Role of laminin and fibronectin in selecting myogenic versus fibrogenic cells from skeletal muscle cells in vitro

Growth of embryonic skeletal muscle occurs by fusion of multinucleated myotubes with differentiated, fusion-capable myoblasts. Selective recognition seems to prevent fusion of myotubes with nonmyogenic cells such as muscle fibroblasts, endothelial cells, or nerve cells, but the nature of the signal is as yet unknown. Here we provide evidence that one of the selection mechanisms may be the enhanced affinity for laminin of myogenic cells as compared to fibrogenic cells. Growing myotubes in myoblast cultures accumulate laminin and type IV collagen on their surface in patches and strands as the first step in assembling a continuous basal lamina on mature myofibers (U. Kühl, R. Timpl, and K. von der Mark (1982), Dev. Biol. 93, 344-359). Fibronectin, on the other hand, assembles into an intercellular fibrous meshwork not associated with the free myotube surface. Over a brief time period (10-20 min) myoblasts from embryonic mouse thigh muscle adhere faster to laminin than do fibroblasts from the same tissue; these adhere faster to fibronectin. When a mixture of the cells is plated for 20 min on laminin/type IV collagen substrates, only myogenic cells adhere, giving rise to cultures with more than 90% fusion after 2 weeks; on fibronectin/type I collagen in the same time primarily fibroblastic cells adhere, giving rise to cultures with less than 10% nuclei in myotubes. The differential affinities of myoblasts for basement membrane constituents and of fibroblasts for interstitial connective tissue components may play a role in sorting out myoblasts from fibroblasts in skeletal muscle development.     

Multispecific DNA methyltransferases from Bacillus subtilis phages. Properties of wild-type and various mutant enzymes with altered DNA affinity

Temperate Bacillus subtilis phages SPR, phi 3T, rho 11 and SP beta code for DNA methyltransferases, each having multiple sequence specificities. The SPR wild-type and various mutant methyltransferases were overproduced 1000-fold in Escherichia coli and were purified by three consecutive chromatographic steps. The stable form of these multispecific enzymes in solution are monomers with a relative molecular mass (Mr) of about 50,000. The methyl-transfer kinetics of the SPR wild-type and mutant enzymes were determined with DNA substrates carrying either none or one of the three recognition sequences (GGCC, CCGG, CCATGG). Evaluation of the catalytic properties for DNA and S-adenosylmethionine binding suggested that the NH2-terminal part of the protein is important for both non-sequence-specific DNA binding and S-adenosylmethionine binding as well as transfer of methyl groups. On the other hand, mutations in the COOH-terminal part lead to weaker site-specific interactions of the enzyme. Antibodies raised against the purified SPR enzyme specifically immunoprecipitated the phi 3T, rho 11 and SP beta methyltransferases, bu failed to precipitate the chromosomally coded enzymes from B. subtilis (BsuRI) and B. sphaericus (BspRI). Immunoaffinity chromatography is an efficient purification step for the related phage methyltransferases.     

Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system

Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.     

Covalent labelling of histones with aurothiomalate. Gold-labelled H2A-H2B dimers assemble to octamers, which form crystalline helical tubes

Histone octamers were covalently labelled with aurothiomalate at amino groups by the method of carbodiimide activation. The labelling procedure was demonstrated to result in the specific covalent coupling through a single bond of the heavy metal atom label to protein amino groups. Such octamers were dissociated to yield soluble H2A-H2B dimers containing three gold atoms per dimer. The dimers were reconstituted with native H3-H4 tetramers to form labelled octamers, which were crystallized to form helical tubes. This strongly suggests that this procedure resulted in minimal changes of protein conformation.     

Energy dependence of protein translocation into chloroplasts

The translocation of in vitro synthesized precursor proteins into intact spinach chloroplasts was investigated with respect to its energy requirement. It was demonstrated that MgATP itself, and not a transmembrane electrochemical gradient across the envelope membrane, promotes protein import. By manipulating the external and the stromal level of MgATP, we provided evidence that MgATP energized the protein import not within the chloroplast but at the outside of the envelope membrane. It is postulated that an MgATP-dependent phosphorylation/dephosphorylation cycle at the outer membrane face was involved in the course of protein translocation into the chloroplast.     

Cardiac sarcoplasmic reticulum contains a low-affinity site for phenylalkylamines

The distribution of the bovine cardiac binding sites for the organic calcium-channel blockers was studied. Crude microsomal membranes were separated into three fractions, which contained mainly membranes derived from sarcolemma, 'junctional' sarcoplasmic reticulum containing transversal tubuli, and free sarcoplasmic reticulum. The high-affinity binding site for the dihydropyridines, determined in the presence of nitrobenzylthioinosine, was enriched 12-fold and 17-fold in sarcolemma and junctional sarcoplasmic reticulum. The binding sites for the phenylalkylamines, determined with [3H]verapamil or [3H](-)desmethoxyverapamil, were enriched 1.5-3.4-fold in sarcolemma and junctional sarcoplasmic reticulum but 6-10-fold in free sarcoplasmic reticulum. The phenylalkylamine-binding site, present in free sarcoplasmic reticulum, was partially destroyed by chymotrypsin or phospholipase A2 and C treatment. Specific binding was proportional to the concentration of the added membrane protein. The binding of (-)desmethoxyverapamil was half-maximally inhibited by 6.5 mM calcium chloride and was optimal in the presence of 5 mM EGTA. In three out of five preparations (-)desmethoxyverapamil bound to a single site with an apparent Kd value of 191 +/- 42.8 nM and a density of 34.5 +/- 7.7 pmol/mg protein. In two out of five preparations an additional high-affinity site (Kd approximately 0.67 nM) was detected. The low-affinity site bound other phenylalkylamines, but stereospecific binding of phenylalkylamines was not observed. Binding of phenylalkylamines to the low-affinity site was inhibited by some but not all calmodulin 'antagonists'. Furthermore dihydropyridines did not affect the binding of (--)desmethoxyverapamil suggesting that the low-affinity site differs considerably from the high-affinity sarcolemmal site. These results suggest that free sarcoplasmic reticulum contains a binding site for phenylalkylamines at a relative high density, which is not related to the high-affinity site present in the voltage-dependent calcium channel.     

Properties of the complexes of riboflavin 3',5'-bisphosphate and the apoflavodoxins from Megasphaera elsdenii and Desulfovibrio vulgaris

Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3',5'-bisphosphate. Several biochemical and biophysical properties of the complexes have been investigated and the results are compared with the properties of the native proteins. The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for the native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone-hydroquinone transition is about 20 mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein-bound artificial prosthetic group and the protein-bound natural prosthetic group are very similar. In both the oxidized and in the fully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15N NMR. 31P-NMR studies show that the 5'-phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3'-phosphate group in riboflavin 3',5'-bisphosphate is monoanionic or even neutral when bound to the protein. The 3'-phosphate group is also close to or on the surface of the protein. Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3',5'-bisphosphate which is 10 times higher as compared to Megasphaera elsdenii apoflavodoxin (Ka = 10(8) M-1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3'5'-bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3',5'-bisphosphate analog gives a value (Kd approximately equal to 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649-655]. The results are discussed in the light of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins and flavoproteins, pp. 493-496, Walter de Gruyter, Berlin].     

Steroid sulfatase. Biosynthesis and processing in normal and mutant fibroblasts

Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis of this enzyme in human skin fibroblasts. Steroid sulfatase is synthesized as a membrane-bound Mr-63 500 polypeptide with asparagine-linked oligosaccharide chains. Within 2 days, newly synthesized steroid sulfatase is processed to a mature Mr-61 000 form. The decrease in size is due to processing of the oligosaccharide chains, which are cleavable by endoglucosaminidase H in both the early and the mature form of steroid sulfatase. The processing involves mannosidase(s) sensitive to 1-deoxy-manno-nojirimycin. The half-life of the steroid sulfatase polypeptides is 4 days. Synthesis of steroid-sulfatase-related polypeptides and steroid sulfatase activity were not detectable in fibroblasts from four patients with X-linked ichthyosis.     

The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits

A binding site for the channel-blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the alpha-, beta- and delta-chains of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata electric tissue. The photolabel was found in homologous positions of the highly conserved sequence helix II, alpha 248, beta 254, and delta 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position delta 262 independent of whether photolabeling was performed with the receptor in its resting, desensitized or antagonist state. A model of the AChR ion channel is proposed, according to which the channel is formed by the five helices II contributed by the five receptor subunits.     

Platelet-activating factor mobilizes intracellular calcium in vascular smooth muscle cells

The effect of platelet-activating factor (PAF) on polyphosphoinositide metabolism and 45Ca2+ efflux was examined in a vascular smooth muscle cell line (A7r5). PAF stimulated a rapid but transient production of inositol trisphosphate and inositol bisphosphate which, in the presence of lithium, resulted in an accumulation of inositol monophosphate. In addition, PAF induced a rapid efflux of 45Ca2+ from preloaded cells, an effect which was concentration-dependent. These data suggest that PAF mobilizes intracellular Ca2+ via the production of inositol trisphosphate.