Characterization of radiolabeled components of human sperm surface and seminal plasma

Externally oriented components on the human sperm cell surface and components in human seminal plasma were labeled by enzymatic iodination with lactoperoxidase and [¹²⁵I] NaI. SDS-7.5% PAGE of labeled sperm surface resolved one minor and four major components with approximate molecular weights of 92, 72, 46, 30, and 20K daltons, respectively. SDS-7.5% PAGE of labeled seminal plasma resolved five components with approximate molecular weights of 74, 51, 43, 28, and 20K daltons. Three of the five moieties seen on the sperm surface and in seminal plasma were similar in molecular weight. This suggested that these surface components were adsorbed from seminal secretions. Because the iodination procedure used labels both proteins and lipids, labeled sperm surface and labeled seminal plasma were subjected to isopycnic density gradient centrifugation to identify the chemical composition of the radioiodinated components. With human sperm surface, two areas of radioactivity were resolved in CsCl gradients, one corresponding to protein and the other to lipid. With human seminal plasma, only one area of radioactivity, corresponding to protein, was identified. Electrophoretic analysis of each peak of radioactivity obtained from the gradients demonstrated that all of the sperm surface and four of five seminal plasma components were in the protein fractions. All three of the seminal plasma components which correspond to sperm surface components were recovered in the protein fraction. This observation supports our hypothesis that some of the proteins labeled on the human sperm cell surface are adsorbed from seminal secretions.     

Molecular nature of human epidermal growth factor (hEGF)-like immunoreactivity in human plasma

To investigate the molecular features of hEGF in the circulatory system, we analyzed hEGF-like immunoreactivity (hEGF-LI) in human serum and plasma by our two-site enzyme immunoassay (EIA), using an aliquot of each fraction obtained by gel filtration of human serum or plasma on Sephadex G-100. The results demonstrated that the majority of the hEGF-LI in the plasma (HMW X hEGF-LI) emerged in the void volume, while a small amount of hEGF-LI (LMW X hEGF-LI) eluted at a position similar with that of standard hEGF. After reduction of HMW X hEGF-LI with 2-mercaptoethanol, hEGF-LI emerged at the same elution position as that of standard hEGF, suggesting that the predominant form of hEGF may circulate as a complex with some macromolecule(s) in human blood. The biochemical properties of the plasma component(s) with high molecular weight which form the complex with hEGF and the physiological significance of this circulating complex still remain to be clarified.     

The isolation and characterisation of a platelet-specific beta-globulin (beta-thromboglobulin) and the detection of antiurokinase and antiplasmin released from thrombin-aggregated washed human platelets.

A protein fraction was isolated from the supernatant of thrombin-aggregated washed human platelets and was shown, by immunodiffusion techniques, to contain a platelet-specific beta-globulin (beta-thromboglobulin) as the major component. A molecular weight of 35 800 was determined for beta-thromboglobin from the measured sedimentation coefficient of3.0 S and Stokes radius of 2.85 nm. Beta-Thromboglobin was detected in the serum from whole blood and the supernatant of 48-h-old platelet-rich plasma and 28-day-old citrated whole blood, but not in platelet-poor plasma. The fraction containing beta-thromboglobulin was shown to possess an antiurokinase activity but was devoid of antiplasmin activity. A further fraction of approximate molecular weight 70 000 was also isolated which contained an antiplasmin but was devoid of antiurokinase activity.     

Drop weight variation in an automated collection procedure and its relationship to apparent surface tension.

A drop counter-regulated fraction collector yields samples containing equal numbers of drops. Such fractions vary slightly in weight depending on experimental conditions such as surface tension. Provided that variables such as flow rate and eluate density remain constant, apparent surface tension may be estimated directly from the weights of eluate fractions obtained from gel filtration experiments. The detergents sodium cholate and sodium lauryl sulphate significantly decreased drop weights in this system. Following gel filtration on Sepharose 4B, sodium cholate eluted in the fractions containing low molecular weight material. It eluted in the same position when pre-mixed with human plasma. Normal plasma was found to contain two surface tension-reducing components with apparent molecular weights of 3-10(6) and 1-10(5). The apparent surface tension of whole human plasma was found to be time dependent and decreased as the flow rate was reduced.     

Drop weight variation in an automated collection procedure and its relationship to apparent surface tension

A drop counter-regulated fraction collector yields samples containing equal numbers of drops. Such fractions vary slightly in weight depending on experimental conditions such as surface tension. Provided that variables such as flow rate and eluate density remain constant, apparent surface tension may be estimated directly from the weights of eluate fractions obtained from gel filtration experiments.The detergents sodium cholate and sodium lauryl sulphate significantly decreased drop weights in this system. Following gel filtration on Sepharose 4B, sodium cholate eluted in the fractions containing low molecular weight material. It eluted in the same position when pre-mixed with human plasma.Normal plasma was found to contain two surface tension-reducing components with apparent molecular weight of 3· 10⁶ and 1·10⁵. The apparent surface tension of whole human plasma was found to be time dependent and decreased as the flow rate was reduced.     

Apical membrane localization of the adenomatous polyposis coli tumor suppressor protein and subcellular distribution of the beta-catenin destruction complex in polarized epithelial cells

The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of beta-catenin as part of a high molecular weight complex known as the beta-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton. To better understand the role of APC and the destruction complex in colorectal cancer, we have begun to characterize and isolate these complexes from confluent polarized human colon epithelial cell monolayers and other epithelial cell types. Subcellular fractionation and immunofluorescence microscopy reveal that a predominant fraction of APC associates tightly with the apical plasma membrane in a variety of epithelial cell types. This apical membrane association is not dependent on the mutational status of either APC or beta-catenin. An additional pool of APC is cytosolic and fractionates into two distinct high molecular weight complexes, 20S and 60S in size. Only the 20S fraction contains an appreciable portion of the cellular axin and small but detectable amounts of glycogen synthase kinase 3beta and beta-catenin. Therefore, it is likely to correspond to the previously characterized beta-catenin destruction complex. Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex. The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.     

New findings on an anti-proteinase in human plasma: l'inter-alpha-trypsin inhibitor]

Inter-alpha-trypsin inhibitor (I alpha I) has been purified from C.N.T.S. fraction III as starting material. The purification procedure includes D.E.A.E. cellulose chromatography and gel filtration on G 150 Sephadex in the presence of EDTA. The purified protein gives one precipitation line in immunoelectrophoresis against anti-whole human sérum. It reacts only with an anti I alpha I immune serum and possesses a strong antitryptic activity. When studied in starch or polyacrylamide gel electrophoresis 2 components are observed, each of them having the same antigenic structure and the same antitryptic activity as the crude preparation. The slower and less important component is dissociated by 0,1% SDS. The molecular weight estimation of I alpha I BY PAA/SDS is about 180,000. This result is not modified by the presence of 1% beta mercaptoethanol indicating that I alpha I consists of one polypeptide chain. Crude preparation reveals under the same electrophoretical conditions small amounts of low molecular weight components (135,000 52,000 and 26,000) which can be due to a proteolytic action on I alpha I. Indeed plasmin is able to produce such fragments having an antitryptic activity as shown by fibrin/polyacrylamide gel electrophoresis. The relationship between small molecular weight inhibitors of human serum and bronchial secretions and those obtained after degradation of I alpha I by plasmin is discussed.     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

PROTEINS OF THE POSTSYNAPTIC DENSITY

An analysis was made of the protein composition of a fraction of postsynaptic densities (PSDs) prepared from rat brain. Protein makes up 90% of the material in the PSD fraction. Two major polypeptide fractions are present, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The major polypeptide fraction has a molecular weight of 53,000, makes up about 45% of the PSD protein, and comigrates on gels with a major polypeptide of the synaptic plasma membrane. The other polypeptide band has a molecular weight of 97,000, accounts for 17% of the PSD protein, and is not a prominent constituent of other fractions. Six other polypeptides of higher molecular weight (100,000–180,000) are consistently present in small amounts (3–9% each). The PSD fraction contains slightly greater amounts of polar amino acids and proline than the synaptic plasma membrane fraction, but no amino acid is usually prominent. The PSD apparently consists of a structural matrix formed primarily by a single polypeptide or class of polypeptides of 53,000 molecular weight. Small amounts of other specialized proteins are contained within this matrix.     

Mechanisms of human plasma protein adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion stabilized with proxanol 268 is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to an increase in the total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and fibrinogen is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been set that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicate considerable conformational rearrangements of this protein during adsorption. It was shown that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction is completely or partially quenched.     

Mechanisms of human plasma proteins adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion, stabilized with proxanol 268, is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to the increase of a total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and immunoglobulin G is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been out that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicates considerable conformational rearrangements of this protein during adsorption. It was shown, that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction, is completely or partially quenched.     

Endothelin in human plasma and culture medium of aortic endothelial cells--detection and characterization with radioimmunoassay using monoclonal antibody

We have developed monoclonal (KY-ET-1-I) and polyclonal (ET-F5) antibodies against endothelin-1 (ET-1) and established sensitive radioimmunoassays (RIAs) with different specificities. The RIA with KY-ET-1-I detected ET-1, ET-2 and ET-3, while the RIA with ET-F5 recognized ET-3 very weakly. Using these RIAs, we have investigated the concentration and molecular forms of ET-1-like immunoreactivity (-LI) in culture medium of bovine aortic endothelial cells and human plasma. Culture medium of endothelial cells contained two major components compatible with big ET and ET-1. ET-1-LI was also detected in human plasma. ET-1-LI in human plasma consisted of apparent two components, the small molecular form emerging at the position of ET-1 and the large form with the peak eluting at the preceding fraction of the elution position of big ET. The concentration of the small form of ET in human plasma was about 5 pg/ml.     

Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein

Cultures of human skin fibroblasts were labeled to high cholesterol specific activity with [3H]cholesterol and incubated briefly (1-3 min) with normal human plasma. The plasma was fractionated by two-dimensional agarose-polyacrylamide gel electrophoresis and the early appearance of cholesterol label among plasma lipoproteins determined. A major part of the label at 1-min incubation was in a pre-beta-migrating apo A-I lipoprotein fraction with a molecular weight of ca. 70,000. Label was enriched about 30-fold in this fraction relative to its content of apo A-I (1-2% of total apo A-I). The proportion of label in this lipoprotein was strongly correlated with its concentration in plasma. Further incubation (2 min) in the presence of unlabeled cells demonstrated transfer of label from this fraction to a higher molecular weight pre-beta apo A-I species, to low-density lipoprotein, and to the alpha-migrating apo A-I that made up the bulk (96%) of total apo A-I in plasma. The data suggest that a significant part of cell-derived cholesterol is transferred specifically to a pre-beta-migrating lipoprotein A-I species as part of a cholesterol transport transfer sequence in plasma.     

Evidence for a low-molecular-weight plasma peptide which stimulates chick chondrocyte metabolism

Plasma contains a number of insulin-like activities (ILA) of molecular weights 7,000 to 90,000 (somatomedins and insulin-like proteins) which stimulate cellular metabolism and may function as growth factors. We have found evidence for the presence of an 800 Dalton peptide in human plasma which markedly stimulates the metabolism of chick chondrocytes. This peptide was extracted from human Cohn fraction IV-1 by procedures similar to those used for somatomedin isolations. At the Sephadex G-50 column separation step, the fraction with molecular weights of 300–1,000 was found to markedly stimulate chick chondrocyte metabolism. Rechromatography on Sephadex G-25 concentrated activity in peptides of molecular weight of about 800. An HPLC separation on a silica C-18 reverse phase column gave elution of the active peptide at 18% acetonitrile in water. This bioactivity appears to be a peptide which is free of lipids, carbohydrates, nucleic acids, metal ions, and immunoreactive insulin. This factor markedly increased the metabolism of cultured chick chondrocytes, but had only marginal activity on rat chondrocytes. When added at 1 μg/ml to chick chondrocytes cultured in F-12 medium plus 1.5% fetal calf serum, the HPLC-purified activity increased DNA synthesis 7.3-fold, lipid synthesis 10.2-fold, and lactate production 2.9-fold after 48 h incubation. However, unlike somatomedins A and C, this factor did not displace insulin from placental membranes. These results suggest that low-molecular-weight peptides, which are smaller than the somatomedins, may contribute to the total ILA of human plasma.     

Glycoproteins containing peanut-agglutinin receptors from human-peripheral-blood T-lymphocyte plasma membranes

A glycoprotein fraction possessing peanut agglutinin receptors has been isolated from detergent extracts of neuraminidase-treated human peripheral blood T-lymphocyte plasma membranes with affinity matrices comprising the peanut agglutinin co-valently immobilised on Sepharose 4B. This fraction could be specifically eluted from affinity columns using buffer solutions supplemented with 0.2 M D-galactose and was shown, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (0.1%), to contain four major glycoprotein components with apparent molecular weights of 200000, 190000, 110000 and 95000. It is suggested, from the observed reactivity of these glycoproteins with various lectins in double-diffusion experiments, that they possess both O-glycosidically and N-glycosidically linked carbohydrates.     

Isolation and identification of HDL particles of low molecular weight

Small particles of high density lipoproteins (HDL) were isolated from fresh, fasting human plasma and from the ultracentrifugally isolated high density lipoprotein fraction by means of ultrafiltration through membranes of molecular weight cutoff of 70,000. These particles were found to contain cholesterol, phospholipids, and apolipoproteins A-I and A-II; moreover, they floated at a density of 1.21 kg/l. They contained 67.5% of their mass as protein and the rest as lipid. Two populations of small HDL particles were identified: one containing apolipoprotein A-I alone [(A-I)HDL] and the other containing both apolipoproteins A-I and A-II [A-I + A-II)HDL]. The molar ratio of apoA-I to apoA-II in the latter subclass isolated from plasma or HDL was 1:1. The molecular weights of these subpopulations were determined by nondenaturing gradient polyacrylamide gel electrophoresis and found to be 70,000; 1.5% of the plasma apoA-I was recovered in the plasma ultrafiltrate.     

Low molecular weight plasma proteins isolated from preparations of human immunoglobulin

Low molecular weight proteins co-purified with IgG constitute 0.22% of the total protein purified from human plasma by ion-exchange chromatography on DEAE-cellulose. We have found that these low molecular weight proteins were obtained free of immunoglobulin by ultrafiltration in 5 M guanidinium chloride. Electrophoresis and isoelectric focusing in polyacrylamide gels demonstrated that this fraction of low molecular weight proteins is remarkably heterogeneous. Chromatography of an Mr 6000 to 12 000 fraction on hydroxyapatite resolved fourteen discrete protein peaks. Three of the peaks contained proteins which appeared to be homogeneous on acid-urea polyacrylamide gels. Two of these proteins were similar in composition to B2 globulin and may represent degradation products of some larger protein. The third protein was found to have an amino-terminal sequence identical to C3a. This population of low molecular weight plasma proteins has previously been shown to contain the cystic fibrosis mucociliary inhibitor and is here shown to contain two proteins similar to B2 globulin, C3a and many proteins remaining to be characterized. The presence of these low molecular weight proteins in measurable concentrations may be insufficiently appreciated in studies using 'purified' immunoglobulins as biological or chemical probes.     

Lectin from rice

N-Acetyl-D-glucosamine-binding lectin was isolated and purified from rice by ammonium sulphate fractionation and affinity chromatography using N-acetyl-D-glucosamine linked Sepharose 6B column. It gave a single hand on Polyacrylamide disc gel. It was identified as a glycoprotein. The purified lectin dissociated into two components on Sephadex G-100 column chromatography,-a higher molecular weight fraction not containing any carbohydrate and a lower molecular weight glycoprotein fraction. The apparent molecular weights of these fractions were 85,000 and 14,500. The lectin agglutinated erythrocytes of human A,B,O groups and of several other mammals and its activity was inhibited only by N-acetyl-D-glucosamine. The glycopeptide isolated by pronase digestion of the lectin was homogeneous and did not possess agglutinating activity. It contained about 10% carbohydrate of which xylose, arabinose and glucose were the major components.     

Immunochemical study of the proteins of various tissues in Crustacea (Decapoda): nature, role, origin

The main proteins of the haemolymph of Crustacea Decapoda have been identified and analysed: haemocyanin, plasma coagulogen, heteroagglutinins, vitellogenins, and molt-related proteins. All these complex components exhibit a high molecular weight and as oligomeric fractions are able to aggregate or dissociate in subunits according to the composition of medium and experimental procedures. Besides their important r?le in the defense mechanism, some proteins are involved in the edification of diverse tissues. They are detected within different compartments: soft integument, calcified carapace and hepatopancreas. They are either in transit or sequestered or synthetized within these tissues. In the crayfish Astacus leptodactylus, some components have been identified in different compartments: --in aqueous extracts from soft integument: the haemocyanin, coagulogen and both fraction F1 (lipoprotein with an approximate molecular weight of 45 kdal) and fraction F2 related to the molt. Both coagulogen and fraction F2 appear sometimes as melanized. These two latter fractions exhibit some glucose-mannose residues and they occur with a higher relative amount than in the blood. --in soluble extracts from calcified cuticle: among the numerous fractions showing a high molecular weight, the haemocyanin and coagulogen are detected. --in aqueous extracts from hepatopancreas: both haemocyanin and coagulogen appear with a little relative amount. Components termed as Fa and Fb are found with a high concentration. One minor fraction is also detected. --in aqueous extracts from eggs: the haemocyanin and fraction Fb are present. Other proteins showing only some antigenic identities with those of the haemolymph are also detected in all these tissues. The haemolymph proteins are not present within these compartments following a passive diffusion. Indeed, their relative amount varies according to the tissue investigated and is different from that found in the blood. Except the haemocyanin detected in all tissues with different aggregation states, the haemolymph proteins identified vary in the organs studied. A qualitative and quantitative selection occurs when the blood proteins enter the other compartments. Perhaps some other proteins are not detected following alterations underwent either in the epithelial barriers or during the tannage process or the chitino-proteic complex formation or due to experimental procedures. On the other hand, each tissue has its own proteins. The integument contains crustacyanins alpha, beta, gamma; the eggs are mainly constituted of lipovitellins and the hepatopancreas is rich in small molecular weight proteins and digestive enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth inhibitors in plasma derived human serum

Summary It was reported previously that plasma derived human serum (PDS) inhibited the growth of cells established from malignant human breast tissues and the MCF-7 cell line but did not inhibit the growth of cells from nonmalignant mammary tissues, including the HBL-100 cell line. Plasma derived human serum was fractionated in the current study by molecular sieve chromatography on Sephadex G-100 in an effort to characterize the factor(s) responsible for inhibition. Plasma derived human serum contained several growth inhibitory fractions, which were designated G-1, G-2, G-3, and G-4. The G-1 was associated with the lipoproteins and immunoglobulins of serum. The lipid portion of G-1 inhibited the growth of both MCF-7 and HBL-100 cells, whereas the protein fraction contained a low activity factor directed against MCF-7 cells only. The G-2 also inhibited MCF-7 cell growth at a low specific activity and was separated in the serum albumin fraction. The MCF-7 inhibitory activity in the G-3 fractions from individual donors fluctuated with the level of activity in the starting sera. The cell specific G-3 components were purified further by Sephadex G-100 superfine chromatography and gel electrophoresis. A tentative molecular weight of 50,000 was assigned to the G-3 inhibitor. The G-4 fraction consisted of small molecular weight materials migrating in advance of the column volume, which inhibited the growth of both cell lines. This investigation was supported by Grant PDT-140 from the American Cancer Society, Inc., and PHS Grant CA30284 awarded by the National Cancer Institute, Bethesda, Maryland.