Purification and structural analysis of a latent form of transforming growth factor-beta from rat platelets

A latent form of transforming growth factor type-beta (TGF-beta) with a high molecular weight was purified to homogeneity from rat platelets by a six-step procedure. The yield of the purified latent TGF-beta from platelets of 2,500 rats was 1.4 mg. The purified latent TGF-beta was activated by treatment with urea at concentrations of over 4M or acidic solutions of below pH 4. SDS-PAGE and gel filtration chromatography showed that the latent TGF-beta consisted of active TGF-beta and glycoproteins of about 200 kDa as masking components, and that under physiological conditions, these components formed a high molecular weight complex of about 400 kDa linked by non-covalent bonds. Here, we found that the masking protein was composed of one large subunit of about 110 kDa and two small subunits of 39 kDa linked by disulfide bridges. The N-terminal amino acid sequence of the small subunit was identical to the N-terminal region of the TGF-beta precursor lacking a signal peptide. From these findings, we proposed a structural model for the latent TGF-beta from rat platelets.     

One of two subunits of masking protein in latent TGF-beta is a part of pro-TGF-beta

A high molecular mass latent form of transforming growth factor type-beta (TGF-beta) was purified to homogeneity from rat platelets by a seven-step procedure involving group-specific affinity chromatographies on Red-Toyopearl and zinc chelating-Sepharose. The purified latent TGF-beta was a complex of TGF-beta (25 kDa) and the binding protein previously named masking protein (approximately 400 kDa) [(1986) Biochem. Biophys. Res. Commun. 141, 176-184]. Analysis of the peptide structure by gel electrophoresis showed that the masking protein consisted of two subunits of 39 kDa and 105-120 kDa linked by disulfide bonds. N-terminal amino-acid sequencing of the 39 kDa subunit indicated that this subunit was identical to the N-terminal part of the TGF-beta precursor.     

Generation of recombinant human large latent transforming growth factor-beta 1 and monoclonal antibodies to it

Transforming growth factor beta 1 (TGF-beta 1) is a regulator of cell growth and differentiation. It is produced in various of cells and tissues as a biologically latent complex, whose significance is still unknown. We established a Chinese hamster ovary cells that produced recombinant human large latent TGF-beta 1. The growth factor was purified from serum-free conditioned medium of the cell line was purified to apparent homogeneity by four steps of column chromatography. The purified protein gave a single band with the apparent molecular weight of 210,000 on SDS-PAGE, and had four subunits, of 12.5, 40, 53, and 150-190 kDa. These components were identical to TGF-beta 1, the N-terminal remnant of pro-TGF-beta 1, pro-TGF-beta 1, and latent TGF-beta 1 binding protein, respectively. The purified growth factor had biological activity similar to that of the growth factor purified from human platelets. We prepared four monoclonal antibodies by immunization of mice with the recombinant protein. In western blotting, two of the antibodies bound to latent TGF-beta 1 binding protein. The two other antibodies reacted with the N-terminal remnant of pro-TGF-beta 1. Recombinant large latent TGF-beta 1 and its monoclonal antibodies could be used for detailed structural and functional studies of the large latent TGF-beta 1 complex.     

Latent transforming growth factor-beta in serum. A specific complex with alpha 2-macroglobulin

The biological latency of serum transforming growth factor-beta (TGF-beta) was shown to be due to the interaction of TGF-beta with a specific serum binding protein. This binding protein was affinity labeled with 125I-TGF-beta, and its Mr and subunit structure were determined using sodium dodecyl sulfate-gel electrophoresis and gel filtration chromatography. Its Mr is reminiscent of that of the serum protease inhibitor, alpha 2-macroglobulin (alpha 2M). Immunoprecipitation of the 125I-TGF-beta-binding protein complex by a specific anti-alpha 2M antibody, and the formation of identical complexes between 125I-TGF-beta and purified alpha 2M, confirmed that alpha 2M is the TGF-beta-binding protein in serum. Immunoblot analysis showed that endogenous serum TGF-beta is also bound to alpha 2M. However, in contrast to added 125I-TGF-beta, the majority of the endogenous TGF-beta is linked to alpha 2M covalently. Alpha 2M and acid-activated TGF-beta co-eluted from a Superose 6 fast protein liquid chromatography column, confirming that the interaction of TGF-beta with alpha 2M accounts for the latency of serum TGF-beta. It is proposed that alpha 2M may serve an important multifunctional role at sites of inflammation by scavenging both active peptides and proteases that are released by platelets at the site of injury.     

A model of visual backward masking

When two successive stimuli are presented within 0-200 ms intervals, the recognition of the first stimulus (the target) can be impaired by the second (the mask). This backward masking phenomenon has a form called metacontrast masking where the target and the mask are in close spatial proximity but not overlapping. In that case, the masking effect is strongest for interval of 60-100 ms. To understand this behaviour, activity propagation in a feedforward network of leaky integrate and fire neurons is investigated. It is found that, if neurons have a selectivity similar to that of V1 simple cells, activity decays layer after layer and ceases to propagate. To combat this, a local amplification mechanism is included in the model, using excitatory lateral connections, which turn out to support prolonged self-sustained activity. Masking is assumed to arise from local competition between representations recruited by the target and the mask. This tends to interrupt sustained firing, while prolonged retinal input tends to re-initiate it. Thus, masking causes a maximal reduction of the duration of the cortical response to the target towards the end of the retinal response. This duration exhibits the typical U-shape of the masking curve. In this model, masking does not alter the propagation of the onset of the response to the target, thus preserving response reaction times and enabling unconscious priming phenomena.     

Purification and characterization of transforming growth factor-beta 2.3 and -beta 1.2 heterodimers from bovine bone.

A unique form of transforming growth factor-beta (TGF-beta), TGF-beta 2.3 heterodimer, has been purified from bovine bone extract. TGF-beta 2.3 migrated as a single 25-kDa band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas under reducing conditions it migrated as a 12.5 kDa band. The TGF-beta 2.3 reacted positively with anti-TGF-beta 2 and anti-TGF-beta 3 antibodies on immunoblots. Equal levels of TGF-beta 2 and TGF-beta 3 sequences were detected by N-terminal sequencing. TGF-beta 2.3 eluted as a single sharp peak by reverse-phase high performance liquid chromatography. However, prior reduction of the protein with dithiothreitol resulted in the protein eluting in two peaks, one containing predominantly TGF-beta 3 and the other containing predominantly TGF-beta 2. TGF-beta 2.3 inhibited proliferation of mink lung epithelial cells and promoted the formation of colonies of normal rat kidney fibroblasts in culture with specific biological activity similar to those of TGF-beta 1 and TGF-beta 2. These results demonstrate that the protein is TGF-beta 2.3 heterodimer, consisting of one polypeptide chain each of TGF-beta 2 and TGF-beta 3 linked by one or more disulfide bonds. In addition, TGF-beta 1.2 heterodimer, previously found only in porcine platelets, has also been purified from bovine bone extract.     

Transforming growth factor-beta complexes with thrombospondin.

Thrombospondin (TSP) was demonstrated to inhibit the growth of bovine aortic endothelial cells, an activity that was not neutralized by antibodies to TSP or by other agents that block TSP-cell interactions but that partially was reversed by a neutralizing antibody to transforming growth factor-beta (TGF-beta). Similar to TGF-beta, TSP supported the growth of NRK-49F colonies in soft agar in a dose-dependent manner, which required epidermal growth factor and was neutralized by anti-TGF-beta antibody. Chromatography of a TSP preparation did not separate the TGF-beta-like NRK colony-forming activity from high molecular weight protein. However, when chromatography was performed at pH 11, this activity was dissociated from TSP. These results suggest that at least some growth modulating activities of TSP are due to TGF-beta associated with TSP by strong non-covalent forces. Most of the active TGF-beta released from platelets after degranulation was associated with TSP, as demonstrated by anti-TSP immunoaffinity and gel permeation chromatography. 125I-TGF-beta binds to purified TSP in an interaction that is specific in the sense that bound TGF-beta could be displaced by TGF-depleted TSP but not significantly by native TSP, heparin, decorin, alpha 2-macroglobulin, fibronectin, or albumin. Hence, TGF-beta can bind to TSP, and the complex forms under physiological conditions. Furthermore, TSP-associated TGF-beta is biologically active, and the binding of TGF-beta to TSP may protect TGF-beta from extracellular inactivators.     

Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane.

We have investigated the characteristics of platelet-activating factor (PAF)-stimulated protein tyrosine phosphorylation in rabbit platelets and its relationship to pp60c-src. 32P-Labeled platelets were challenged with PAF (10(-7) M) for 15 s, the reaction was killed by lysis at 4 degrees C, and samples were loaded onto a phosphotyrosine monoclonal antibody (Tyr(P)-mAb)-agarose column. The column was eluted with 10 mM phenyl phosphate, and the fractions were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography of the column fractions, showed that PAF increased the radioactivity of about a dozen protein bands with predominant ones of approximate molecular masses of 50, 60, 71, 82, and 300 kDa. When Tyr(P)-mAb-agarose column fractions were subjected to immunoblotting with pp60v-src mAb, it was observed that PAF treatment increased the reactivity of 50- and 60-kDa protein species. Immunoprecipitation with pp60v-src mAb further confirmed that PAF treatment increased phosphorylation of the 60- and 50-kDa proteins. Polyclonal antibody to G-protein (alpha-subunit) did not exhibit any reactivity to the column fractions and thus ruled out this protein as substrate for the tyrosine kinase. We next attempted to localize the pp60c-src. Platelet membrane particulate and cytosol fractions were separated from control and PAF-treated platelets, and it was observed that the immunoreactivity to pp60v-src mAb dramatically increased in the particulate membrane fraction from PAF-treated platelets. A concomitant decrease in the immunoreactivity in the cytosol fraction of PAF-treated platelets was also noted. It is concluded that PAF stimulates phosphorylation of pp60c-src tyrosine kinase and causes its rapid translocation from cytosol to membranes in rabbit platelets.     

TGF-beta in blood: a complex problem

The cytokine transforming growth factor-beta (TGF-beta) was initially purified from human platelets, a rich source of this protein. In addition to platelets, TGF-beta1 is also found in other blood fractions, including plasma and the circulating leukocytes. However, more than 15 years after the initial isolation of TGF-beta1, there remains no consensus on how much TGF-beta1 is present in normal human plasma. Here we review the difficulties associated with measuring TGF-beta concentrations in complex biological fluids, and discuss the current state of knowledge on the distribution of TGF-beta isoforms in various blood fractions as well as the nature of the TGF-beta-containing protein complexes.     

A transforming growth factor-beta like growth factor in mouse embryonal carcinoma cells

Our previous study indicated that polypeptides isolated from acid/ethanol extracts of solid tumors of a cloned F9-3 embryonal carcinoma cells by Bio-Gel P60 column chromatography were found to be able to stimulate anchorage independent growth of either NIH 3T3 cells or NRK 49 F cells in soft agar. The major peak of active elute had a molecular weight of about 15 kDa as determined by SDS-polyacrylamide gel electrophoresis. In the present report we further isolated and purified the active compound corresponding to molecular weight of 15 kDa by gel filteration on Bio-Gel P10 column (Fig. 1) and then by high pressure liquid chromatography (Fig. 2). It was found that the purified 15 kDa molecules showed some properties similar to transforming growth factor-beta (TGF-beta): 1. Colony-stimulating activity in soft agar can be induced in NRK 49 F cells only in the presence of mouse epidermal growth factor (EGF) (Plate I); 2. Increase in relative uptake of 3H-thymidine in NRK 49 F cells occurred in the presence of EGF, but with the same amount of EGF, not much change in 3H-thymidine incorporation could be found with further increasing amounts of purified 15 kDa molecules (Fig. 3); 3. Like human blood platelets derived TGF-beta, inhibition effect on the growth of mink lung epithelial cells (CCL/64) can also be exhibited by purified 15 kDa molecules (Fig. 4). In addition, using ELISA procedure, we have also demonstrated that the 15 kDa molecules had immunological reactivity with the antibody raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta 1 from human blood platelets (Fig.5). Thus, the 15 kDa molecules isolated from mouse F9-3 embryonal carcinoma cells appeared to share some common antigenic determinants with human TGF-beta 1 molecule. These results taken together provide strong support for the existence of TGF-beta like growth factor in mouse embryonal carcinoma cells.     

The Studies of the Stabilizing Factor of the Nitrate Reductase(E. C. 1.6.6.2) Activity from the Tomato(Lycopersicum esculentum)Leaves

A stabilizing factor of NR activity was isolated from tomato leaf homogenate by (NH4)2SO4 precipitation, boiling water treatment, Sephadex G-75 column and DEAE 52 column and dialysis against water. This factor appeared as band on polyacryl amide gel electrophoresis. This factor protected NR activity of tomato leaves from inactivation by temperature. It delayed the inactivation of N-R activity in wheat seedlings. when mixed with stabilizing factor and stored at 0 ℃ for nine days NR from wheat seedlings still kept its activity; without stabilizing factor, its activity was lost completely. Stabilizing factors both treated in boiling water in the process of isolation and isolated below 4 ℃ could stabilize and increase NR activity of tomato leaves. This factor existed in two forms, one free and one combined with some protein. It could be separated from the protein by heating during extraction. The stabilizing factor is different from FAD, haem, and proline.     

An improved method of purification of transforming growth factor, type beta from platelets

Platelets contain high concentrations of TGF-beta and for this reason have been the primary source of materials for its purification. The yield from acid-ethanol extracts of platelets has been reported to be 0.5 microgram/unit of platelets. We have developed a method which yields approximately 2 micrograms/unit of platelets from acid-ethanol extracts. The use of an ion-exchange column followed by RPLC results in a homogeneous product in less time and with fewer manipulations than the previously published methods. A tritiated thymidine incorporation assay using the CC164 cell line is described for the quantitation of TGF-beta.     

Characterization of a protein:glucosyltransferase activity in human platelets

Human platelets exhibited significant glucosyltransferase activity, that transfer [14C]glucose from UDP-Glc to an endogenous protein acceptor. The enzyme protein:glucosyltransferase responsible for the catalysis was characterized and compared with glycogen:glucosyltransferase. We describe a partial separation of both activities, the ratio of protein:glucosyltransferase/glycogen:glucosyltransferase varied from 7:1 in a crude homogenate of platelets to 36:1 in the Sephadex G-100 column. This procedure failed to separate the protein:glucosyltransferase from its endogenous acceptor. Glucosylation of protein demonstrated dependence with respect to time and both protein and UDP-Glc concentration, and was saturated by very low concentration of donor and acceptor substrates. It was inhibited 76% by 5 mM Mn2+ concentration and was activated 23 and 11% by 5 mM concentrations of Ca2+ and Mg2+, respectively. With respect to glycogen:glucosyltransferase, when the effect of time, protein, and substrate concentration were determined under identical conditions, it did not show the same dependence. At 5 mM concentration, Mn2+, Ca2+, and Mg2+ were activators of the enzyme 43, 80, and 200%, respectively. On the basis of these characteristics, we conclude that the synthesis of glucoprotein and glycogen are catalyzed by two distinct enzymes. Addition of exogenous glycogen (range 0.002-1%) inhibited the protein:glucosyltransferase, whereas at 0.001-0.007% concentration it was acceptor substrate for glycogen:glucosyltransferase activity. At higher concentrations this activity was strongly inhibited. The concentration of glycogen in platelets could play a regulatory role in forming the glucoprotein and the glycogen molecules.     

Measurement of alpha-ketoglutarate dehydrogenase activity in tissue extracts and human platelets using reversed-phase high-performance liquid chromatography

A new method for the determination of the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) in mouse brain and liver mitochondria and in human platelets using reversed-phase high-performance liquid chromatography is described. This method is based on the quantification of succinyl-CoA formed in the reaction catalyzed by KGDHC. Succinyl-CoA was separated using a YMC-Pack C8 column employing isocratic elution and detected spectrophotometrically at 254 nm. The detection limit of succinyl-CoA was 0.05 nmol. Succinyl-CoA in the supernatant of the assay mixture was stable for several hours at 4 degrees C and for a week when stored at -20 degrees C. The KGDHC assay showed good linearity with time and added protein, and all tissues demonstrated an absolute requirement for added alpha-ketoglutarate, nicotinamide dinucleotide, and coenzyme A and partial or no requirement for thiamine pyrophosphate, magnesium chloride, and dithiothreitol. The specific activities in liver and brain mitochondria and platelet homogenates determined by the present method were 19.2 +/- 0.9, 18.1 +/- 2.8, and 2.6 +/- 0.3 nmol/min/mg protein, respectively. In human platelets, the present method gives higher specific activity and lower blank values than a prior method using 14CO2 and may be useful in the diagnosis of KGDHC deficiency. This method is simple, rapid, and can be readily employed for the determination of KGDHC activity in various animal tissues and human platelets.     

Human platelet alpha granules contain a nonspecific inhibitor of megakaryocyte colony formation: its relationship to type beta transforming growth factor (TGF-beta)

Whole blood serum (WBS) and platelet-poor plasma-derived serum (PDS) from the same normal subject were compared for their abilities to support human megakaryocyte (MK) colony formation. In all cases, PDS promoted the growth of a higher number (20-50%) of MK colonies than did WBS. Increasing amounts of WBS decreased the number of colonies, whereas increasing concentration of PDS had no marked effects. Crude platelet extracts or platelet secretory products from thrombin-activated platelets also elicited an inhibition of MK colony formation in a dose-dependent manner. A complete inhibition was found for a dose equivalent to 1.10(9) platelets/ml and a 50% inhibition in a range of 1.10(7)-1.10(8) platelets/ml. These platelet products were also inhibitory for erythroid progenitor growth. Platelets from two patients with gray platelet syndrome elicited only a minor inhibition of MK growth, suggesting that the platelet alpha granule is the origin of this inhibition. When platelet extracts were acid-treated, the biological activity of the inhibitor on CFU-MK and CFU-E growth was 20-50-fold higher. In addition, a potent stimulatory activity on the growth of day 7 CFU-GM was observed. The enhancement of biological activities by acid treatment suggests that type beta transforming growth factor (TGF-beta) could be involved in this platelet inhibitory activity. The homogeneous native TGF-beta (from 1 pg to 1 ng/ml) produced the same effects previously induced by platelet products. It totally inhibited CFU-MK growth (at a 500 pg/ml), it inhibited CFU-E growth, and it stimulated growth of day 7 CFU-GM in the presence of a colony-stimulating factor. The inhibition of CFU-MK growth was also observed on purified progenitors. In conclusion, these results suggest that TGF-beta may be implicated in negative autocrine regulation of megakaryopoiesis. However, since this molecule has ubiquitous biological activities, its physiologic relevance as a normal regulator of megakaryopoiesis requires further investigation.     

Ca(2+)-independent, phospholipid-activated protein kinase in 3Y1 cells.

We obtained a Ca(2+)-independent but 12-O-tetradecanoyl phorbol ester (TPA).phospholipid-activated protein kinase from rat embryo fibroblast 3Y1 cells by succeeding steps of DEAE-cellulose, H-9 affinity, and hydroxylapatite chromatography. This kinase was separated chromatography. This kinase was separated from a conventional PKC (Type III), by H-9 affinity column chromatography. The major peak from H-9 affinity column was eluted at 0.4 M of arginine and on the following step of hydroxylapatite column chromatography, at the KPO4 concentration of 0.1 M. The enzyme could be stimulated by phospholipids and by the tumor promoter TPA, but did not respond to calcium. The Ca(2+)-independent, phospholipid-activated protein kinase activity was susceptible to the protein kinase C inhibitors H-7 and K252a, but showed a phospholipid dependency and substrate specificity distinct from the conventional types of PKC. This protein kinase did not react with monoclonal antibodies against Types I, II, and III PKC. The activity of this enzyme was specifically reduced by immunoprecipitation, depending on the concentration of the polyclonal antibody, PC-delta, which was raised against a peptide synthesized according to a sequence of rat brain nPKC delta. The enzyme had a Mr of 76,000 as estimated by Western blotting. These results provide evidence for a unique type of Ca(2+)-independent, phospholipid-activated kinase, as expressed in 3Y1 cells.     

Conformational change of the haemolymph juvenile-hormone-binding protein from Galleria mellonella (L)

A juvenile-hormone-binding protein (juvenile-hormone carrier), isolated from Galleria mellonella haemolymph, was treated with trypsin, chymotrypsin, carboxypeptidase A and subtilisin. Among these enzymes, only subtilisin was able to affect juvenile-hormone-binding activity of this protein. With SDS/PAGE it was shown that juvenile-hormone-binding protein, a 32-kDa peptide, is first slowly converted into a 30-kDa molecule, then into two or three smaller-molecular-mass species (20-25 kDa), which in turn were further digested to small peptides undetectable in PAGE. The 30-kDa peptide has a 2.4-times-higher dissociation constant for juvenile hormone than the native protein. No binding activity was detected for 20-25-kDa peptides. The rate of proteolysis of juvenile-hormone-binding protein was decreased by more than twofold in the presence of hormone, however, the overall cleavage pattern was unchanged. Under non-denaturing conditions, free binding-protein molecules could be separated from juvenile-hormone-binding-protein complex due to a slower electrophoretic mobility of the complex. As judged from ultracentrifugation and cross-linking experiments, binding of the hormone to its haemolymph carrier does not induce formation of oligomers, but shifts the sedimentation coefficient from 2.30S to 2.71S. It is concluded that juvenile-hormone binding induces a conformational transition of its carrier protein. This hormone-induced change might have a physiological significance for signal transmission.     

Expression of soluble recombinant TGF-beta type II receptor fused with the Fc portion of human IgG1 (sTbetaRII-Fc) in NS0 cells

We have constructed and expressed recombinant chimeric soluble TGF-beta type II receptor fused with the Fc portion of human IgG1 (sTbetaRII-Fc) in NS0 mouse myeloma cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The GS-NS0 expression system takes advantage of the strong human cytomegalovirus immediate early promoter expression vector and glutamine synthetase as a selectable marker. The recombinant chimeric receptor could be produced in high amounts and efficiently purified by one step chromatography on a protein A column. Biochemical studies revealed that recombinant sTbetaRII-Fc binds native TGF-beta1 and TGF-beta3 isoforms and neutralizes their activity in vitro.     

Humoral and formed elements of blood modulate the response of peripheral blood monocytes. I. Plasma and serum inhibit and platelets enhance monocyte adherence.

Human and rabbit peripheral blood monocytes normally adhere to plastic tissue culture plates in vitro when they are suspended in Hanks' media. Increasing amounts of autologous serum or heat-inactivated plasma in the cell suspensions prevented the adherence of both monocytes and lymphocytes. The inhibitory effect of plasma was separated into three areas of activity by chromatography on Sephacryl S-200. The profile of inhibitory activity did not coincide with the protein elution profile, suggesting that inhibition was not a nonspecific protein effect. A layer of adherent platelets overcame the inhibitory effect of plasma on monocyte adherence. Platelets selectively increased monocyte as opposed to lymphocyte adherence and this was specific for platelets in that neither neutrophils nor fibroblasts could substitute for platelets. Both plasma and platelets acted directly on monocytes.     

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%.