Hydroxyproline-rich cell wall protein (extensin): role in the cessation of elongation in excised pea epicotyls

The synthesis and accumulation of cell wall hydroxyproline increases coincident with the cessation of elongation growth in pea epicotyls. We examined the relationship between these biochemical and physiological events by using epicotyl sections challenged with α, α′-dipyridyl. This chelator blocked hydroxyproline biosynthesis without affecting overall protein synthesis. Epicotyl sections mimicked elongation growth in situ when placed in indoleacetic acid. Elongation was blocked by the addition of benzimidazole or Ethrel. These latter compounds acted independently as judged by their kinetics of action and the inhibition of Ethrel's effect only by CO₂.During rapid elongation growth in indoleacetic acid, there was no increase in cell wall hydroxyproline. However, incubation in either growth-inhibitory agent increased hydroxyproline 3-fold. When this increase was blocked by dipyridyl incubation, growth was not inhibited in benzimidazole or Ethrel, but proceeded at the maximal rate. During long-term incubations in buffer, cell wall hydroxyproline increased and the sections eventually became unable to grow. However, if dipyridyl was added to block the hydroxyproline increase, growth potential remained. Elongation was inhibited by supraoptimal concentrations of indoleacetic acid. However, such inhibition did not occur in the presence of dipyridyl.These results indicate that an hydroxyproline-containing component is necessary in rendering the cell wall inextensible when elongation growth ceases.     

Biosynthesis of alkanes by particulate and solubilized enzyme preparations from pea leaves (Pisum sativum)

Enzymatic activity responsible for the conversion of fatty acids to alkanes catalyzed by pea leaf homogenate was found to be mainly in the microsomal fraction. This particulate preparation catalyzed alkane formation from n-C18, n-C22, and n-C24 acids at rates comparable to that observed with n-C32 acid with O2 and ascorbate as required cofactors. In each case the major alkane contained two carbon atoms less than the precursor acid. Since the preparation also catalyzed alpha-oxidation, it was suspected that some alpha-oxidation intermediate, with one less carbon atom than the substrate acid, might lose another carbon to generate the alkane. Thin-layer and radio-gas-liquid chromatographic analysis of the products generated from [U-14C]stearic acid by the particulate preparation after different periods of incubation showed that, at all time periods, alpha-hydroxy C18 acid, C17 aldehyde, and C17 acid were the major products. Since C16 alkane was the major product even after short periods of reaction, the C17 aldehyde might have been the immediate precursor of the alkane. Exogenous labeled C18 and C24 aldehyde were converted to alkanes. The alkane-synthesizing activity was solubilized from the microsomal preparation using Triton X-100. The solubilized preparation was retarded in a Sepharose 6-B column, but the hydrocarbon-forming activity was not resolved from alpha-oxidation. The solubilized preparation produced alkane with two carbon atoms less than the parent acid in a time- and protein-dependent manner. The soluble preparation also required O2 and ascorbate and, like the microsomal preparation, was inhibited by dithioerythritol and metal ion chelating agents.     

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.     

Evidence for peptidoglycan-associated protein(s) in Neisseria gonorrhoeae.

Growth pH markedly influenced the composition of the cell envelope of $\text{Neisseria gonorrhoeae}$. The composition of the peptidoglycan from cells grown at pH 7.2 and 8.0 consisted primarily (91%) of muramic acid, glutamic acid, alanine, $\text{meso}$-diaminopimelic acid, and glucosamine in approximate molar ratios of 1:1:2:1:1. The peptidoglycan from cells grown at pH 6.0 contained an accessory protein(s) which accounted for 42% of the weight of the isolated complex.     

Biosynthesis of glycoproteins in human placenta: Processing of oligosaccharides

The processing of the high-mannose asparagine-linked oligosaccharides synthesized by first-trimester human placenta has been investigated. Tissue was pulsed for 1 h with [2-³H]mannose and chased for zero, 45, 90, and 180 min in media containing unlabeled mannose. Glycopeptides, prepared by Pronase digestion of the delipidated membrane pellets at each time point, were treated with endo-β-N-acetylglucosaminidase-H to release the high-mannose asparagine-linked oligosaccharides. The largest major processing intermediate isolated was Glc₁Man₉GlcNAc, which was converted into Man₉GlcNAc, and then into Man₈GlcNAc, Man₇GlcNAc, Man₆GlcNAc, and Man₅GlcNAc. There was also a minor pathway in which mannosyl residues were removed prior to the glucose. By carrying out the detailed structural characterization of the individual processing intermediates, it was possible to demonstrate that processing of the Man₉GlcNAc to Man₅GlcNAc proceeded by the nonrandom removal of the α1,2-linked mannosyl residues. Specifically, of 12 possible sequences of removal of the four α1,2-linked mannosyl residues present in Man₉GlcNAc, first-trimester human placenta utilized only two of these in the processing of asparagine-linked oligosaccharides. It is suggested that the limited number of processing pathways reflects a high degree of specificity of these reactions in human placenta.     

Regulation of protein synthesis and accumulation during oogenesis in Xenopus laevis

The tissue and developmental distribution of the various myosin subunits has been examined in bovine cardiac muscle. Electrophoretic analysis shows that a myosin light chain found in fetal but not in adult ventricular myosin is very similar and possibly identical to the light chain found in fetal or adult atrial and adult Purkinje fiber myosins. This light chain comigrates on two-dimensional gels with the bovine skeletal muscle embryonic light chain. Thus, this protein appears to be expressed only at early developmental stages in some tissues (cardiac ventricles, skeletal muscle) but at all stages in others (cardiac atria). The heavy chains of these myosins have been examined by one- and two-dimensional polypeptide mapping. The ventricular and Purkinje fiber heavy chains are indistinguishable. They are, however, different from the heavy chain found in cultured skeletal muscle myotubes, in contrast to the situation concerning the embryonic/atrial light chain.     

Malonyl-coenzyme A:isoflavone 7-O-glucoside-6"-O-malonyltransferase from roots of chick pea (Cicer arietinum L.)

A malonyltransferase which catalyzes the malonylation of isoflavone 7-O-glucosides in position 6 of the glucose moiety using malonyl-coenzyme A as acyl donor has been purified 157-fold from 4-day-old roots of chick pea (Cicer arietinum L.). The enzyme showed a pH optimum of 8.0 and a molecular weight of 112,000. The Km for malonylcoenzyme A was 48 microM and, for the chick pea isoflavones biochanin A and formononetin, 36 and 24 microM, respectively. Various other isoflavone, flavone, and flavonol 7-O-glucosides and chalcone 4'-O-glucosides were much poorer substrates. Flavonol 3-O-glucosides and isoflavone 4'-O-glucosides were not malonylated by the malonyltransferase.     

Amino acid transport in kidney epithelial cell line (MDCK): characteristics of Na+/amino acid symport in membrane vesicles and basolateral localization in cell monolayers

Na+-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na+-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na+-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na+-stimulated MeAIB transport activity and Na+,K+,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na+-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na+-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na+ dependence of amino acid transport in vesicles suggested a Na+ symport mechanism with a 1:1 stoichiometry between Na+ and amino acid.     

Enhanced natural killer (NK) cell activity and NK-sensitive thymic cells in murine muscular dystrophy

Studies have shown that there is an abnormality in the thymus of dystrophic mice with respect to age-dependent thymus weight changes and altered morphology (T. DeKretser and B. Livett, Nature (London), 263, 682, 1976). Recently, others have shown that natural killer (NK) cells can lyse cells of a large, immature, rapidly dividing cell subpopulation within the thymus of normal young (3 weeks of age) mice (M. Hansson, K. Karre, R. Kiessling, J. Roder, B. Anderson, and P. Hayry, J. Immunol., 123, 765, 1979). The NK susceptibility of dystrophic mouse thymocytes as targets was therefore studied. Spleen cells from normal (+/+) and dystrophic ($\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$) male C57BL/6J mice 8–10 weeks old were passed over nylon wool and the nonadherent cells were incubated with ⁵¹Cr-labeled YAC-1 lymphoma target cells or thymocytes in a ⁵¹Cr-release assay. Spleen cells from dystrophic mice killed twofold more YAC-1 target cells than did spleen cells from normal mice. Thymocytes from 3- to 4-week-old dystrophic mice were three to four times more susceptible to NK lysis by dystrophic mouse spleen cells as compared with normal mouse spleen cells. Spleen cells from dystrophic mice had the same NK activity against dystrophic and normal mouse thymocytes as targets. Normal mouse spleen cells killed three- to fourfold more dystrophic mouse thymocytes than that of normal mouse thymocytes as targets. Target cellbinding studies revealed that conjugate-forming cells from nylon nonadherent dystrophic mouse spleen cells were found to be two- to fourfold greater than for normal mouse spleen cells using YAC-1 tumor cells as targets. The number of lymphocytes bound per YAC-1 target cell ranged from 2 to 5 for dystrophic mouse spleen cells as compared with 1 to 2 for the normal control group. Using both normal and dystrophic mouse thymocytes as targets, the conjugate-forming cells from dystrophic mouse spleen cells were also found to be twofold greater than in the normal control group. Cold target inhibition studies revealed that the natural killing of dystrophic mouse thymocytes was due to a YAC-1-reactive NK cell. Effector cell depletion studies using monoclonal anti-Thy-1.2 plus complement treatment and plastic petri dish adherence also revealed that the natural killing of dystrophic mouse thymocytes was not due to either T lymphocytes or macrophages. Taken together, these results show an increase in NK-sensitive thymocyte targets in dystrophic mice, in combination with an increase in splenic NK activity.     

Rise and fall of protein phosphorylation during meiotic maturation in oocytes of Sabellaria alveolata (polychaete annelid)

Incorporation of [32P]phosphate into proteins was monitored, in preloaded Sabellaria oocytes, during meiosis. After a fourfold increase during the transition from prophase to metaphase I, the incorporated radioactivity decreased steadily by 25% during completion of meiosis, while it slowly increased in metaphase I-blocked oocytes. Measurements of the amount and specific activity of nucleotide pools showed no variation, while total alkali-labile protein-bound phosphate was found to increase and then decrease during meiosis. Autoradiography of sodium dodecyl sulfate slabgels showed that some proteins have peculiar phosphorylation-dephosphorylation kinetics. The changes in the level of phosphorylation of proteins may be related to similar changes in maturation-promoting factor (MPF) activity.     

Identification of enzymatic activities which process protein bound mono(ADP-ribose)

Enzymatic activities have been identified in extracts of cultured mouse cells which catalyze the removal of intact mono(ADP-ribosyl) residues linked to proteins at arginine. Activities that sequentially remove AMP and ribose 5-phosphate have also been identified. These results suggest that mono(ADP-ribosylation) of proteins is a reversible post translational modification.     

Biosynthesis of monoterpenes. Partial purfication and characterization of a bicyclic monoterpenol dehydrogenase from sage (Salvia officinalis)

Galactose 1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose 1-phosphate uridylyltransferase, EC 2.7.7.12) was isolated from human red cells by DEAE-cellulose and hydroxylapatite chromatography. The enzyme consists. of two similar subunits of molecular weight 44,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was found to be 67,000 by Sephadex G-200 chromatography and 88,000 by ultracentrifugation studies in sucrose density gradients. The specific activity of the purified enzyme was about 40 μmoles per min per mg of protein.     

Studies on bacterial cell wall inhibitors: II. Inhibition of peptidoglycan synthesis in vivo and in vitro by amphomycin

Amphomycin has been reported by the present authors to be a selective inhibitor of cell wall peptidoglycan synthesis in Bacillus cereus T (ōmura, S., Tanaka, H., Shinohara, M., ōiwa, R. and Hata, T. (1975) Chemotherapy 5, 365–369). Investigations were carried out to clarify the target of amphomycin.Amphomycin (10 μg/ml) lysed growing cells of B. cereus T, and inhibited peptidoglycan synthesis, accompanied by accumulation of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc) peptides. The nucleotide precursors that accumulated in cells of Staphylococcus aureus FDA 209P in the presence of amphomycin were identified as UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala, UDP-MurNAc-L-Ala and UDP-MurNAc. In the experiments using a particulate enzyme system of Bacillus megaterium KM, amphomycin inhibited the polymerization of UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine, and also inhibited the formation of lipid intermediates, but did not inhibit the cross-linking, the last step of peptidoglycan synthesis. Unlike bacitracin, amphomycin did not lyse protoplasts of B. megaterium KM.We conclude that the site of action of amphomycin is the formation of MurNAc-(pentapeptide)-P-P-lipid from MurNAc-pentapeptide and undecaprenol (lipid) phosphate.     

In vitro modulation of mouse natural killer (NK) cell activity by the serum thymic factor (FTS)

Previous studies indicated that the serum thymic factor (FTS) could modulate in vivo the level of splenic natural killer (NK) cell activity in mice. The present report shows that such an effect is also observed after a short term in vitro incubation of the effector cells with FTS. The regulatory effects of FTS result in an increase or a decrease of the splenic NK cell cytotoxicity depending upon the age and the mouse strain. Furthermore, FTS is able to enhance the NK cell activity of thymus and bone marrow cells which are known to be weakly reactive in NK cytotoxicity. Depletion experiments demonstrated that the FTS-induced increase of NK cell activity was not mediated by Thy 1+ cells nor macrophages, thus suggesting a direct action of FTS on the effector cells. Comparative studies using other thymic hormones revealed similar patterns of reactivity. These results favor the hypothesis of a close relationship between the thymus and NK cells.     

Biosynthesis of medium chain fatty acids in Drosophila melanogaster

Adult Drosophila melanogaster synthesizes dodecanoic and tetradecanoic acids in vivo, along with the more common 16- and 18-carbon fatty acids. The radiolabeled C12 and C14 fatty acids synthesized from sodium [1-14C]acetate are found primarily in the diacylglycerol and triacylglycerol fractions. Partially purified fatty acid synthetase (FAS) synthesizes C14, C16, and C18 fatty acids (as the free acids) at 0.2 M ionic strength. Increasing the ionic strength to 2.0 M causes partially purified FAS to synthesize primarily C12 and C14 fatty acids. Addition of aliquots of the microsomal pellet and other soluble protein fractions does not alter the pattern of fatty acids synthesized by FAS. The percentage of C12 and C14 fatty acids synthesized at high ionic strength by individual fractions from the FAS peak (Sepharose 6B column) is constant across the peak. None of the soluble protein fractions is able to relieve the inhibition of FAS by phenylmethylsulfonyl fluoride. These results indicate that the FAS of D. melanogaster has the inherent capability to form C12 and C14 fatty acids and that no other soluble protein appears to be involved in their synthesis.     

Kinetic studies of fructokinase I of pea seeds

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.     

Interaction between heparin and human pregnancy-associated plasma protein A (PAPP-A): a simple purification procedure

Human pregnancy-associated plasma protein A (PAPP-A) binds to heparin-Sepharose. This affinity chromatography preceded by molecular sieve chromatography provides a simple two-step purification procedure of PAPP-A from late pregnancy plasma. One hundred percent of the applied PAPP-A was recovered, with more than 40% being electrophoretically homogeneous after the two procedures. The remaining PAPP-A could be purified by negative affinity chromatography on anti-total human serum immobilized on agarose.