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...

A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857). Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products. Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins. The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA.

y-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified-y-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of-y-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 x 10-M. Enzyme activity was insensitive to the glutamate analog Lmethionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a y-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline. Proline biosynthesis in Escherichia coli occurs through a series of three enzymatic reactions catalyzed by the gene products of the proB, proA, and proC loci: through feedback inhibition of-y-glutamyl kinase by proline (1, 3). E. coli mutants which excrete proline can be obtained by selection for'resistance to the toxic proline analog 3,4-mg2 L-glutamate + ATP-)-y-glutamyl phosphate + ADP phosphate+ NADPH proA ,y-glutamyl phosphate + NADPH L-glutamic acid 5-semialdehyde + NADP+ + Pi L-glutamic acid 5-semialdehyde) A1-pyrroline-5-carboxylate roC P+ A1pyrroline-5-c'arboxylate + NADPH-L-proline + NADP' Reaction 1 is catalyzed by-y-glutamyl kinase (ATP:L-glutamate 5-phosphotransferase, EC 2.7.2.11) and most likely results in the production of an extremely labile intermediate, y-glutamyl phosphate (1, 8). Due to the lability of this compound, it has been proposed that-y-glutamyl kinase functions in a complex with the second enzyme of the pathway,-y-glutamyl phosphate reductase (1) (GP-reductase; L-glutamate 5-semialdehyde:NADP+ oxidoreductase [phosphorylating], EC 1.2.1.41). The product of reaction 2, Lglutamic acid 5-semialdehyde, undergoes a spontaneous cyclization to A-pyrroline-5-carboxylate, which is converted to proline through the action A1-pyrroline-5-carboxylate reductase (L-proline:NAD[P]+ 5-oxidoreductase, EC 1.5.1.2), the third enzyme in the pathway (1, 22). The second and third enzymes of proline biosynthesis have been purified and characterized (5, 9, 19); however, the first enzyme,-y-glutamyl kinase, has only been studied in cell-free extracts or crude preparations (1, 2). Regulation of proline biosynthesis is thought to be exerted primarily

The sequence of the Escherichia coli proC gene which encodes for delta 1-pyrroline-5-carboxylate (PCA) reductase was determined. Overproduction of the proC gene product via an expression plasmid carrying the bacteriophage lambda PL promoter allowed the purification to homogeneity of PCA reductase by affinity adsorption chromatography. NH2 and COOH-terminal analysis and amino acid composition of the purified proC protein is consistent with the gene sequence reported. The molecular weight of the proC monomer is 28,112.

Eco R, restriction endonuclease makes two cuts in each repeating unit of amplified ribosomal DNA (rDNA) from Xenopus laevis. The locations of these cuts have been established by comparison of the secondary structure of single-stranded Eco RI fragments (as visualized in the electron microscope) with that of X. laevis rRNAs and of long strands of uncut rDNA. Of the two classes of fragments generated, the smaller one contains 90% of the 28S rRNA gene, has a duplex molecular weight of 3.0 X 106 and is homogeneous in size. Eukaryotic cells contain families of closely related repetitious DNA sequences. Some of these nucleotide sequences are highly species specific, such as those present in satellite DNAs of unknown function or the spacer sequences which separate genes of known function. A variety of evolutionary mechanisms has been proposed to account for the tandem evolution of genes and their spacers (1). One means to distinguish these evolutionary mechanisms depends upon the ability to detect heterogeneity within a family of DNA repeats and to map the location of this heterogeneity. The DNA containing the genes for 18S and 28S rRNA (rDNA) in Xenopus laevis has been characterized by thermal denaturation criteria (2, 3) and by the regularity of its denaturation map (3,4) Eco R1 Restriction Endonuclease was purified from E. coli strain RY 13 carrying the RI drug resistance plasmid (a generous gift from D. Nathans). The purification procedure followed the method of Greene et al. (7) through step F (second hydroxyl apatite concentration). Purity and specificity of the enzyme were demonstrated by its ability to make a single duplex cut in closed circular SV40 DNA; no singlestrand breaks were observed by electron microscopy of denatured SV40 DNA. Restriction endonuclease Hind III was prepared by the method of Smith (8), from a streptomycin supernate generously provided by H. Smith.Digestion of rDNA was carried out in a final volume of 0.5 ml containing 10 mMiMgCl2, 50 mM\ Tris-HCl (pH 8), 0.5-2 ug of rDNA with enough enzyme to give a limit digest. Reactions were stopped by the addition of EDTA (ethylenediaminetetraacetate), extracted with phenol, and DNA was precipitated from the aqueous phase with ethanol.Electrophoresis. DNA fragments were electrophoresed in 1% agarose disc gels and photographed after staining with ethidium bromide (9). Phage X DNA fragments produced by restriction endonuclease Hind III were run in parallel as molecular weight standards. Hind III makes six cuts in linear X DNA; the molecular weights of the seven resulting fragments were determined by electron microscopy to be 14.6, 5.84, 4.05, 2.67, 1.40, 1.21, and 0.34 X 106. The 14.6 and 2.67 X 106 fragments contain the ends of the X DNA with their short single-stranded termini.Electron Mlicroscopy. Duplex DNA was spread for electron microscopy from 50% formamide (10). Single-stranded DNA was prepared for electron microscopy as follows: The DNA was denatured in 0.1 N NaOH for 30 min, then neutralized with 0.1 N HCl. Concentrated stock solutio...