Engineering and mechanistic studies of the Arabidopsis FAE1 beta-ketoacyl-CoA synthase, FAE1 KCS.

The Arabidopsis FAE1 beta-ketoacyl-CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N-terminal membrane-spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His6-tag at their N-terminus, and expressed in Saccharomyces cerevisiae. The membrane-bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild-type recombinant FAE1 KCS was active with several acyl-CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl-CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3-(14)C]malonyl-CoA, indicating that it requires binding of the acyl-CoA for decarboxylation activity. Site-directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl-CoA even when 18:1-CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1-KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane-bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase.

A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals.     

Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.     

Modulation of adenylate cyclase activity of fibroblasts by free fatty acids and phospholipids

The effect of certain lipids on adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from fibroblasts in culture has been investigated. The unsaturated fatty acids, as well as lysolecithin, were found to act as potent inhibitors of fibroblast adenylate cyclase activity. Increasing the degree of unsaturation increases the extent of inhibition noted at a given fatty acid concentration. The inhibitory effect of the unsaturated fatty acids or lysolecithin is not selective for a specific function of the adenylate cyclase system since basal, and hormone- or fluoride-stimulated cyclase activities are inhibited to the same extent. The fatty acid-inactivated state of fibroblast adenylate cyclase is not readily reversed for enzyme activity is not restored when arachidonate-treated membranes are washed with Tris buffer containing 10 mm EDTA, 0.15 mm albumin, or 0.15 m KCl. Previous studies have shown that the adenylate cyclase system from Moloney sarcoma virus-transformed NRK (MNRK) cells is not stimulated by the addition of GTP or hormones. Of interest is the present finding that the addition of unsaturated fatty acids, or lysolecithin, over a narrow concentration range (0.1 – 0.2 mm) leads to partial restoration of GTP activation of MNRK cyclase activity. Hormonal responsiveness to l-epinephrine or prostaglandin E₁ is not restored to the MNRK enzyme with fatty acid or lysolecithin treatment.     

Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo.

The influence of topoisomerase I and gyrase mutations in Escherichia coli on the supercoiled density of recombinant plasmids and the stability of left-handed Z-DNA was investigated. The formation of Z-DNA in vivo by dC-dG sequences of different lengths was used to determine the effective plasmid supercoil densities in the mutant strains. The presence of Z-DNA in the cells was detected by linking number and EcoRI methylase inhibition assays. A change in the unrestrained superhelical tension in vivo directly effects the B- to Z-DNA transition. Alterations in the internal or external environment of the cells, such as the inactivation of gyrase or topoisomerase I, a gyrase temperature-sensitive mutant, or starvation of cells, have a dramatic influence on the topology of plasmids. Also, E. coli has significantly more superhelical strain than Klebsiella, Morganella, or Enterobacter. These studies indicate that linking deficiency and effective supercoil density are mutually independent variables of plasmid tertiary structure. A variety of factors, such as protein-DNA interactions, activity of topoisomerases, and the resulting supercoil density, contribute to the B to Z transition inside living cells.     

Cytosine methylation enhances Z-DNA formation in vivo.

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Metabolism of polyadenylic acid sequences during germination of blastocladiella emersoni: Zoospores.

The metabolism of the poly(A) sequences isolated from Blastocladiella emersonii was followed during the first hour of germination. Poly (A) sequences synthesized during the first 30 min of germination do not undergo detectable changes in size. During the first 45 min of germination, poly(A) sequences synthesized during zoosporogenesis decrease in size to the extent that there is essentially no size overlap between poly(A) fragments which were present in the zoospore and newly synthesized poly(A) sequences. The results presented indicate that during germination, polyadenylation occurs in RNA molecules which were present in the zoospore but lacked poly(A) sequences. No detectable size differences were observed between poly(A) sequences added to newly synthesized RNA compared to those added to the nonpolyadenylated RNA present in the zoospore during germination. Cycloheximide did not prevent the observed decrease in size of the poly(A) sequences during germination.