Molecular forms of acetylcholinesterase present in the white and grey matter of pig brain

Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100 gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove acetylcholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter.The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter.Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge.Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed.A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.     

Characterization of acetylcholinesterase in Caco-2 cells

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-beta-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.     

Multiple forms of acetylcholinesterase from human erythrocytes

1. Acetylcholinesterase from human erythrocytes was solubilized with Triton X-100 in strong salt solution and partially purified by (NH(4))(2)SO(4) fractionation. This preparation showed three main bands of enzyme activity after electrophoresis on polyacrylamide gel and incubation with either alpha-naphthyl acetate or acetylthiocholine as enzyme substrate. Two of the multiple forms were completely inhibited by 10mum-eserine and one only partially. Treatment with neuraminidase had no effect on the electrophoretic pattern; therefore sialic acid does not appear to determine or affect the ratios of the acetylcholinesterase multiple forms, unlike those of the serum cholinesterase. 2. Chromatography of the preparation on Sephadex G-200 revealed one major peak of enzyme activity and a suggestion of two minor zones of mol.wt. 546000, 184000 and 93000 (i.e. in the proportion 6:2:1). The main peak was almost completely separated from the Triton X-100 and the overall purification was about 600-fold. Further attempts to purify the enzyme by absorption on calcium phosphate gels were unsuccessful. 3. Electrophoresis of the enzyme preparation on a polyacrylamide gradient for 24h revealed three main bands that corresponded to the three values for molecular weights obtained by column chromatography. After 70h of electrophoresis a further three zones of activity developed making six molecular entities, the molecular weights of which were simple multiples of a monomer, thus resembling the cholinesterase found in serum.     

EFFECT OF FATTY ACIDS ON RAT BRAIN PARTICULATE HEXOKINASE

Abstract— The effect of free fatty acids on rat brain particulate hexokinase was studied in vitro. Hexokinase bound with brain mitochondrial fraction was found to be sensitive to the action of free fatty acids, resulting in the solubilization of at least part of bound enzyme activity into the supernatant. The decrease of total enzyme activity observed at the highest free fatty acid concentration was probably due to the inhibition of hexokinase. The physiological consequence of hexokinase solubilization by low concentrations of free fatty acids, similar to that observed in vivo , is discussed in relation to activity changes of soluble and particulate enzyme forms demonstrated previously under hypoxic conditions.     

Solubilization of membrane-bound acid alpha-glucosidase by proteolysis

The membrane-bound acid alpha-glucosidase was purified partially (400-fold) from human placenta by solubilization with trypsin, concanavalin A-Sepharose chromatography, Ultrogel AcA-34 gel filtration, and Sephadex G-100 affinity chromatography. Two molecular forms of the enzyme were found in the final preparation of the purified enzyme. They were identical in molecular weight with a precursor (110 kDa) and an early intermediate form (105 kDa) of this enzyme. Also direct incubation of the membrane fraction without trypsin resulted in a release mainly of the 105 kDa form, which was inhibited by N-ethylmaleimide, but not by leupeptin, pepstatin or phenylmethylsulfonylfluoride. It was concluded that the precursor of acid alpha-glucosidase is an intrinsic membrane protein, which is transported into lysosomes after solubilization by proteolysis.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.     

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.     

Partial Purification and Some Properties of the Staphylococcus aureus Cytoplasmic Nitrate Reductase

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

Solubilization of frog brain and retina cholinesterase and studies of different molecular forms.

1. The cholinesterase (ChE) of frog brain and retina could be easily solubilized. About 10% of the brain and 20% of the retina ChE were found to be soluble in 0.05 M phosphate buffer. After treatment with 0.5% (v/v) Triton X-100, about 30% of the total ChE activity of the brain and only 10% for retina was left particle bound. NaCl by itself did not solubilize ChE. Use of higher NaCl concentrations in combination with Triton X-100 as well as higher detergent concentrations alone seemed to cause an inhibiting effect of the solubilized ChE from retina. 2. The solubilized ChE from brain as well as retina were electrofocused as one main activity peak, corresponding to isoelectric points of pH 6.1 and 6.0, respectively. A second molecular form at pH 5.9 was distinguishable for the brain, but not for retina ChE. 3. Sucrose gradient centrifugation indicated that the ChE solubilized from the brain and retina consists of two molecular forms exhibiting S values of 5.1 +/- 0.24, 10.9 +/- 0.33 and 6.1 +/- 0.30, 10.9 +/- 0.43, respectively. After solubilization by higher Triton X-100 concentrations the soluble extracts from brain and retina seemed to contain the activity of these forms in different proportions. 4. Polyacrylamide gel electrophoresis separated three molecular forms of the brain ChE. One of these forms was found to have a molecular weight of 394,000 +/- 20,000. The others were found to have an identical molecular weight of 550,000 +/- 10,000. Two molecular forms exhibiting molecular weights of 292,000 +/- 10,000 and 470,000 +/- 10,000, could be separated for retina.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of alkyldihydroxyacetone-P synthase from Ehrlich ascites cell microsomal membranes.

The enzyme, alkyldihydroxyacetone-P synthase, has been solubilized and partially purified from microsomal preparations of Ehrlich ascites cells after treatment with Triton X-100 and phospholipase C, followed by chromatography on Sepharose 4B. When the Triton X-100 was removed after solubilization the enzyme was still active but eluted in the void volume of the Sepharose 4B column, whereas in the presence of detergent it eluted much later as a single peak of activity, indicating that the solubilized enzyme tends to aggregate unless detergent is present. The lower molecular weight form of alkyldihydroxyacetone-P synthase (in detergent) had an estimated molecular mass of 250,000–300,000 daltons.     

Comparison of the association and orientation of gamma-glutamyltranspeptidase in lecithin vesicles and in native membranes.

gamma-Glutamyltranspeptidase purified following solubilization with Triton X-100 can associate with single-layered [14C]lecithin vesicles. Enzyme activity and radiolabeled vesicles were shown to co-migrate during Sepharose 4B chromatography and isopycnic sucrose gradient centrifugation. The enzyme-vesicle complex exhibits a density corresponding to that of a single enzyme molecule bound to a single vesicle, gamma-Glutamyltranspeptidase purified following a solubilization with papain does not bind to vesicles. In addition, papain treatment of vesicles containing the Triton-purified transpeptidase results in the release of 95% of the transpeptidase activity without release of internally trapped [3H]sucrose. The released transpeptidase is chromatographically identical to the papain-purified transpeptidase. gamma-Glutamyltranspeptidase activity associated with both native membranes and with lecithin vesicles exhibits a temperature-induced transition in its energy of activation. In contrast, the proteolytic- and detergent-solubilized forms of the enzyme exhibit a single energy of activation over the entire temperature range. These results suggest that gamma-glutamyltranspeptidase binding to vesicles is due to a papain sensitive sequence of amino acids and that the enzyme.vesicle complex closely approximates the interaction and orientation of gamma-glutamyltranspeptidase with brush border membranes.     

Distribution of acid and alkaline deoxyribonucleases in white and grey matter regions of growing and aging chick brain

The activities of acid and alkaline deoxyribonucleases in the white and grey matter areas of growing and old chick cerebrum were measured. Two marker enzymes for glial cells, butyrylcholinesterase and carbonic anhydrase were also measured in these regions. Higher specific activities of both butyrylcholinesterase and carbonic anhydrase were found in the white matter region at all the stages studied. Acid and alkaline deoxyribonuclease activities were observed in both white and grey matter. The decrease in the specific activity of acid deoxyribonuclease with advancement of age was more pronounced as compared to the alkaline deoxyribonuclease Marked reduction in total acid deoxyribonuclease activity in white matter, beyond the age of 130 days, was observed. On the other hand, total alkaline deoxyribonuclease activity in both white and grey matter continued to increase with age Further, the activity per mg of DNA also increased in white matter of the old brain. These results indirectly suggest a continued role for alkaline deoxyribonuclease in glial cells formed at a later age.     

Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis.

A sedimentable form of acid phosphatase (EC 3.1.3.2) from Tetrahymena pyriformis was found to be solubilized by Triton X-100. The total enzyme activity in the insoluble cell fraction increased almost 200% upon solubilization with Triton X-100 or Nonidet P-40. Removal of membrane lipids and Triton X-100 from the particulate wash solution with a chloroform extraction resulted in non-specific enzyme-protein aggregation which was reversible upon addition of Triton X-100. The results indicate that this acid phosphatase is an integral membrane protein. The pH optima for this particulate bound acid phosphatase was 3.5 with o-carboxyphenyl phosphate and 4.0 with p-nitrophenyl phosphate as substrates. The Km values of each substrate were 3.1 and 0.031 mM, respectively.     

Degradation of heme by a soluble peptide of heme oxygenase obtained from rat liver microsomes by mild trypsinization.

A tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The purified peptide gave only a single protein band with a molecular mass of 28 kDa on SDS/PAGE. The tryptic peptide, like the native heme oxygenase, readily bound with substrate heme forming a hemeprotein transiently. The absorption spectra of the ferric, ferrous, ferrous-CO and ferrous-O2 forms of the resulting complex resembled those of the corresponding forms of the complex of heme and the native enzyme. Ferric heme bound to the tryptic peptide was quantitatively decomposed to biliverdin on incubation with a mixture of ascorbic acid and desferrioxamine, indicating that the tryptic peptide still retained catalytic activity. These observations suggest that heme oxygenase has two domains, a hydrophilic and a hydrophobic domain, and that the two domains are folded almost independently of each other. An NADPH-cytochrome-P-450 reductase system composed of NADPH and detergent-solubilized NADPH-cytochrome-P-450 reductase readily reduced the ferric heme bound to the tryptic peptide, but failed to transfer the second electron required for rapid heme degradation, suggesting that the hydrophobic domain of heme oxygenase is important for receiving the second electron from the reductase.     

THE REGIONAL DISTRIBUTION OF CYTIDINE 5''-MONOPHOSPHO-N-ACETYL-NEURAMINIC ACID SYNTHETASE IN CALF BRAIN

—The enzyme cytidine 5′-monophospho-N-acetylneuraminic acid synthetase was studied in different parts of the calf brain. Characterization of partial purified enzyme preparations from cortical grey matter and corpus callosum by means of pH optima and K_m values, showed the enzyme of grey and white brain areas to be identical. Unexpectedly the regional differences of the enzyme activities per g wet tissue and per mg protein were very slight. From the presence of the enzyme in pure white brain areas, which are known to be poor in neuronal perikarya, and the fact that the enzyme is localized in the cell nucleus, we concluded that cytidine 5′-monophospho-N-acetylneuraminic acid is produced in glia cell nuclei and that it is very likely that biosynthesis of sialo-glycoproteins and/or ganglio-sides occurs within glia cells. The enzyme activity per μmol DNA-P is somewhat higher in grey than in white regions, indicating a slightly higher activity per neuronal than per glial nucleus. The regional differences of lipid and protein-bound sialic acid and RNA show a striking similarity and contrast to those of the enzyme. These differences are interpreted in terms of a differential content in neurons and glia cells.     

Sphingomyelinase activity at pH 7.4 in human brain and a comparison to activity at pH 5.0.

A hitherto undescribed sphingomyelinase (sph'ase 7.4) of human brain has been studied in crude and partially purified (3- to 4- fold) extracts of grey matter, and compared to the known sphingomyelinase with an acid pH optimum (sph'ase 5.0). Its specificity for sphingomyelin as substrate is similar to that of sph'ase 5.0, but it differs from sph'ase 5.0 in its pH optimum (7.4 vs 5.0) and in a requirement for Mg2+ for optimal activity. Other properties of sph'ase 7.4 that distinguish it from sph'ase 5.0 include (a) its lack of appreciable solubilization during dialysis of crude homogenates (b) a more marked concentrations in grey matter than in white matter (9- to 13- fold vs 1.5- to 2-fold for sph'ase 5.0); (c) inhibition by Ca2+ and Cd2+ ions, and by EDTA; (D) stimulation by dithiothreitol, and inhibition by cysteine, N-ethylmaleimide, and p-hydroxymercuribenzoate; (e) lack of inhibition by nucleotides (AMP.ADP, and ATP) and by NAD plus NADH; and (f) relative instability to storage or manipulation between -20degrees C and 40degrees C. These differences indicate the SPH'ASE 7.4 is a different enzyme protein from sph'ase 5.0. Unlike sph'ase 5.0, which is widely distributed in mammalian tissues, sph'ase 7.4 occurs predominantly in grey matter and little activity was observed is spleen, liver, or leukocytes. The high levels of this enzyme in brain suggest a role related to the specific functions of this organ or to the need for a more stringent control of sphingomyelin catabolism in brain as compared to other organs.     

Calcium-activated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms

Three forms of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) were extensively purified from rat liver homogenate. Subcellular fractionation analysis indicated that the majority (approximately 85%) of the activity was associated with particulate fractions of the liver. Among these, the microsomal and nuclear fractions accounted for approximately 63% and approximately 10% of total activity. The remaining 15% of protein kinase C was recovered in the soluble fraction following differential centrifugation. It was also found that most of the membrane-associated protein kinase C was latent, with 4-6-fold stimulation with detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, octyl beta-glucoside, or Triton X-100. The activity of both the bound form and the soluble enzyme was enhanced by the addition of Ca2+ and phosphatidylserine, when histone H1 was used as substrate. The bound protein kinase C activity was dissociated by homogenization of liver in buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,-N,N',N'-tetraacetic acid, ethylenediaminetetraacetic acid, and various proteolytic inhibitors, and the solubilized extract was used to purify multiple forms of the enzyme. The purification procedure sequentially utilized (NH4)2SO4 fractionation, ion-exchange chromatography on DEAE-cellulose, gel permeation chromatography on Fractogel TSK HW-55 (F), ion-exchange chromatography on hydroxylapatite, gel permeation chromatography on Ultrogel AcA34, and affinity chromatography on polyacrylamide-immobilized phosphatidylserine. On hydroxylapatite columns, protein kinase C activity was resolved into three isoenzymic forms designated C-I, C-II, and C-III. The molecular weights of the three isoenzymic forms were in the range of 208,000-225,000 as shown by chromatography on calibrated Ultrogel AcA34 columns and sucrose density gradient centrifugation. Furthermore, all three isoenzymes demonstrated a single peak with a sedimentation coefficient (s20.w) in the range of 9.0-9.2. However, with polyacrylamide gel electrophoresis, all the forms showed a single protein component with average molecular weight of 64K, suggesting that the native isoenzymes may be composed by subunits. Finally, all three isoenzymes exhibited nearly identical enzymatic properties.     

Catechol-O-methyltransferase from rat liver: two forms having different meta:para methylation ratios.

Cytoplasmic catechol-O-methyltransferase activity from rat liver was resolved by gel filtration into two enzymes: a major form having an estimated molecular weight of 23,000 and a minor one of 45,500. The relative abundance of these forms in liver is about 5:1, respectively. Microsomal catechol-O-methyltransferase constituted only 2% of the total liver activity. After solubilization by sonication most of the microsomal enzyme showed a molecular weight in excess of 100,000, but some 23,000 - enzyme was also released. The bound enzyme thus may represent an aggregate form of the soluble activity. The two cytoplasmic enzymes differ in several properties, including pH optima and thermal stability. The two forms also differ in the extent of methylation of the $\text{para}$ hydroxyl group, the larger enzyme having a $\text{meta}$:$\text{para}$ methylation ratio twice that obtained with the smaller form.     

Carboxylic acid reductase: a new tungsten enzyme catalyses the reduction of non-activated carboxylic acids to aldehydes

An enzyme which we call carboxylic acid reductase (aldehyde dehydrogenase) seems to be the first which is able to reduce non-activated carboxylic acids to aldehydes at the expense of reduced viologens. There is no further reduction of the aldehydes to the corresponding alcohols. In the presence of oxidized viologens aldehydes can be dehydrogenated to carboxylic acids roughly 20 times faster than the latter are reduced. The specific enzyme activity in crude extracts is about 100 times increased if 10 microM tungstate and a sulphur source in addition to sulphate is given to the growth medium of Clostridium thermoaceticum. Carboxylic acid reductase seems to be present in two forms. One has an apparent molecular mass of about 240 kDa and is bound to red-Sepharose, whereas, the other, a form of an apparent molecular mass of about 60 kDa, is not bound. SDS gel electrophoresis shows a higher complexity. The very labile enzyme has been enriched by a factor of about 145 by binding to octyl-Sepharose and further chromatographic separation by red-Sepharose and FPLC using Mono-Q and phenyl-Superose columns. After cell growth in the presence of [185W]tungstate, radioactivity coincides with the two forms of enzyme activity during all purification steps. This is also the case when the enzyme is electrophoretically separated on polyacrylamide slab gels.