Purification and Properties of Putrescine Hydroxycinnamoyl Transferase from Tobacco (Nicotiana tabacum) Cell Suspensions

The enzyme putrescine hydroxycinnamoyl transferase (PHT) was purified 400-fold in 7.1% yield from tobacco (Nicotiana tabacum L. cv Xanthi) cell suspensions to a final specific activity of 45 nanokatal per milligram protein. The purification procedure involved conventional chromatography techniques (anion exchange chromatography, gel permeation, and hydroxylapatite chromatography) followed by chromatography on caffeoyl-cysteamine-Sepharose. This procedure led to considerable enrichment of a 50 kilodalton protein that could be further purified to near homogeneity by chromatofocalization (apparent isoelectric point = 8). PHT activity was repeatedly found associated with this protein, although approximately 66% of the enzymic activity was lost during chromatofocalization. Purified PHT exhibited the same properties as in the unpurified extract. It was not specific for putrescine and used other aliphatic diamines (mainly diaminopropane and cadaverine) as substrates. The most efficient phenolic substrate was caffeoyl-CoA, but cinnamoyl-, feruloyl-, sinapoyl-, and p-coumaroyl-CoA were also conjugated to putrescine, in decreasing order of activity. PHT could also use the artificial substrate p-fluorocinnamoyl-CoA.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

Immunological studies of the fragments of bovine cartilage proteoglycan produced by chondroitinase-trypsin digestion.

The peptidyl transferase activity of polysomes from Escherichia coli, rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to 10-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.     

Lactate Dehydrogenase in Oryza sativa L. Seedlings and Roots: Identification and Partial Characterization

A lactate dehydrogenase activity is present in rice (Oryza sativa L.) seedlings and roots. Under aerobic conditions, lactate dehydrogenase activity is barely detectable in rice seedlings and is very low in rice roots. In 30 day old roots, the activity is increased two to three times by an anoxic or hypoxic treatment and can be detected on immunoblots by an antiserum raised against barley lactate dehydrogenase. The activity present in aerobic seedlings was partially purified. The native enzyme has a molecular mass of 160 kilodaltons, and is a tetramer of 2 subunit (38 and 39 kilodaltons) randomly associated. Studies of substrate specificity, native gel electrophoresis, and immunoblot analysis indicate that the partially purified enzyme is a typical lactate dehydrogenase. However, no increase of lactate dehydrogenase activity or protein was observed in seedlings transferred to anoxia.     

Biosynthesis and Characterization of 4-Fluorotryptophan-Labeled Escherichia coli Arginyl-tRNA Synthetase

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher K _m values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of ¹⁹F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.     

Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function

The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3 L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function.     

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA^Leu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA^Leu (CAG)—a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.     

Characterization of Starch-Debranching Enzymes in Pea Embryos

Two distinct types of debranching enzymes have been identified in developing pea (Pisum sativum L.) embryos using native gel analysis and tests of substrate preference on purified or partially purified activities. An isoamylase-like activity capable of hydrolyzing amylopectin and glycogen but not pullulan is present throughout development and is largely or entirely confined to the plastid. Activities capable of hydrolyzing pullulan are present both inside and outside of the plastid, and extraplastidial activity increases relative to the plastidial activity during development. Both types of debranching enzyme are also present in germinating embryos. We argue that debranching enzymes are likely to have a role in starch metabolism in the plastid of the developing embryo and in starch degradation during germination.     

The Biosynthetic Pathway of (Z)-11-Hexadecenal,the Sex Pheromone Component of the Rice Stem Borer,Chilo sappressalis WALKER (Lepidoptera: Pyralidae)

Lipoxygenase-3, the major component of the enzyme in rice grain, was purified 2980-fold with a yield of 7% from embryos. The purified enzyme had a specific activity of 280 μmol O₂ formed/min per mg protein. This enzyme was inactivated by SH compounds, such as cysteine and glutathione. The inactivation was prevented by the addition of catalase or replacement of the air by N₂ gas. These two treatments were also effective for the stable storage of the purified enzyme. The molecular weights measured by sodium dodecyl sulfate gel and gradient gel electrophoresis were 93,000 and 89,000, respectively, indicating that the enzyme is a single polypeptide chain. The purified enzyme contained 0.73 Fe atom per molecule. The absorption spectrum suggested that the enzyme is a non-heme iron protein. Some similarities in amino-acid composition were observed between rice, soybean, and pea lipoxygenases. The purified enzyme specifically produced 9-d-hydroperoxy-10,12(E,Z)-octadecadienoic acid when linoleic acid was used as a substrate.     

Action of Corn and Rice-inactivating Proteins on a Purified Nitrate Reductase from Chlorella vulgaris

When nitrate reductase (NR) purified from Chlorella was incubated with NR-inactivating proteins purified from corn roots and rice cell suspension cultures or with trypsin there was a loss in NADH-NR and NADH cytochrome c reductase (NADH-CR) activities with time whereas the reduced methylviologen NR (MV-NR) remained active. When NADH-NR and NADH-CR activities were inactivated completely by the incubation with corn protein, the major protein band obtained by polyacrylamide gel electrophoresis shifted from an R_F value of 0.12 to an R_F of 0.25 and reduced MV-NR activity moved to the new position on the gel. When NADH-NR and NADH-CR activities were partially inactivated by the corn protein, NADH-NR activity was detected in an intermediate position (R_F value of 0.18). Incubation with trypsin also caused a change in the NR protein migration pattern (R_F value of 0.20). This protein band also had reduced MV-NR activity. Thus, the corn inactivator degrades NR in a fashion similar to but not identical with trypsin. The incubation of NR with rice inactivating protein resulted in a loss of NADH-NR but had no effect on the migration of NR protein or on the reduced MV-NR activity or mobility suggesting that the rice protein binds to Chlorella NR.     

Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins

A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, K_d and B_max values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein.     

Induction of acid phosphatase in cotton seedlings: Enzyme purification,subunit structure and kinetic properties

About a 16-fold rise in acid phosphatase (EC 3.1.3.2) activity was observed during the early stages of germination of cotton embryos. Administration of cyclobeximide to the germinating embryos significantly blocked the enhancement of acid phosphatase activity. This indicated that translational activity was essential for the induction of enzyme activity. Conclusive proof for the de novo synthesis of the enzyme was obtained by showing the incorporation of ³⁵S from ³⁵SO²⁻₄ into the cysteine residues of the purified acid phosphatase. The enzyme was purified (1046-fold) to electrophoretic homogeneity by ammonium sulphate fractionation, CM-Sephadex C-50 and affinity chromatography on concanavalin A-Agarose. PAGE gave two isozyme bands. The M, of the phosphatase was 200 k as determined by molecular sieving on Sephadex G-200. SDS-PAGE of acid phosphatase revealed a single band of M 55 k. Thus the native enzyme is a tetramer of four identical subunits. The K_m of the enzyme with p-nitrophenyl phosphate was 0.5 mM. Optimal enzyme activity was observed at pH 5.0, using p-nitrophenyl phosphate as substrate. The enzyme activity remained linear for 105 min at 37° and was proportional to the concentration of protein within the range 0.6–2.4 μg.     

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) I: Endogenous Angiotensin Converting Enzyme (ACE) Inhibition

Angiotensin-converting enzyme (ACE) inhibitors represent the fifth most often prescribed drugs. ACE inhibitors decrease 5-year mortality by approximately one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors, which endogenous inhibitory effects are much less characterized than that for the clinically administered ACE inhibitors. Here we aimed to investigate this endogenous ACE inhibition in human sera. It was hypothesized that ACE activity is masked by an endogenous inhibitor, which dissociates from the ACE when its concentration decreases upon dilution. ACE activity was measured by FAPGG hydrolysis first. The specific (dilution corrected) enzyme activities significantly increased by dilution of human serum samples (23.2±0.7 U/L at 4-fold dilution, 51.4±0.3 U/L at 32-fold dilution, n = 3, p = 0.001), suggesting the presence of an endogenous inhibitor. In accordance, specific enzyme activities did not changed by dilution when purified renal ACE was used, where no endogenous inhibitor was present (655±145 U/L, 605±42 U/L, n = 3, p = 0.715, respectively). FAPGG conversion strongly correlated with angiotensin I conversion suggesting that this feature is not related to the artificial substrate. Serum samples were ultra-filtered to separate ACE (MW: 180 kDa) and the hypothesized inhibitor. Filtering through 50 kDa filters was without effect, while filtering through 100 kDa filters eliminated the inhibiting factor (ACE activity after <100 kDa filtering: 56.4±2.4 U/L, n = 4, control: 26.4±0.7 U/L, n = 4, p<0.001). Lineweaver-Burk plot indicated non-competitive inhibition of ACE by this endogenous factor. The endogenous inhibitor had higher potency on the C-terminal active site than N-terminal active site of ACE. Finally, this endogenous ACE inhibition was also present in mouse, donkey, goat, bovine sera besides men (increasing of specific ACE activity from 4-fold to 32-fold dilution: 2.8-fold, 1.7-fold, 1.5-fold, 1.8-fold, 2.6-fold, respectively). We report here the existence of an evolutionary conserved mechanism suppressing circulating ACE activity, in vivo, similarly to ACE inhibitory drugs.     

An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.     

Processing in vitro of pronapin, the 2S storage-protein precursor of Brassica napus produced in a baculovirus expression system

The maturation of the 2S albumin, napin, in Brassica napus L. involves removal of an amino-terminal and an internal propeptide. Pulse-chase experiments with B. napus embryos showed that intermediates are detectable during the pronapin processing. Intact pronapin was expressed by baculovirus in Spodoptera frugiperda insect cells in order to obtain substrate for studying the processing event. Processing of pronapin with a crude B. napus embryo protein extract resulted in several fragments of similar sizes to those of napin heavy and light chains. The character of the major processing activity in the B. napus extract suggested that it was due to an aspartic proteinase. A secondary activity indicated an additional endo-proteinase involved in the pronapin processing. Limited proteolysis of pronapin with a purified aspartic proteinase from Hordeum vulgare showed that cleavage occurred exclusively in the prosequences. The cleavage products formed in-vitro requires additional trimming of the propeptides in order to obtain the subunits of mature napin.     

The treatment of purified maize oil bodies with organic solvents and exogenous diacylglycerol allows the detection and solubilization of diacylglycerol acyltransferase

In spite of its importance in the biosynthesis of reserve oils in plants, diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) has not been purified to homogeneity, and its study has remained incomplete. We found that the microsomal preparations from developing maize embryos contained substantial amounts of endogenous diacylglycerol (DAG). A solubilization procedure for extracting DAGAT from the microsomes (D. Little, R. Weselake, K. Pomeroy, S.T. Furukawa, J. Bagu, Biochem. J. 304 (1994)) was ineffective in eliminating the endogenous DAG, even after gel filtration. DAG removal through the preparation of acetone powders from the embryos led to the loss of DAGAT activity. Labelled triacylglycerol (TAG) was produced in the standard DAGAT assay when labelled DAG was supplied in benzene solution to the freeze-dried microsomes and the sample was dried and resuspended in an aqueous buffer. In contrast, no labelled TAG was produced when a similar sample supplied with non-labelled DAG was assayed with emulsified labelled DAG and acyl-CoA. Repeated washing of the microsomal freeze-dried fraction with benzene resulted in a complete loss of DAGAT activity in the standard assay, but the activity was restored by the addition of DAG plus phosphatidylcholine or Tween 20 in benzene. Although DAGAT has been reported to be confined mainly to the endoplasmic reticulum, we found that DAGAT activity was high in the purified oil bodies from both developing and mature maize embryos and was not removed by repeated washing with 6 M urea. The DAGAT activity was restored from delipidated oil bodies and from microsomes after the preparations had been resuspended in methanol/acetic acid/water (1:1:1, v/v). Although most of the proteins in the suspension were eluted as a single peak at the void volume after gel filtration chromatography, DAGAT activity was found in later fractions. SDS–PAGE of the peak activity fraction revealed no protein bands after silver staining, and the finding suggest that DAGAT protein is of low abundance and has a high k_cat.     

Phosphatidylcholine biosynthesis in castor bean endosperm : purification and properties of cytidine 5'-triphosphate:choline-phosphate cytidylyltransferase

Cytidine 5′-triphosphate:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been purified to near homogeneity (3350-fold) from castor bean (Ricinus communis L. var Hale) endosperm. The steps of purification included a differential solubilization of this enzyme with n-octyl β-d-glucopyranoside (OGP) and column chromatography on sequential DEAE-sepharose, sepharose-6B, and second DEAE-sepharose columns. The uses of appropriate concentrations of the detergent, OGP, in each step were crucial to obtain the highly purified enzyme. The purified enzyme gave a single protein band on nondenaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed one major protein band of 40 kilodaltons. Gel filtration chromatography indicated that native cytidylyltransferase was approximately 155 kilodaltons, suggesting that it exists naturally as a tetramer. The purified enzyme used methylethanolamine-phosphate as a substrate but not ethanolamine-phosphate and dimethylethanolamine-phosphate. ATP and other nucleotides tested showed little effect on the purified enzyme. The purified enzyme activity was stimulated by both phospholipids extracted from castor bean endosperm and phosphatidylcholineoleate vesicles.     

Glycosyltransferases with endogenous acceptor activity in plasma membranes isolated from rat liver

Plasma membrane fractions from rat liver exhibited glycosyltransferase activity with endogenous membrane-associated acceptors and either UDP-galactose, UDPglucose, UDP-N-acetylglucosamine, or GDPmannose donors. Of these, incorporation into non-lipid acceptors was most active with UDP-galactose and only with UDPgalactose and UDPmannose was there incorporation into endogenous lipid acceptors. CMP-N-acetylneuraminic acid was inactive as a donor with the isolated plasma membranes. In order to demonstrate transferase activity, low concentrations of substrate sugar nucleotides and short incubation times were used as well as sulfhydryl protectants and a phosphatase inhibitor (NaF) in the reaction mixtures. The findings support the concept of surface localization of at least a galactosyl transferase in cells of rat liver.