Abstract. Thrombospondin (TSP), a major plateletsecreted protein, has recently been shown to have activity in tumor cell metastasis, cell adhesion, and platelet aggregation. The type 1 repeats of TSP contain two copies of CSVTCG and one copy of CSTSCG, per each of the three polypeptide chains of TSP and show homology with peptide sequences found in a number of other proteins including properdin, malarial circumsporozoite, and a blood-stage antigen of Plasmodium falciparum . To investigate whether these common sequences functioned as a cell adhesive domain in TSP, we assessed the effect of peptides corresponding to these sequences and an antibody raised against one of these sequences, CSTSCG, in three biological assays which depend, in part, on the cell adhesive activity of TSP These assays were TSP-dependent cell adhesion, T HROMBOSPONDIN (TSP)l is a 450,000-dalton glycoprotein secreted by platelets in response to such physiological activators as thrombin and collagen (9). TSP comprises 3% of the total platelet protein and 25% of the total platelet-secreted protein (26) . Although the precise biological role of TSP has yet to be fully established, it is generally accepted that TSP plays a major role in cell adhesion and cell-cell interactions . For example, TSP was found to promote the cell-substratum adhesion of a variety ofcells, including platelets, melanoma cells, muscle cells, endothelial cells, fibroblasts, and epithelial cells (24) . In addition, cells with very specialized functions such as keratinocytes (27) and osteoblasts (16) attach to TSP. Finally, TSP promotes the irreversible aggregation of platelets (23) .Thrombospondin has been postulated to play a role in malarial infection induced by only one strain of malaria, Plasmodiumfalciparum, and tumor cell metastasis induced by small cell carcinoma and mouse sarcoma. During malarial infection, TSP promotes adhesion of parasitized red cells to endothelial cells (15) and during tumor cell metastasis platelet aggregation, and tumor cell metastasis. We found that a number of peptides homologous to CSV TCG promoted the adhesion of a variety of cells including mouse B16-F10 melanoma cells, inhibited platelet aggregation and tumor cell metastasis, whereas control peptides had no effect . Anti-CSTSCG, which specifically recognized TSP, inhibited TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis, whereas control IgG had no effect. These results suggest that CSVTCG and CSTSCG present in the type I repeats function in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation, and tumor cell metastasis. Peptides, based on the structure of these repeats, may find wide application in the treatment of thrombosis and in the prevention of cancer spread .TSP promotes adhesion of mouse sarcoma cells to the vascular bed (20) and expression of the malignant phenotype of small cell carcinoma (3). Furthermore, TSP has been shown to share sequence homologies with malarial circumsporozoite proteins (8), which function in the c...

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Abstract. We have previously shown that peptides derived from the thrombospondin sequence, CSVTCG, promoted tumor cell adhesion. To further investigate this observation, the CSVTCG-tumor cell adhesion receptor from A549 human lung adenocarcinoma cells was isolated and characterized. A single protein peak was isolated by CSVTCG affinity chromatography which also analyzed as a single peak by anion exchange chromatography. The purified protein had a pI of 4.7 and analyzed on SDS-gels as a single band of Mr = 50,000 under nonreducing conditions and as two protein bands of Mr = 50,000, and 60,000 under reducing conditions. Purified CSVTCG binding protein (CBP) bound either CSVTCG-or TSP-Sepharose but showed little interaction with either VCTGSC-or BSA-Sepharose. CBP was cell surface exposed. CSVTCG derivatized with [1251] Bolton-Hunter reagent was taken up by cells in a dose-dependent manner and the cell association was inhibited with a monospecific polyclonal anfi-CBP antibody. Examination of the cell proteins crosslinked to labeled CSVTCG by SDS-gel electrophoresis revealed one band that comigrated with purified CPB. Using an in vitro binding assay, purified CBP bound mannose, galactose, and glucosamine-specific lectins. CBP bound TSP saturably and reversibly. The binding was Ca+2/Mg +2 ion dependent and inhibited with fluid phase TSP and anti-CBP. Little or no binding was observed on BSA, fibronectin, GRGES, and GRGDS. Heparin, but not lactose, inhibited binding. Anti-CBP IgG and anti-CSVTCG peptide IgG inhibited A549 cell spreading and adhesion on TSP but not on fibronectin and laminin. These results indicate that CBP and the CSVTCG peptide domain of TSP can mediate TSP-promoted tumor cell adhesion.T HROMBOSPONDIN (TSP) 1 is a large multi-domain protein that was originally purified from platelets (15) but has since been found in many tissues including bone (23), muscle (39), skin (38, 39), and brain (20). A major physiological function of this protein appears to involve cell-extracellular matrix interaction because purified preparations of TSP promote cell-substratum interaction of a variety of cell types (34), including platelets (30, 33). Support for a role of TSP in matrix-cell interaction was demonstrated in a recent study by Arbeille et al. (3) who showed that TSP was localized in microfibrils at the junction between basement membrane and connective tissue in sections of human placenta, porcine arteries and skin. Additional support comes from several in vitro studies which show that TSP has a high binding affinity for components of the basement membrane such as collagen (8), fibronectin (13), and heparansulfate containing macromolecules (9).Recently, our group (32) and two others (21, 22) independently demonstrated that peptides containing the sequence VTCG promoted cell attachment. Rich et al. (22) that several peptides containing VTCG from region H of the circumsporozoite protein isolated from the malarial parasite Plasmodium vivax strongly promoted the attachment of T cells and myeloid cells. Prater et ...

EnzymesLineweaver-Burk plats of reaction rate data obtained with immobilized enzymes need not be liqear even when intrinsic enzyme kinetics follow the simple Michaelis-Menten rate expression. Theoretical calculations show that mass transfer effects mqy cause curvature which is concave or convex to the abscissa, depending upon experimental conditions. Consequently, graphical procedures commonly employed for analysis of soluble enzyme kinetics may yield misleading regults when applied to immobilized enzymes. Three approaches which follow from the behavior of numerical and asymptotic solutions to the problem are proposed for extraction of intrinsic kinetic information. Practical application of enzymatic catalysis often requires that enzymes be immobilized, thereby permitting recovery and continuops use. It is important that the kinetics of immobilized enzymes be well understood to facilitate their economic utilization. In particular, it is necessary to account for the effect of diffusional limitations and to develop procedures which permit extraction of intrinsic kinetic parameters from observed reaction rates. BRUCENative enzymes in sdution typically follow hyperbolic Michaelis-Menten kinetics, Equation (l), which are formally analogous to Langmuir-Hinshelwood kinetics in heterogeneous catalysis. Graphical analysis of kinetic data is commonly carried out on transformed coordinates such as a Lineweaver-Burk plot of reciprocal reaction rate versus reciprocal substrate concentration. This serves to linearize the data so that the maximum reaction rate and the Michaelis constant qan be evaluated directly from the intercept and slope. The same approach is often used with immobilized enzymes, but little attention has been paid to the precise physical meaning of the parameters so determined. Nonlinear Lineweaver-Burk plots have been reported in several experimental studies with enzymes immobilized in porous supports (Lilly and Sharp, 1968;Kay and Lilly, 1970;Bunting and Laidler, 1972); in all cases, the enzymes in solution gave Michaelis-Menten kinetics. It is unclear whether the curvature observed with immobilized enzymes stems from diffusional limitations, from changes in intrinsic enzyme kinetics after immobilization, or from other factors.In this paper we examine through theoretical analysis the effect of diffusional limitations on the observed kinetics of immobilized enzymes. Of particular interest i s the behavior of Lineweaver-Burk plots and the extraction of intrinsic kinetic parameters when diffusional effects are large. The problem considered is substrate diffusion in a homogeneous porous support in the form of a one-dimensional slab or membrane in which an enzyme that catalyzes a reaction following irreversible Michaelis-Menten kinetics is uniformly immobilized. Both internal and external diflusional resistances are considered, and emphasis is placed upon the asymptotic solution which is valid when diffusional effects are important and the modified Thiele modulus is large. CONCLUSIONS A N D SIGN IF ICANCELinewea...

Daunorubicin (daunomycin; NSC 82151) is a fermentation-derived anthracycline antibiotic that is clinically useful in the treatment of human leukemias. Daunorubicin itself is found rarely in microbial fermentations, but is present normally in the form of glycoside derivatives that yield the free drug on simple acid hydrolysis. A major by-product of daunorubicin fermentations is usually the structurally related anthracycinone e-rhodomycinone. We have used mutants of a daunorubicin-producing Streptomyces species to study the biosynthetic relationship between E-rhodomycinone and daunorubicin. We found that exogenously added E-rhodomycinone can be converted to daunorubicin glycosides by a nonproducing mutant and by a mutant that produces daunorubicin glycosides but not E-rhodomycinone. Molar conversion efficiencies were in the 15 to 30% range.The latter mutant was also shown to convert exogenous "C-labeled E-rhodomycinone to "C-labeled daunorubicin glycosides, again at conversion effitiencies of about 25%. The same biotransformation was observed with daunorubicin production strain C5, which normally accumulates both E-rhodomycinone and daunorubicin glycosides. A significant percentage (16 to 37%) of exogenously added e-['4C]rhodomycinonewas metabolized by strain C5, and 22 to 32% of the metabolized radioactivity could be recovered as daunorubicin glycosides. A mathematical model of E-rhodomycinone metabolism was constructed based on plausible assumptions concerning the kinetics of E-rhodomycinone accumulation and catabolism. When analyzed according to this model, our data indicate that most (63 to 73%), but not all, of the daunorubicin glycosides accumulated in the experiments with production strain C5 derived from e-rhodomycinone. A pathway network for the biosynthesis of daunorubicin glycosides is proposed that is in agreement with these data. In this proposed pathway network, e-rhodomycinone is an intermediate in one of at least two pathways which yield daunorubicin glycosides.Daunorubicin (Fig. 1) is an anthracycline antibiotic that is elaborated by several Streptomyces species