DE-52 Cellulose Column Research Articles

Partial purification and characterization of a superoxide distimutase (SOD1) from black tiger shrimps <i> Penaeus monodon </i>

Superroxide dismutase (SOD, EC.1.15.1.1) is the enzyme which dismutates superoxide radicals and plays an important role in protection of living cells against oxidative stress. SOD is also involved in immune response in shrimps. In this study, it was found that the total SOD activity of black tiger shrimp muscular tissues is 10 fold higher than that of the haemolymph, however, the specific activity of SOD in the shrimp haemolymph is 9.2 fold higher than that of muscular tissues. By using active gel electrophoresis, 2 different SOD forms were found in black tiger shrimps (one in muscular tissues and two in haemolymph).Using DE-52 cellulose and Q-Sepharose ion exchange column chromatography, one SOD (SOD1) from black tiger shrimp haemolymph was partially purified, and its purity was 31.2 times higher than that of the starting haemolymph. The SOD1 was shown to have mainly one protein band of approximately 24 kDa on SDS-PAGE. SOD1 was most active at 45oC and pH of 5.5. At a concentration of 5 mM, Mn2+ strongly activated SOD1 (up 200% activity), Ca2+ và Zn2+ could increase approximately 20% activity while Cu2+ inhibited more than 60% ativity of the enzyme.

Ultrasonic-assisted Extraction, Antioxidant Activity and Structural Characterization of Polysaccharides from Oenothera Biennis L. Leaves

This paper studies the optimization of ultrasonic-assisted extraction of polysaccharides from Oenothera biennis L. leaves using Box-Behnken design, and their in vitro antioxidant activity and structure. It is demonstrated that the optimum extraction conditions are: the ultrasonic power at 400 W, solvent-tosample ratio of 23:1 (mL/g), ultrasonication temperature at 53 °C, and time for 36 min, under which the yield of polysaccharides from Oenothera biennis L. leaves, namely 3.54%, remains close to the predicted value in the model. The assays of the scavenging effect on DPPH and hydroxyl free radicals, and of the reducing power indicate that polysaccharides from Oenothera biennis L. leaves possess strong antioxidant activity and certain relationship exists between their dose and effect in the concentration range of the experiment; Fourier IR spectroscopy reveals that characteristic absorption peak of polysaccharides can be seen from the sample; the DE-52 cellulose column chromatography, HPLC profiles, and GC-MS chromatogram identify the obtained OCP-2 as heteropolysaccharides, whose major components, including arabinose, mannose, galactose, and talose, have an average molecular weight of 5435 Da.

Characterization of a Novel Thrombin-like Enzyme, Globlase from the Venom of Gloydius blomhoffii (Japanese Mamushi)

Aims: To elucidate the coagulation mechanisms of a novel clotting factor isolated from Gloydius blomhoffii venom, its hydrolytic activity on various substrates were examined. Furthermore the primary structure was determined and compared with the other snake venom components.
 Methodology: A thrombin-like enzyme was isolated from the crude venom of G. blomhoffii by DE52 Cellulose and CM52 Cellulose column chromatography. Enzyme activity was measured by using synthetic substrates (arginine esters, MCA-substrates and 3-(Acyloxy)-4-nitrobenzoic acid). Effect on fibrinogen was detected with bovine and human fibrinogen. Isoelectric point and molecular mass were measured by polyacrylamide gel electrophoresis and MALDI-TOF-MS. Amino acid sequence was decided with a protein sequencer by analyzing enzymatically cleaved peptides.
 Results: A clotting factor was found to be homologous as indicated by a single band on SDS-PAGE, and the final preparation was named as globlase. Molecular mass of this enzyme was determined to be 13,876.36 Da and the isoelectric point was 8.8. Globlase showed arginine ester hydrolytic activity, and specificity for substrates of thrombin. Proteolytic activity and phospholipase A2 (PLA2) activity were not detected. Complete amino acid sequence analysis indicated that the primary structure of globlase is similar to PLA2. However the aspartic acid which exists in the active site of PLA2 was found to be substituted by glutamine.
 Conclusion: It was shown in our current investigation that globlase is a novel thrombin-like enzyme isolated from G. blomhoffii venom. It was revealed that this enzyme had structure unlike the serine-protease such as the other thrombin-like enzymes.

Partial Purification and Thermal Stability of Two Peroxidases from Pithecellobium dulce (Roxb.) Benth. Aril

Two peroxidase (POX) forms, named PdPI and PdPII, were partially purified from the Pithecellobium dulce aril, using ammonium sulfate precipitation and anion exchange chromatography. Zymography of the proteins obtained after precipitation in the range of 60-90% ammonium sulfate (F6090 fraction) showed two bands with peroxidase activity. In DE-52 cellulose column, the peroxidase activity was detected in unadsorbed peak (PdPI) and adsorbed peak eluted with 0.2 mol L -1 NaCl (PdPII) suggesting that PdPI (~114 kDa) and PdPII (~77 kDa) are basic and acidic proteins, respectively, being PdPI stable at 80oC. This is the first report of partial purification of POX from P. dulce aril, indicating that this species may be a new source of enzymes for use in biosensors and other biotechnological applications.

Isolation and characterization of a c-type lysozyme from the nurse shark

Lysozyme is a ubiquitous antibacterial enzyme that occurs in numerous invertebrate and vertebrate species. Three forms have been described c-type, g-type and i-type which differ in primary structure. Shark lysozyme has not been characterized; here we report on the isolation and characterization of lysozyme from unstimulated shark (Ginglymostoma cirratum) leukocytes and provide amino acid sequence data across the highly conserved active site of the molecule identifying it to be a c-type lysozyme. A leukocyte lysate was applied either (a) to the first of two sequential DE-52 cellulose columns or alternatively, (b) to a DEAE-Sepharose column. Lysozyme activity in lysate and active fractions was identified by zones of lysis of Micrococcus lysodeikticus cell walls on lysoplates and zones of growth inhibition in agar diffusion assays using Planococcus citreus as the target organism. SDS-PAGE analysis revealed a 14 kDa protein which was identified as lysozyme by mass spectroscopic analysis of peptides, reactivity against anti-HEWL antibodies on a Western blot, hydrolysis of M. lysodeikticus cell walls, and inhibition of growth of P. citreus on AU-gel blots in which the area of growth inhibition correlated to a 14 kDa protein.

Chemical Analysis and Antioxidant Activities <i>In Vitro</i> of Polysaccharide Extracted from Corn Silk

Corn silk polysaccharide (CSP) was investigated for the treatment of different kinds of diseases. In order to characterize the chemical properties and antioxidant activities of CSP, the CSP was isolated from corn silk and purified by DE-52 cellulose column chromatography. Four components were separated, and the highest one named CSP-A. The CSP-A was characterized by FTIR and the monosaccharide components were analyzed by HPLC. The FTIR spectra indicated that CSP-A was characteristic of β-glucosidic bond and α-glycosidic bond. The CSP-A mainly comprised of glucose, galactose, arabinose, mannose, rhamnose. The antioxidant activities of CSP were determined by hydroxyl radical (•OH) and DPPH radicals scavenging assays. When the concentration of CSP was 10mg/mL, the scavenging capacity of •OH and DPPH could reach to 40% and 48%, respectively.

Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4

A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.

Characterization and Free Radical Scavenging Activity of Rapeseed Meal Polysaccharides WPS-1 and APS-2

Two major polysaccharide fractions, WPS-1 and APS-2, were isolated from water-soluble and alkali-soluble extracts of Huaza No. 4 rapeseed meal with a stepwise procedure of D3520 macroporous adsorption resin column chromatography, ethanol precipitation, and DE-52 cellulose column chromatography. Physicochemical properties of the polysaccharides were determined by chemical methods, high -performance liquid chromatography (HPLC), gel permeation chromatography (GPC), and Fourier transform infrared spectrometry (FT-IR). The chemiluminescence (CL) method was used to investigate the free radical scavenging activity of the polysaccharide fractions. The polysaccharides were primarily polymers of arabinose, galactose, and glucose, associated with protein portions consisting of 13 different amino acids. The average molecular masses of WPS-1 and APS-2 were 7.20 x 10(5) and 1.61 x 10(5) Da, respectively. Compared with APS-2, WPS-1 was more effective at scavenging superoxide radical (O(2)(*-)) and hydroxyl radical (HO(*)), but less effective at scavenging hydrogen peroxide (H(2)O(2)). In decreasing order, the free radical scavenging activity of WPS-1 and APS-2 toward reactive oxygen species (ROS) was H(2)O(2) > HO(*) > O(2)(*-).

A new female-specific fat body protein of Antheraea pernyi: purification, immunological properties, developmental profile and synthesis

A new female-specific protein has been discovered to be a major component of fat body proteins of the last larval instar of Antheraea pernyi and Antheraea yamamai. The new female-specific protein was purified to homogeneity by gel-permeation chromatographies, DE-52 cellulose column chromatography and hydroxyapatite column chromatography. The molecular weight of the protein was estimated to be 24,000 by SDS-PAGE and 43,000 by gel-permeation chromatography, suggesting that the protein is a homodimer protein. The protein was temporarily named “24 kDa peptide” (24K). Developmental changes in 24K titer were followed from the final larval ecdysis to adult emergence by immunoblotting with a specific antiserum against 24K. 24K appeared in the fat bodies at day 9 fifth instar and remained at an almost constant level up to adult emergence. Immunoblot analysis using antisera against Bm 30K proteins and BmLSP showed that the immunological properties of 24K were different from those of these proteins. In vitro culture to label fat body proteins with [ 35S]-methionine showed that the synthesis of 24K occurred in only female fat bodies. The function of 24K remains unknown.

Formate dehydrogenase from the methane oxidizer Methylosinus trichosporium OB3b

Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were detected. No other metals were found in stoichiometric amounts. When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture. Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN. Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity. The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN. The evolutionary significance of this similarity is discussed.

Characterization of a phosphomonoesterase from Brucella abortus

Brucellae are facultative intracellular bacterial pathogens that reside primarily in cells of the reticuloendothelial system. The high-speed supernatant obtained after centrifuging a suspension of Brucella abortus that had been frozen-thawed and sonicated contained abundant phosphomonoesterase activity, determined by using 4-methylumbelliferylphosphate as the substrate; this enzyme was purified 2,900-fold (yield, 570%) by chromatography on DE-52 cellulose and hydroxylapatite columns and high-performance liquid chromatography-gel filtration. The native enzyme had a molecular mass of 120,000 daltons (+/- 10,000 daltons), as determined by gel filtration chromatography, and resolved into two bands (60,000 and 66,000 daltons) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The B. abortus phosphomonoesterase had the following properties: pH optimum, 6.0 to 6.5; isoelectric point, 3.0; substrate specificity, 5'-AMP greater than 3'-AMP greater than 3'-GMP greater than 5'-GDP greater than 5'-CDP greater than 5'-CTP greater than 5'-UPT greater than phosphotyrosine greater than phosphoserine greater than phosphothreonine. The Km for 5'-AMP was 0.37 mM. Phosphatidylinositol 4,5-bisphosphate and myo-inositol 1,3,4-trisphosphate were poor substrates for the B. abortus enzyme. The phosphomonoesterase did not inhibit superoxide anion production by human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine. The phosphomonoesterase may be one of the bacterial enzymes in the pathway leading to the production of adenine, which is secreted by B. abortus and blocks the activation of neutrophils.

Characterizations of Two Distinct Ca2+-dependent Phospholipid-binding Proteins of 68-kDa Isolated from Human Placenta

Two distinct 68-kDa proteins, named 68K-I (pI 6.4) and 68K-II (pI 5.6), were solubilized from human placenta by treatment with 5 mM EGTA. On DE52 cellulose column chromatography at pH 7.4, 68K-I in the EGTA eluate was recovered in the unadsorbed fractions, whereas 68K-II was retained on the column and eluted with 0.2 M NaCl. The 68K-I protein was obtained in more than 95% purity by further hydroxylapatite and cation exchange chromatographies, while the 68K-II protein was purified further by gel filtration and hydroxylapatite chromatographies. Partial amino acid sequence data showed that 68K-I protein was a novel protein which shared the same sequences as lipocortin I and that 68K-II was the same as human p68/67-kDa calelectrin (Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21-27; Südhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664-668). The two proteins bound to acidic phospholipids, phosphatidylserine, and/or phosphatidylinositol, but not to phosphatidylcholine, in the presence of micromolar levels of Ca2+. 68K-I bound to phosphatidylinositol preferentially to phosphatidylserine, whereas 68K-II bound only to phosphatidylserine. Both 68K-I and 68K-II inhibited phospholipase A2 activity, and the inhibition by 68K-II was detectable only in the presence of 100 mM KCl. 68K-I, but not 68K-II, was found to bind to F-actin in a Ca2+-dependent (1 mM) manner. Moreover 68K-I, but not 68K-II, was phosphorylated in vitro at tyrosine residues by fps kinase and by epidermal growth factor receptor/kinase, the latter reaction being dependent on Ca2+ and epidermal growth factor. Western blot analysis with affinity purified anti-68K-I and anti-68K-II antibodies showed that 68K-I was located in only certain tissues, especially human placenta, whereas 68K-II was present in many human and rat tissues.

Electrophoretic and immunochemical characterization of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues.

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.

Protein kinase C activity in a freshwater planarian ( Dugesia gonocephala)

1. 1. Protein kinase activity was detected in cytosol and membrane fractions of freshwater planarian. 2. 2. This activity was partially purified by ion-exchange chromatography on DE52 cellulose column after elution with NaCl (0.09–0.14 M). 3. 3. It was strictly dependent on the presence of phospholipid and was increased by Ca 2+. 4. 4. Diacylglycerol and tumor promoter triggered enzyme activity, but stimulation was no longer dependent on Ca 2+. 5. 5. In addition, the enzyme was inhibited by some phospholipid-interacting drugs such as chlorpromazine, dibucaine, mellitine and alkyl lysophosphatidylcholine in a dose-dependent manner. 6. 6. Taken as a whole, the above characteristics demonstrated the presence of protein kinase C in planarian.

THE ALTERNATIVE COMPLEMENT PATHWAY IN CHICKENS

Restoring the haemolytic activity of heat-treated chicken serum was used to monitor an initial purification of chicken complement component factor B of the alternative complement pathway. The partially purified factor B was used to prepare a monospecific rabbit antiserum against factor B, and this antiserum was now used to monitor and establish a purification scheme for chicken factor B which could be used for large-scale purifications. The final purification involved precipitation with polyethyleneglycol, ion exchange chromatography on CM 52-cellulose, gel-filtration on Sephadex G 200, elution from a Con A-Sepharose column, and finally ion exchange chromatography on a DE 52-cellulose column. 2.8 mg of factor B was purified from 100 ml of chicken plasma with a yield of 28%. Purity was high as judged from crossed immunoelectrophoresis, SDS-PAGE, and from immunizations of rabbits with the factor B preparation. The purification gave preliminary indications that chicken factor B is a glycoprotein with beta-mobility and with a molecular weight around 95 Kd.

Calcium-dependent, phospholipid-activated protein kinase in plants

A phospholipid-stimulated protein kinase has been demonstrated in plant extracts which is similar to protein kinase C in its behaviour on a DE-52 cellulose column, its substrate specificity and its calcium dependence. Nonspecific enzyme activation has been discounted by the inability of sodium dodecyl sulphate to mimic the effect of phophatidylserine plus diacylglycerol. However the lack of specific phorbol ester binding by the enzyme fractions makes a complete identity with protein kinase C doubtful.

Alterations in hepatic protein kinase activity induced by triiodothyronine.

Quantitative and qualitative alterations in protein kinase (PK) activity in preparations of rat liver from hypothyroid (H) and T3-treated hypothyroid animals (T) were analyzed in comparison to enzyme from normal (N) animals. Qualitative variations in type of protein kinase were assessed by chromatography of cytosol preparations on DE-52 cellulose columns. Cytosol kinases resolved into a small fraction I containing a catalytic subunit, fraction II containing a type 1 holoenzyme, fraction III containing a cyclic-AMP independent PK, and fraction IV containing a type 2 holoenzyme. A cytosolic kinase active with casein as substrate was also identified. In H the type 1 holoenzyme was reduced in comparison to N. There were no other qualitative changes. Nuclear PKs were extracted with solvents containing 0.3 M KCl. Qualitative changes were evaluated by chromatography on phosphocellulose columns, but available methodology did not give reproducible evidence of changes in individual PKs. H had significantly more cytosolic PK active with protamine substrate than did normal animals, and by administration of T3, the H level was reduced progressively over 48 h to the N level. The activation ratio of total PK in cytosol was higher in H, and was also reduced to the N level by T3 administration. This suggests a higher steady state level of cyclic-AMP may be present in H rat liver cytosol. Cytosolic protein kinase reactive with casein as substrate increased gradually over 48 h after T3 administration from the H to the higher N level. It was significantly elevated to 110% of the H value by 48 h after T3 administration. The behavior of nuclear PK was entirely different. Nuclear PK reactive with protamine as substrate was increased within 1-1/2 h after T3 administration, reaching a peak of 130% of the control value at 5 h and returning to the normal level by 48 h. In contrast, nuclear PK reactive with casein as substrate also increased by 1-1/2 h after T3 administration and remained elevated at 110-115% of the control value throughout 48 h after T3 administration. The early changes in nuclear PK activity were prevented by administration of cycloheximide or alpha-amanitin. The observed changes in cytosolic PK, including the increment induced by T3 in type 1 enzyme, reduction in

Purification and characterization of rat plasma kallikrein.

Kallikrein enzyme initially was isolated from rat plasma by passage of citrated plasma through a DEAE-Sephadex column at pH 7.2. The active fraction was purified to electrophoretic apparent homogeneity by precipitation to 60% ammonium sulfate saturation, sequential passage through DE-52 cellulose, Sephadex G-200 and SP-Sephadex columns and finally by chromatofocusing on a PBE-94 column. The kallikrein content of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME) and by its ability to form kinin from heat-treated rat plasma. The molecular weight was estimated by gel filtration to be 50,000 and by SDS-gel electrophoresis 41,000. Multiple isozymic forms were obtained with pI values ranging from 4.2 to 5.0. The enzyme has a pH optimum of 8.3. The Km and Vmax values for TAME, Bz-pro-phe-arg-pNA and H-D-val-leu-lys-pNA were 1.6, 0.16 and 1.7 mM and 3.09, 0.96 and 0.25 microM/mg/min respectively. The enzyme was inhibited by soybean trypsin inhibitor but not by lima bean trypsin inhibitor.

The development of an immobilized lactate oxidase system for lactic acid analysis

An enzyme designated as lactate oxidase was purified from Acetobacter peroxydans by using the partition methods of separation. A DE-52 cellulose column was used for the primary purification of lactate oxidase, and the purified enzyme was covalently bound to a porous cellulose bead matrix in which benzoquinone was used as the coupling reagent. The physicochemical properties of the native and immobilized enzymes were determined including molecular weight, cofactor requirements, and optimal reaction conditions. Lactate oxidase was shown not to be subject to product inhibition, and to require Mg(2+) as a metal cofactor. Analysis of an immobilized lactate oxidase packed-bed reactor indicated that this system may not be subject to internal diffusional limitations. Molecular oxygen appeared to be a cosubstrate of the enzyme, and a reaction mechanism was postulated to predict the kinetic behavior of the immobilized reactor system. Applications of the immobilized lactate oxidase reactor for the pulse-flow analysis of lactic acid in whole milk and in a yeast fermentation system were considered.

Multiple forms of isopentenyl pyrophosphate isomerase of avian liver.

Four kinds of isopentenyl pyrophosphate isomerase were separated from avian liver homogenates by DE-52 cellulose column chromatography. These enzymes were similar in terms of optimum pH (pH 6.6) and metal ion requirement (Mn2+). They all catalyzed the specific elimination of the pro-R hydrogen at C-2 of isopentenyl pyrophosphate. However, they showed different susceptibilities to iodoacetamide and beta-mercaptoethanol.