Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.     

Surface-enhanced resonance Raman scattering spectroscopy of bacterial photosynthetic membranes. The carotenoid of Rhodospirillum rubrum

Resonance Raman scattering by the carotenoid, spirilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodospirillum rubrum, is greatly enhanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510, 1150-1155, and 1000-1005 cm-1 with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast surface, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit membrane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e., it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin-pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B880 is presented. This work demonstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.     

Signal peptidases recognize a structural feature at the cleavage site of secretory proteins

The cloning of the gene for staphylococcal nuclease A in the pIN-III-OmpA secretion vector results in a hybrid protein which is processed by signal peptidase I, yielding an active form of the nuclease that is secreted across the cytoplasmic membrane (Takahara, M., Hibler, D., Barr, P. J., Gerlt, J. A., and Inouye, M. (1985) J. Biol. Chem. 260, 2670-2674). Using oligonucleotide-directed site-specific mutagenesis, we have constructed a set of mutants at the cleavage site area of the precursor hybrid protein designed to alter progressively the predicted secondary structure of the cleavage site. Our results show that processing becomes increasingly defective as the turn probability decreases. These results are consistent with the structural requirement that we found for the processing of lipoprotein by signal peptidase II (Inouye, S., Duffaud, G., and Inouye, M. (1986) J. Biol. Chem. 261, 10970-10975). We conclude that secretory precursor proteins have a distinct secondary structural requirement at their cleavage site for processing by signal peptidase I, as well as by signal peptidase II.     

Thrombospondin is a substrate for blood coagulation factor XIIIa

Thrombospondin (TSP) is released from alpha granules of activated platelets, binds to platelet surfaces, and copolymerizes with fibrin. In the present experiments, we investigated the action of factor XIIIa (plasma transglutaminase) on TSP. Factor XIIIa catalyzed incorporation of [14C]putrescine into soluble TSP and ligation of TSP to itself and to fibrin intermediates. Proteolytic digestion of [14C]putrescine-labeled TSP with trypsin or thrombin yielded a labeled disulfide-bonded core of 90 or 120-130 kilodalton (kDa) subunits, labeled fragments of less than 10 kDa, and an unlabeled 30-kDa heparin-binding fragment, indicating the presence of multiple factor XIIIa reactive glutaminyl residues located in several domains of the molecule. TSP became ligated in fibrin clots formed from amidinated fibrinogen, i.e., fibrin that could not contribute lysyl residues to factor IIIa catalyzed cross-links. The disulfide-bonded core of TSP formed upon thrombin digestion copolymerized with fibrin as efficiently as intact TSP. However, a lower proportion of the disulfide-bonded core became ligated. These results indicate that TSP, both in clots and in solution, contributes glutaminyl and lysyl residues to factor XIIIa catalyzed ligation. Cross-linking may be important in stabilizing interactions among TSP, fibrinogen, or fibrin and other molecules in hemostatic plugs.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

Thiol reactivity of histone H3 in soluble and DNA-associated histone complexes: evidence for allosteric and torsional regulation

The reactivity of chick erythrocyte and calf thymus histone H3 thiol groups toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been investigated both in the soluble, DNA-free state and in various nucleohistone complexes. We have found that the thiol reactivity of both tetramers and octamers decreases continuously as the ionic strength of the assay is increased, up to and beyond 2.0 M NaCl. Upon association of dimers with tetramers, there is loss of labeling by DTNB at one site, suggesting the existence of allosteric regulation [see also Godfrey, J. E., Eickbush, T. H., & Moudrianakis, E. N. (1980) Biochemistry 19, 1339-1346] of dimer-tetramer interfaces emanating from within the tetramer complex. Comparison of the thiol reactivities of chick and calf tetramers indicates that the thiol groups at amino acid positions 96 and 110 are not chemically equivalent. When the histones are associated with DNA, in either reconstituted complexes, core particles, or long soluble chromatin, the thiol reactivity is greatly diminished, and this "DNA effect" overwhelms any influence of dimers. However, if single-strand nicks are introduced into the DNA backbone of core particles and other chromatin-like complexes by the action of DNase I, the influence of the DNA double helix upon thiol reactivity is reduced, and the effect of dimers can be detected once again. We can therefore conclude that the DNA effect derives from intranucleosomal torsional strain of the continuum of the double helix in equilibrium with coupled protein conformational changes. These observations support the concept that the octamer complex is a dynamic tripartite structure whose properties can be modulated through its interactions with DNA and by changes occurring in the dimer-tetramer interfaces.     

Sulfate-chloride exchange transport in a glioma cell line

Transport of SO4(2-) was studied in the glioma cell line LRM55 to determine whether it is mediated by the Cl-/HCO3- exchanger or the K+/Cl- cotransporter previously described in these cells (Wolpaw, E.W. and Martin, D.L. (1984) Brain Res. 297, 317-327). 35SO4(2-) influx was saturable with SO4(2-). External SO4(2-) stimulated 35SO4(2-) efflux, indicating an exchange mechanism. External Cl- was a competitive inhibitor of 35SO4(2-) influx. Internal Cl- stimulated 35SO4(2-) influx and external Cl- stimulated 35SO4(2-) efflux, indicating that Cl- is an exchange substrate for the SO4(2-) carrier. Also, SO4(2-) flux was sensitive to SITS, DIDS and furosemide. However, saturating external SO4(2-) did not inhibit 36Cl- influx and did not inhibit 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 35SO4(2-) influx as it does Cl- influx. These findings indicate that SO4(2-) transport into these cells is mediated by an exchange carrier distinct from both the Cl-/HCO3- exchanger and the K+/Cl- cotransporter. While Cl- is an alternative substrate for the SO4(2-) porter, this carrier is responsible for only a minor fraction of total Cl- flux in these cells.     

Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 1. Effect of exogenous sterols

Chinese hamster ovary cells grown in medium containing low-density lipoprotein (LDL) express high acyl coenzyme A:cholesterol acyltransferase (ACAT) activity as measured by an [3H]oleate pulse. Removal of LDL from the medium causes rapid inactivation of ACAT activity; the t1/2 for the initial inactivation rate is 0.8 h. Preincubation with protein synthesis inhibitors (cycloheximide or emetine) for 2 h or longer lengthens the t1/2 for the initial inactivation rate to approximately 2.1 h. When LDL is removed for more than 10 h, the cells contain only 3% of the original ACAT activity. Cycloheximide under this condition causes an 8-fold increase in ACAT activity; the increase approaches a maximum in 6-8 h. The extent of ACAT activation by cycloheximide inversely depends on exogenous sterol present in the medium; LDL diminishes the activation, while cationized LDL or 25-hydroxycholesterol completely abolishes the activation. Adding LDL back to the sterol-free medium causes a 40-70-fold increase in ACAT activity; however, the activation of LDL is not further augmented if the cells are pretreated with cycloheximide. The above observations are qualitatively confirmed by ACAT assays in vitro with cell homogenates. LDL or cycloheximide has no effect on the rates of 3H-labeled triglyceride and 3H-labeled polar lipid synthesis. Efflux of prelabeled cholesterol from cells is cycloheximide-insensitive. Rates of degradation of [3H]-leucine-pulse-labeled total protein in cells grown with or without LDL are identical. The above results imply the existence of at least one specific short-lived factor that directly or indirectly inhibits ACAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chelating agents on the Ca2+-stimulated ATPase of rat liver plasma membranes

Using strictly controlled ionic conditions we have demonstrated, in agreement with previous findings (Lotersztajn et al. (1981) J. Biol. Chem. 256, 11209-11215; Lotersztajn, S. and Pecker, F. (1982) J. Biol. Chem. 257, 6638-6641) a Ca2+-stimulated ATPase in rat liver plasma membranes which is detectable at low free Mg2+ concentrations (normally fulfilled by endogenous levels) but not at free Mg2+ concentrations greater than about 10(-5) M. The findings reported here also suggest that this (Ca2+ + Mg2+)-ATPase is activated by EGTA or one of its liganded species. Furthermore, this is probably an intrinsic property of the enzyme as it was found to be independent of the isolation technique. The stimulation by EGTA appears to be a function both of free Ca2+ concentration and of one or more liganded species of EGTA and it is also inhibited at high free Mg2+ concentrations (approx. 10(-5) M). The specificity of the EGTA effect on ATPase activity is studied with respect to other, widely used, chelating agents namely HEEDTA, EDTA and CDTA. Of these, only CDTA shares the effect, although the concentration dependence of the activation is different from EGTA, suggesting that there is some degree of structural specificity involved rather than a generalised effect of complexed Ca2+.     

Phenylalanine ammonia-lyase immobilized in microcapsules for the depletion of phenylalanine in plasma in phenylketonuric rat model

Microencapsulation of the enzyme phenylalanine ammonia-lyase was developed for in vivo depletion of systemic phenylalanine in phenylketonuric rats. Compared to normal rats, systemic phenylalanine blood levels in phenylketonuric rats was increased by 15-20-fold. Daily oral administration of 1 unit of phenylalanine ammonia-lyase-loaded artificial cells to phenylketonuric rats lowered the systemic phenylalanine level to 58% +/- 18% (mean + S.D.) in 7 days (P less than 0.010), while 5 units lowered the systemic phenylalanine level to 25% +/- 8%. 5 units of the immobilized enzyme lowered the systemic phenylalanine level to normal levels within 6 days. Phenylketonuric treated rats showed no signs of abnormal behavior and weight loss compared to phenylketonuric non-treated rats. The immobilized enzyme within artificial cells is therefore protected against low gastrointestinal pH and proteolytic enzymes.