonine/tyrosine). Capacitation increased the intensity of serine/threonine phosphorylation per sperm cell, increased the number of sperm cells that were phosphorylated, and induced a subcellular shift in the serine/threonine-specific fluorescence. These findings indicate that protein serine/threonine phosphorylation is involved and may have a physiological role in sperm capacitation.     

Fibronectin receptors of mononuclear phagocytes: Binding characteristics and biochemical isolation

Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, we have investigated the hypothesis that proteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. We report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. The Ki for inhibition of attachment (0.5 microM) was consistent with the Kd for binding of the 3H-labeled 80-kDa fragment (0.34 microM) to U937 cells in suspension. There were 4-5 x 10(5) 80-kDa binding sites per cell. The relatively high affinity of the 80-kDa fragment for the monocyte surface permitted the isolation and characterization of fibronectin-binding proteins from U937 cells and peripheral blood monocytes by affinity chromatography. When octylglucoside lysates of lactoperoxidase iodinated cells were applied to 80-kDa-Sepharose columns, a polypeptide complex of 152/125 kDa was eluted with the synthetic peptide GRGDSPC, but not with GRGESP. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.     

Purification and properties of a novel broad substrate specific alcohol oxidase from Aspergillus terreus MTCC 6324

An alcohol oxidase was isolated from the microsome of n-hexadecane grown Aspergillus terreus and purified by ion exchange chromatography. The oxidase was found to act on short chain-, long chain-, secondary-, and aromatic-alcohol substrates with highest affinity for n-heptanol (K(M)=0.498 mM, K(cat)=2.7x10(2) s(-1)). The native protein molecular mass was 269+/-5 kDa and the subunit molecular masses were 85-, 63-, 43-, 27-, and 13-kDa. The isoelectric point of the proteins was within 8.3-8.5. High aggregating property of the protein was demonstrated by AFM, DLS and TEM analyses. Chemical analysis showed the presence of oleic acid and palmitic acid at a ratio of 2:1 in the purified protein. This lipoidic nature of the protein particles was correlated to the high aggregating property. In this flavoenzyme, flavin was non-covalently but avidly associated. Peptide mass fingerprinting studies showed the presence of two FAD binding domains in 63 kDa protein. Among these two FAD binding domain sequences only the YPVIDHEYDAVVVGAGGAGLR peptide shows 45-50% sequence homology with the reported N-terminal sequences of other known alcohol oxidases. Non-redundant database search of 63- and 43-kDa subunits peptide sequences showed no sequence similarity with the other alcohol oxidase protein reported till now.     

Muscarinic Acetylcholine Receptors from Avian Retina and Heart Undergo Different Patterns of Molecular Maturation

Muscarinic acetylcholine receptors (mAChRs) from the avian CNS exist in two molecular weight forms whose concentrations change during development. Here, we have compared the development of mAChRs from embryonic hearts with those of the CNS. Analysis of [3H]-propylbenzilylcholine mustard (PrBCM)-labeled retina and heart mAChRs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two atropine-sensitive peaks for each tissue. Apparent molecular masses of retina mAChRs, 86 +/- 0.7 kilodaltons (kDa) and 72 +/- 0.7 kDa, were different from those of heart mAChRs, 77 +/- 1.0 kDa and 52 +/- 0.9 kDa. During retina development, the major receptor type changed from 86 kDa to 72 kDa. No such change occurred during heart development. Furthermore, the 52-kDa species appeared to be generated by endogenous proteolysis, as prolonged incubation of heart membranes at 37 degrees C increased the amount of 52-kDa peptide with a decrease of 77-kDa peptide. Protease inhibitors blocked this conversion. Incubation of retina membranes at 37 degrees C did not result in a conversion of the 86-kDa peptide into the 72-kDa peptide, but it did cause the appearance of a minor amount of 52-kDa peptide. The proteolysis of retina mAChRs was not enhanced by cohomogenizing them with heart tissue, arguing against the presence of releasable proteases in heart. Membrane-bound retina and heart mAChRs displayed similar sensitivity to exogenous (Staphylococcus aureus V8) protease, indicating that heart receptors were not unusually susceptible to proteolytic attack; analysis of the labeled polypeptides with the V8 protease showed different patterns of digestion for the retina and heart receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation by protein kinase C inactivates an inositol 1,4,5-trisphosphate 3-kinase purified from human platelets

An inositol 1,4,5-trisphosphate 3-kinase purified from human platelets contains two major components, 53 and 36 kDa polypeptides. Each polypeptide expresses Ca2+/calmodulin-dependent enzymatic activity and is phosphorylated by an unidentified protein kinase in the enzyme preparation. The 36-kDa polypeptide may be further phosphorylated on serine residues by protein kinase C to a stoichiometry of 0.8 mole phosphate per mole of protein. Phosphorylation of the 36-kDa component is correlated with inhibition of the kinase activity; the inhibitory effect is dependent upon Ca2+ and phosphatidylserine/diolein and may be blocked by a selective peptide inhibitor of protein kinase C. Phosphorylation by protein kinase C decreases the Vmax of the enzyme from 160 to 28 nmol/mg/min; the Km (0.76 microM) is not altered. These data suggest that protein kinase C may negatively regulate inositol 1,4,5-trisphosphate 3-kinase activity in the human platelet.     

Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis.

To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T3 action we examined the effects of T3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T3 alone and T3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.     

LRIG1 protein in human cells and tissues

We have recently cloned the human LRIG1 gene (formerly LIG1). LRIG1 is a predicted integral cell-surface protein showing similarities to Kekkon-1, the Drosophila melanogaster epidermal growth-factor-receptor antagonist. A specific peptide antibody, LRIG1-151, was raised in rabbits and used to study the LRIG1 protein. LRIG1 migrated in denaturing polyacrylamide gel electrophoresis under reducing conditions as two species with apparent molecular weights of 143 kDa and 134 kDa, and as two fragments corresponding to an N-terminal 111-kDa species and a C-terminal 32-kDa species. Under non-reducing conditions, both apparent monomers and apparent higher molecular weight complexes were evident. Immunoblotting analysis of cell-surface-biotinylated lysates and confocal microscopy revealed that LRIG1 was localized to the cell surface in ZR-75 cells expressing endogenous LRIG1 and in COS-7 cells expressing a synthetic LRIG1-GFP fusion protein. Immunohistochemical analysis of normal human tissues showed staining for LRIG1 in epithelia in various organs, scattered neurons, and muscles. Immunoblotting demonstrated LRIG1 protein in tissue lysates from normal human prostate, mammary epithelial cells, ileum, stomach, lung, and cerebral cortex. These results demonstrate that LRIG1 is an integral cell-surface membrane protein that is expressed by specific cells in various human tissues and that its 143-kDa form might be cleaved into 111-kDa and 32-kDa fragments.     

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.     

Endoplasmic reticulum of rat liver contains two proteins closely related to skeletal sarcoplasmic reticulum Ca-ATPase and calsequestrin

Rat liver endoplasmic reticulum (ER) membranes were investigated for the presence of proteins having structural relationships with sarcoplasmic reticulum (SR) proteins. Western immunoblots of ER proteins probed with polyclonal antibodies raised against the 100-kDa SR Ca-ATPase of rabbit skeletal muscle identified a single reactive protein of 100 kDa. Also, the antibody inhibited up to 50% the Ca-ATPase activity of isolated ER membranes. Antisera raised against the major intraluminal calcium binding protein of rabbit skeletal muscle SR, calsequestrin (CS), cross-reacted with an ER peptide of about 63 kDa, by the blotting technique. Stains-All treatment of slab gels showed that the cross-reactive peptide stained metachromatically blue, similarly to SR CS. Two-dimensional electrophoresis (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326) of ER proteins showed that the CS-like component of liver ER, similarly to skeletal CS, fell off the diagonal line, as expected from the characteristic pH dependence of the rate of mobility of mammalian CS. In addition, the CS-like component of liver ER was released from the vesicles by alkaline treatment and was found to be able to bind calcium, by a 45Ca overlay technique. From these findings, we conclude that a 100-kDa membrane protein of liver ER is the Ca-ATPase, and that the peripheral protein in the 63-kDa range is closely structurally and functionally related to skeletal CS.     

Insulin-like growth factor-binding protein from human plasma. Purification and characterization

Insulin-like growth factor (IGF)-binding protein (BP) has been purified from Cohn fraction IV of human plasma by acidification, ion exchange to remove endogenous ligands, and affinity chromatography on agarose-IGF-II. The pure protein appeared as a single peak by high performance reverse-phase and gel permeation chromatography (molecular mass, 45-50 kDa), but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band at 53 kDa and a minor band at 47 kDa, unreduced, or 43 and 40 kDa, respectively, reduced. The two bands stained for both protein and carbohydrate. After storage at 2 degrees C for 5 months at pH 3, two additional bands, at 26 and 22 kDa on unreduced gels, were also present. Autoradiography after affinity labeling with IGF-I or IGF-II tracer revealed a single labeled band of 61 kDa. BP, quantitated using a specific radioimmunoassay, was retained by agarose-immobilized IGF-I, IGF-II, concanavalin A, and wheat germ lectin, but not Helix pomatia lectin. Competitive binding curves using pure BP and human IGF-I and IGF-II as both labeled and unlabeled ligands indicated association constants of 2-3 X 10(10) liters/mol for both peptides, with a slightly higher affinity for IGF-II than IGF-I, and 0.9 binding sites for either peptide per 53-kDa protein. The exact relationship of this acid-stable IGF BP to the 150-kDa complex from which it is derived remains to be determined.     

Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells

Calcium-dependent phospholipid binding and phospholipase A2 inhibitory proteins were isolated from human mononuclear cells. Lipocortins I and II were present whereas lipocortin IV (endonexin I) was not. The other proteins were purified to homogeneity and shown to have molecular masses of 35, 36, 32 and 73 kDa. The 36-kDa and 73-kDa proteins are related, the smaller appears to be part of the larger. The 73-kDa protein is related to the 67-kDa calelectrin and to lipocortin VI; the 32-kDa protein is different from endonexin I but related to chromobindin 7 and to lipocortin V. The 35-kDa protein has been identified by tryptic peptide sequencing as lipocortin III. All these proteins inhibit phospholipase A2 activity in vitro and the three smaller ones inhibit the [3H]arachidonic acid release from prelabelled monocytes induced by the calcium ionophore A23187 in a dose-dependent manner.     

Precursor-product relationship between the 26-kDa and 18-kDa fragments formed by iodination of human thyroglobulin

At moderate iodination levels (20 iodine at atoms/molecule), human thyroglobulin (hTgb) produces after reduction a thyroxinyl-peptide of 26 kDa which represents the N-terminal part of the protein. At higher iodination levels, the 26-kDa peptide is accompanied by another T4-containing peptide of 18 kDa. A precursor-product relationship between the 26- and 18-kDa fragments was demonstrated by the study of the tryptic fragments of both hormonopeptides. In addition, comparison with the protein sequence deduced from the nucleotide sequence of the 5'-end of hTgb mRNA demonstrated that the N-terminal region of Htgb from which are issued the 26-kDa peptide and its 18-kDa derivative is especially sensitive to proteolysis. This character is possibly related with a facilitated release of thyroid hormones in vivo.