Different Sets Of Enzymes Research Articles

Single Neuron Ubiquitin-Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Disease

The accumulation of mutant protein in intracellular aggregates is a common feature of neurodegenerative disease. In Huntington disease, mutant huntingtin leads to inclusion body (IB) formation and neuronal toxicity. Impairment of the ubiquitin-proteasome system (UPS) has been implicated in IB formation and Huntington disease pathogenesis. However, IBs form asynchronously in only a subset of cells with mutant huntingtin, and the relationship between IB formation and UPS function has been difficult to elucidate. Here, we applied single-cell longitudinal acquisition and analysis to monitor mutant huntingtin IB formation, UPS function, and neuronal toxicity. We found that proteasome inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB formation, the UPS is more impaired in neurons that go on to form IBs than in those that do not. After forming IBs, impairment is lower in neurons with IBs than in those without. These findings suggest IBs are a protective cellular response to mutant protein mediated in part by improving intracellular protein degradation.

Stanley James Holt

Stanley James Holt, professor emeritus of experimental biochemistry of the University College and Middlesex School of Medicine, London, died from cardiorespiratory insufficiency on 30 October 2008 at the age of...

Cell Wall-Related Genes Involved in Supporting Tissue Formation and Transcriptional Regulation in Arabidopsis thaliana

The characteristic growth pattern of vascular plants largely depends on the intrinsic properties of their cell walls, which are flexible, but strong enough to support the plant body. The plant body is composed of various tissues each with a specific cell wall type. Different sets of enzymes are required for the construction of these individual cell wall types. The cell wall type-specific enzyme-set hypothesis has been described to explain the mechanisms underlying cell wall construction. This hypothesis suggests that specific sets of transcription factors are required for the construction of each of the cell-wall types. Recent reverse genetic studies investigating secondary wall formation in Arabidopsis thaliana have demonstrated the existence of a hierarchical transcriptional network that governs the regulation of secondary wall formation in cell wall types. The examination of the effects of mechanical stimuli on the expression of genes encoding a particular set of cell wall-related enzymes and transcriptional factors has shown that A. thaliana is able to perceive subtle changes in self-weight of the aerial portions, and use this information as a signal to regulate formation of cell walls in the supporting tissues. However, the mechanisms by which mechanical signals are perceived via sensors presumably located at the cell surface remain unknown. In addition, the pathways through which the signal is transmitted and integrated into the transcriptional network that governs the coordinated actions of cell wall-related genes are also yet to be described. Current reverse genetic approaches based on comprehensive expression analysis of cell wall-related genes may aid in the elucidation of the regulatory mechanisms underlying supporting tissue formation via mechanical signals. Such information may contribute not only to a further understanding of the molecular basis underlying evolution of the plant vascular system, but may also provide us with the knowledge required for the future development and utilization of plant cell walls as a sustainable resource.

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

Enzymatic desymmetrization of 2,5-dideoxystreptamine precursors

The stereoselective enzymatic acylation of meso-6,7-diazabicyclo[3.2.1]oct-6-ene-2,4-diol 4 and meso-4,6-diazidocyclohexane-1,3-diol 5 in organic media gave the corresponding monoesters in high enantiomeric excess. The hydrolysis of the corresponding diacetate derivatives in the presence of a different set of enzymes provided the same monoesters. The products are precursors of dideoxystreptamine, an aminocyclitol found in synthetic aminoglycoside antibiotics.

A Novel Polymorphic Cytochrome P450 Formed by Splicing of CYP3A7 and the Pseudogene CYP3AP1

The cytochrome P450 3A7 (CYP3A7) is the most abundant CYP in human liver during fetal development and first months of postnatal age, playing an important role in the metabolism of endogenous hormones, drugs, differentiation factors, and potentially toxic and teratogenic substrates. Here we describe and characterize a novel enzyme, CYP3A7.1L, encompassing the CYP3A7.1 protein with the last four carboxyl-terminal amino acids replaced by a unique sequence of 36 amino acids, generated by splicing of CYP3A7 with CYP3AP1 RNA. The corresponding CYP3A7-3AP1 mRNA had a significant expression in liver, kidney, and gastrointestinal tract, and its presence was found to be tissue-specific and dependent on the developmental stage. Heterologous expression in yeast revealed that CYP3A7.1L was a functional enzyme with a specific activity similar to that of CYP3A7.1 and, in some conditions, a different hydroxylation specificity than CYP3A7.1 using dehydroepiandrosterone as a substrate. CYP3A7.1L was found to be polymorphic due to a mutation at position -6 of the first splicing site of CYP3AP1 (CYP3A7_39256T-->A), which abrogates the pseudogene splicing. This polymorphism had pronounced interethnic differences and was in linkage disequilibrium with other functional polymorphisms described in the CYP3A locus: CYP3A7*2 and CYP3A5*1. Therefore, the resulting CYP3A haplotypes express different sets of enzymes within the population. In conclusion, a novel mechanism, consisting of the splicing of the pseudogene CYP3AP1 to CYP3A7, causes the formation of the novel CYP3A7.1L having a different tissue distribution and functional properties than the parent CYP3A7 enzyme, with possible developmental, physiological, and toxicological consequences.

High Level Oxacillin and Vancomycin Resistance and Altered Cell Wall Composition in Staphylococcus aureus Carrying the Staphylococcal mecA and the Enterococcal vanA Gene Complex

Recently, for the first time in the history of this bacterial species, methicillin-resistant Staphylococcus aureus (MRSA) carrying the enterococcal vanA gene complex and expressing high level resistance to vancomycin was identified in clinical specimens (CDC (2002) MMWR 51, 565-567). The purpose of our studies was to understand how vanA is expressed in the heterologous background of S. aureus and how it interacts with the mecA-based resistance mechanism, which is also present in these strains and is targeted on cell wall biosynthesis. The vanA-containing staphylococcal plasmid was transferred from the clinical vancomycin-resistant S. aureus (VRSA) strain HIP11714 (CDC (2002) MMWR 51, 565-567) to the methicillin-resistant S. aureus (MRSA) strain COL for which extensive genetic and biochemical information is available on staphylococcal cell wall biochemistry and drug resistance mechanisms. The transconjugant named COLVA showed high and homogeneous resistance to both oxacillin and vancomycin. COLVA grown in vancomycin-containing medium produced an abnormal peptidoglycan: all pentapeptides were replaced by tetrapeptides, and the peptidoglycan contained at least 22 novel muropeptide species that frequently showed a deficit or complete absence of pentaglycine branches. The UDP-MurNAc-pentapeptide, the major component of the cell wall precursor pool in vancomycin-sensitive cells was replaced by UDP-MurNAc-depsipeptide and UDP-MurNAc-tetrapeptide. Transposon inactivation of the beta-lactam resistance gene mecA caused complete loss of beta-lactam resistance but had no effect on the expression of vancomycin resistance. The two major antibiotic resistance mechanisms encoded by mecA and vanA residing in the same S. aureus appear to use different sets of enzymes for the assembly of cell walls.

Microtubule sensors and sensor array based on polyaniline synthesized in the presence of poly(styrene sulfonate).

Microtubule sensors for glucose, urea, and triglyceride were fabricated based on poly(styrene sulfonate)-polyaniline (PSS-PANI) composites synthesized within the pores of track-etched polycarbonate membranes. The synthesis of a sufficiently thick and conducting PSS-PANI film at pH 5 provided the advantage of immobilizing enzymes during polymerization. This resulted in the improvement of sensor response for urea and triglyceride by a factor of approximately 10(2) with a significant increase in the linear region of response compared to polyaniline-based sensors, where the enzymes were immobilized by physical adsorption after the polymerization. The sensors based on urea and triglyceride were found to have a higher linear range of response, better sensitivity, improved multiple use capability, and faster response time compared to the potentiometric and amperometric sensors based on polyaniline. A microtubule sensor array for glucose, urea, and triglyceride based on PSS-PANI was fabricated by immobilization of three different sets of enzymes on three closely spaced devices and its response was found to be free from cross-interference when a sample containing a mixture of the above analytes was analyzed in a single measurement.

Chain initiation on the soraphen-producing modular polyketide synthase from Sorangium cellulosum

Background: Polyketides are structurally diverse natural products with a wide range of useful activities. Bacterial modular polyketide synthases (PKSs) catalyse the production of non-aromatic polyketides using a different set of enzymes for each successive cycle of chain extension. The choice of starter unit is governed by the substrate specificity of a distinct loading module. The unusual loading module of the soraphen modular PKS, from the myxobacterium Sorangium cellulosum, specifies a benzoic acid starter unit. Attempts to design functional hybrid PKSs using this loading module provide a stringent test of our understanding of PKS structure and function, since the order of the domains in the loading and first extension module is non-canonical in the soraphen PKS, and the producing strain is not an actinomycete. Results: We have constructed bimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only extension modules 1 and 2 and a thioesterase (TE) domain, by substituting one or more domains from the soraphen PKS. A hybrid PKS containing the soraphen acyltransferase domain AT1b instead of extension acyltransferase domain AT1 produced triketide lactones lacking a methyl group at C-4, as expected if AT1b catalyses the addition of malonyl-CoA during the first extension cycle on the soraphen PKS. Substitution of the DEBS1-TE loading module AT domain by the soraphen AT1a domain led to the production of 5-phenyl-substituted triketide lactone, as well as the normal products of DEBS1-TE. This 5-phenyl triketide lactone was also the product of a hybrid PKS containing the entire soraphen PKS loading module as well as part of its first extension module. Phenyl-substituted lactone was only produced when measures were simultaneously taken to increase the intracellular supply of benzoyl-CoA in the host strain of Saccharopolyspora erythraea. Conclusions: These results demonstrate that the ability to recruit a benzoate starter unit can be conferred on a modular PKS by the transfer either of a single AT domain, or of multiple domains to produce a chimaeric first extension module, from the soraphen PKS. However, benzoyl-CoA needs to be provided within the cell as a specific precursor. The data also support the respective roles previously assigned to the adjacent AT domains of the soraphen loading/first extension module. Construction of such hybrid actinomycete–myxobacterial enzymes should significantly extend the synthetic repertoire of modular PKSs.

Old antiseptic, new use

Rima McLeod, et al (Int. J. Parasitol., February) report on the use of Triclosan, a common antiseptic used in household products such as toothpaste, skin creams and deodorants, to kill the parasites responsible for malaria and toxoplasmosis. Triclosan has been used as an antiseptic since the 1960s and blocks an enzyme, known as ‘FabI’ that bacteria need to manufacture the fatty acids used in cell membranes. Because animals possess a very different set of enzymes, triclosan does not interfere with this process in humans. The malarial FabI has the same pattern of critical amino acids that bind triclosan as the plant and bacterial variants. D.M.

Repair of oxidative DNA damage: mechanisms and functions.

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2-6-nucleotide patch size) pathways need AP endonuclease to generate a 3' hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.

Polyphosphate and phosphate pump.

In microbial cells, inorganic polyphosphate (polyP) plays a significant role in increasing cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. polyP is a polyfunctional compound. The most important of its functions are the following: phosphate and energy reservation, cation sequestration and storage, membrane channel formation, participation in phosphate transport, involvement in cell envelope formation and function, gene activity control, regulation of enzyme activities, and a vital role in stress response and stationary-phase adaptation. The functions of polyP have changed greatly during the evolution of living organisms. In prokaryotes, the most important functions are as an energy source and a phosphate reserve. In eukaryotic microorganisms, the regulatory functions predominate. Therefore, a great difference is observed between prokaryotes and eukaryotes in their polyP-metabolizing enzymes. Some key prokaryotic enzymes are not present in eukaryotes, and conversely, eukaryotes have developed new polyP-metabolizing enzymes that are not present in prokaryotes. The synthesis and degradation of polyP in each specialized organelle and compartment of eukaryotic cells are mediated by different sets of enzymes. This is consistent with the endosymbiotic hypothesis of eukaryotic cell origin.

TCDD Induces CYP1A4 and CYP1A5 in Chick Liver and Kidney and Only CYP1A4, an Enzyme Lacking Arachidonic Acid Epoxygenase Activity, in Myocardium and Vascular Endothelium

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other Ah receptor ligands, species differences in sensitivity and the relationship of CYP1A induction to the toxicity, are poorly understood. Ah receptor ligands induce formation of CYP1A1 and 1A2 in mammals and of a different set of enzymes, CYP1A4 and 1A5, in chicks. We examined induction by TCDD of CYP1A4 and 1A5 mRNA and protein in chick embryo liver, heart, kidney, lung, intestine, bursa, spleen, thymus, brain, and muscle by in situ hybridization and immunohistochemistry and verified the histochemical findings by CYP-specific assays, 7-ethoxyresorufin deethylase for CYP1A4 and arachidonic acid epoxygenation for CYP1A5. CYP1A4 alone was extensively induced in the cardiovascular system, in cardiac myocytes, in perivascular cells having the same location as impulse-conducting Purkinje cells, and like CYP1A1, in vascular endothelium in every organ examined. Unlike mammalian CYP1A, CYP1A4 and 1A5 were both substantially induced in kidney proximal tubules as well as liver, and neither enzyme was induced in kidney glomeruli or lung or brain parenchymal cells. The findings demonstrate (a) a route for CYP1A4 to affect cardiac function, (b) that vascular endothelium is a major site of CYP1A induction across species, and (c) that CYP1A induced in heart or endothelial cells cannot affect cardiac or vascular function via generation of arachidonic acid epoxides because the CYP1A enzymes induced in those organs are not arachidonic acid epoxygenases. Further, the specificity of CYP1A induction sites and of the catalytically active enzymes induced at each site support a significant role for CYP1A induction in Ah receptor ligand toxicity and species differences in sensitivity.

Partial characterization of a major autolysin from Mycobacterium phlei.

Autolytic enzyme profiles of fast- and slow-growing mycobacteria were examined using SDS-PAGE zymography with incorporated mycobacterial peptidoglycan sacculi as substrate. Each species tested (Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium kansasii) appeared to produce a different set of enzymes on the basis of differing number and molecular masses. A major autolysin from M. phlei was purified to apparent homogeneity by DEAE-cellulose chromatography, preparative gel electrophoresis and Mono Q FPLC. This enzyme had an estimated molecular mass of 38 kDa, an isoelectric point of 5.5 and a pH optimum of pH 7.5. Digestion of purified peptidoglycan by the enzyme resulted in the appearance of reducing sugars, suggesting that the 38 kDa autolysin is a beta-glycosidase. Partial internal amino acid sequence of the autolysin was determined and should facilitate identification, cloning and overexpression of the encoding gene.

Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase.

Modular polyketide synthases (PKSs) catalyse the biosynthesis of complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic diketides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide synthase (DEBS 1-TE) to study chain extension starting from a variety of diketide analogues and, in some cases, from the alternative acyl-CoA thioester substrates. Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural variation in the starter unit of the synthetic diketide, but other changes completely abolished activity. Interestingly, a racemic beta-keto diketide was found to be reduced in situ on the PKS and utilised in place of its more complex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading module. Significantly higher concentrations of diketide N-acetylcysteamine analogues than their corresponding acyl-CoA thioesters are required to achieve comparable yields of triketide lactones. Although a broad range of variation in the starter residue is acceptable, the substrate specificity of module 2 of a typical modular PKS in vitro is relatively intolerant of changes at C-2 and C-3. This will restrict the usefulness of approaches to synthesise novel erythromycins using synthetic diketides in vivo. The use of synthetic beta-keto diketides in vivo deserves to be explored.

DIVERSITY OF HALOGENATED SECONDARY METABOLITES IN THE RED ALGA LAURENCIA NIPPONICA (RHODOMELACEAE, CERAMIALES)1,2

ABSTRACTMany morphologically similar, but chemically distinct, populations have been found in the marine red alga Laurencia nipponica Yamada (Rhodomelaceae, Ceramiales) growing in Japan. Each chemical type is characterized by a specific end‐product of halogenated secondaly metabolite synthesis: chamigrane‐type sesquiterpenoids such as prepacifenol and halochamigrene epoxide and C15 bromoethers such as laurencin, laureatin, isoprelaurefucin, epilaurallene, and kumausallene. These seven types of secondary metabolite syntheses remained the same in the wild and under various culture conditions. Because bromoethers and terpenoids are probably synthesized by different metabolic pathways, it is virtually certain that different sets of enzymes participate in their synthesis. Prepacifenol‐ and laureatin‐producing populations were selected as representatives of terpenoid and bromoether groups, respectively. F1 tetrasporophytes derived from crosses between reciprocal, female and male gametophytes of prepacifenol‐ and laureatin‐producing strains bore both types of metabolites, suggesting that the genes Producing these enzyme systems are encoded by nuclear genomes. The F1 gametophytes resulting from the reciprocal crosses produced either prepacifenol or laureatin, and the four individuals derived from spore tetrads (a set of tetraspores derived from a single tetrasporangium) produced either prepacifenol or laureatin in a 1:1 ratio, indicating that genes participating in terpenoid synthsis and those participating in bromoether synthesis are on different loci of homologous chromosomes and are segregated at meiosis (tetrasporogenesis). One individual of this interpopulational F1 gamtophyte produced both parental types of metabolite, perhaps indicating the occurrence of a recombination type. Natural hybrid individuals, including such recombination‐type gametophytes, were found in a sympatric locality at which these two chemical types occur. F1 tetrasporophytes derived from crosses between respective prepacifenol‐ and laureatin‐producing strains and their F1 gametohytes produced only parental‐type metabolite‐producing plants. These results indicate that the diverse chemical types can be referred to as races (chemical races).

Differences in the Acute Actions of sGnRH and cGnRH-II on Gonadotropin Release in Goldfish Pituitary Cells

The signal transduction mechanisms mediating the acute actions of salmon gonadotropin (GTH)-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II) on GTH release from goldfish pituitary cells were compared. In cell column perifusion experiments, treatment with inhibitors of phospholipase A 2 (50 μ M quinacrine or 50 μ M bromophenacylbromide) or the lipoxygenase enzyme (50 μ M nordihydroguaiaretic acid) reduced the GTH response to 100 n M sGnRH, but not the response to 100 n M cGnRH-II. These results suggest that AA mobilization through phospholipase A 2 and the subsequent metabolism of AA through the lipoxygenase pathway are involved in rapid sGnRH-induced GTH secretion, but not in acute cGnRH-II action. Consistent with the idea that calcium entry through voltage-sensitive calcium channels is involved in acute GnRH action, perifusion with 1 μ M verapamil, a voltage-sensitive calcium channel inhibitor, reduced both 100 n M sGnRH- and 100 n M cGnRH-II-induced GTH secretion. However, the response to cGnRH-II was decreased to a greater extent compared to sGnRH-elicited release, suggesting a greater dependence on extracellular calcium entry for acute cGnRH-II-stimulated GTH secretion. The metabolism of inositol phosphates (InsPs) following acute sGnRH and cGnRH-II administration was also investigated by monitoring the levels of [ 3H]InsPs in [ 3H]inositol-prelabeled goldfish pituitary cells. Incubation with 100 n M sGnRH increased [ 3H]InsP 1 by 5 min and [ 3H]InsP 2, [ 3H]InsP 3, and other higher [ 3H]InsPs by 10 min. In contrast, following 10 min of stimulation by 100 n M cGnRH-II, only [ 3H]InsP 2 levels were elevated, suggesting that cGnRH-II may activate a different set of enzymes in the phosphoinositide metabolic pathways compared to sGnRH. The lack of an InsP 3 response may also reflet the relative ineffectiveness of cGnRH-II to mobilize calcium from intracellular stores. Taken together, these results strongly indicate that the mechanisms mediating rapid sGnRH-induced and cGnRH-II-elicited GTH responses are different as in the case for prolonged GnRH action.

Chlorophyll Biosynthetic Reactions during Senescence of Excised Barley (Hordeum vulgare L. cv IB 65) Leaves.

The chlorophyll (Chl) biosynthetic reactions were monitored during senescence of dark-incubated excised barley (Hordeum vulgare L. cv IB 65) leaves floated in double-distilled water or kinetin solution. Kinetin abolished the degradation of Chl but failed to check the net degradation of protochlorophyllide (Pchlide), suggesting that different sets of enzymes, i.e. kinetin sensitive and insensitive, are responsible for the degradation of Chl and Pchlide, respectively. Upon exposure of the leaves to light, the dark-accumulated Pchlide was efficiently phototransformed to chorophyllide (Chlide), even on the 7th d of dark incubation, demonstrating that the activity of Pchlide reductase, one of the late enzymes of the Chl biosynthetic pathway, is not substantially affected during senescence. The senescing leaves continued to synthesize Pchlide and Chlide until the 7th d, although at a reduced rate (20% of the 1st d). The decline of the rate of synthesis of Pchlide and Chlide is due to the loss of activity of two early enzymes of the Chl biosynthetic pathway, i.e. 5-aminolevulinic acid dehydratase and porphobilinogen deaminase. Kinetin substantially checked the loss of activity of these two enzymes.

Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells.

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N-acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans-Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.

Partial purification and properties of a microsomal lysophosphatidic acid acyltransferase from oilseed rape

Storage oils in seeds are synthesized via the Kennedy pathway localized in the endoplasmic reticulum (Gurr, 1980). Triacylglycerol is formed by the sequential addition of three acyl groups to a glycerol 3-phosphate backbone. The esterifications are catalysed by three separate acyltransferase enzymes. It is believed that the enzyme catalysing esterification of acyl groups to the sn-2 position of glycerol, acyl-CoA: lysophosphatidic acid acyltransferase (LPA.AT ), plays an important role in regulating the acyl composition of seed oils. LPA.AT has the greatest degree of substrate specificity for acyl-CoAs of the three acyltransferases. Selectivity is a measure of substrate preference shown when the enzyme is incubated with a mixture of fatty acyl-CoAs. When incubated with a mixture of saturated and unsaturated fatty acylCoAs (Stobart & Stymne, 1985), safflower LPA.AT shows the following preference: linoleate > linolenate > oleate, with no incorporation of saturated fatty acids at position sn-2. However, if the levels of unsaturated fatty acids are limiting, the LPA.AT can use any available saturated fatty acids. It does not show absolute specificity for unsaturated fatty acids. Therefore the fatty acyl composition of lipids depends on a complex balance of selectivities and specificities. Complete exclusion of some fatty acyl-CoAs from position sn-2 has been demonstrated (Sun et al., 1988). Maturing seeds of palm, maize and rapeseed were incubated with lauroyl( 12:0), oleoyl( 18: 1 ) and erucoyl(22: 1 j CoAs. No 22:l could be incorporated at position sn-2 in any of the plants tested, even in rapeseed, which can contain up to 65% erucic acid in triacylglycerol. Also, only the palm was capable of esterifying 129 CoA to sn-2. The other two acyltransferases showed much lower substrate preferences. It is important to know the substrate preference of these acyltransferase enzymes, before strategies for modifying oil composition of seed storage oils by genetic manipulation can be worked out. If the composition of the cellular fatty acylCoA pool is modified, it is necessary to ensure that the existing acyltransferases can utilize the available fatty acids. Introducing acyltransferases with different substrate preferences from other plant species is a possible solution to this problem. Preliminary results on substrate specificity showed that the LPA.AT from crude rapeseed microsomal preparations had a four-fold preference for oleoyl-CoA over palmitoylCoA. This indicates that the acyltransferase activity in the membrane preparation was localized mainly in the endoplasmic reticulum. A second Kennedy pathway exists in the plastid, having a different set of enzymes from that of the endoplasmic reticulum. The LPA.AT activity associated with the inner membrane of the plastid has a preference for unsaturated over saturated acyl groups (Cronan & Roughan, 1987). This difference in the substrate specificities provides a means of testing that the purified protein does in fact originate in the endoplasmic reticulum, and that the plastid enzyme has not been inadvertently purified. After sucrose density gradient centrifugation, most of the LPA.AT activity was found in fractions with the same buoyant density as that of the endoplasmic reticulum. This supports the results on