Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.     

Assay,properties, and regulation of rat ovarian δ-aminolevulinic acid synthetase activity

δ-Aminolevulinic acid synthetase (EC 2.3.1.37) has been detected in homogenates of rat ovaries. Optimal substrate and coenzyme concentrations, and parameters for assay of ovarian δ-aminolevulinic acid synthetase have been determined. Subcellular fractionation studies have shown that enzyme activity is predominantly localized in the mitochondrial fraction. Fasting, which is known to increase enzyme activity in the adrenal and to have no effect on activity in the testis, had no effect on enzyme activity in the ovary. Administration of the hepatic inducer allylisopropylacetamide or the hormone progesterone failed to alter activity of the ovarian enzyme. The activity of the enzyme was significantly increased during the diestrus-1 phase of the estrus cycle, during pregnancy, and by human chorionic gonadotropin at 24 and 48 h, suggesting that ovarian δ-aminolevulinic acid synthetase and the synthesis of heme may be under hormonal control.     

2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.     

Selective inhibition of cell growth and associated changes in glycolipid metabolism induced by monovalent antibodies to glycolipids

Monovalent antibodies directed against N-acetylhematoside are growth inhibitory for BALB/3T3 and NIL hamster fibroblasts but not their transformed counterparts. Within a similar dose range antibodies directed against globoside have no effect on cell growth. Inhibition of 3T3 cell growth by anti-hematoside correlates with a specific change in the metabolism of hematoside within the cell membrane. Following antibody treatment the radiolabeling of hematoside is elevated for cell in logarithmic growth but reduced relative to control at final saturation density. This effect is not observed for 3T3 cells transformed by Kirsten murine sarcoma virus. It is suggested that cell surface glycolipids may play a role in the control of normal cell growth in vitro.     

Purification and characterization of regenerating mouse L929 karyoplasts

Within 72–96 hr after preparation, about 10% of the karyoplasts made from mouse L929 cells regenerated to reform whole viable cells. As soon as 30 hr after preparation, however, nearly all of the remaining 90% of karyoplasts were dead. By separating living and dead karyoplasts at 30 hr, therefore, that fraction destined to complete regeneration was effectively purified. Complete separation was accomplished by sedimentation through Ficoll-paque (Pharmacia), a patented preparation originally developed for the separation of monocytes from whole blood. With the addition of this technique to the previously reported purification scheme for karyoplasts, various biochemical and morphological studies were attempted. Of particular importance are results indicating that karyoplasts that regenerate do not initially contain any more cytoplasm than the average karyoplast in a preparation—that is, about 10% of the cytoplasm within a whole cell. Electron microscopy of karyoplasts immediately after preparation indicated an unequal partitioning of cytoplasmic organelles at the time of enucleation. For example, karyoplasts initially contained about 11.4% of the mitochondrial volume of whole cells, but only 2.9% of the Golgi apparatus. The size of the karyoplasts and the volume occupied by a variety of organelles was followed throughout the process of regeneration. Although there was an approximately linear increase in the diameter of regenerating karyoplasts, there appeared not to be a simple concordant increase in the volume occupied by all cellular organelles. An extensive investigation was performed to determine whether or not karyoplasts contained centrioles. Immediately after enucleation, 15,000 random thin sections through karyoplasts, which represented about 100 complete bodies, were examined for the presence or absence of centrioles. No centrioles were observed. Examination of the cytoplasts revealed that they contained a sufficient number of centrioles to account for all of the centrioles that were present in the whole cells before enucleation. Centrioles were first detected in karyoplasts at 24 hr after preparation, about the same time that karyoplasts regained the ability to adhere to the surface of tissue culture dishes. At this time, however, the average karyoplast had less than one centriole. By 72 hr, the regenerated karyoplasts had approximately the same number of centrioles as whole cells.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

Methylxanthine inhibition of neurosecretion in the brain and corpus cardiacum of Cecropia

The fine-structure of the median neurosecretory cells and corpora cardiaca of the Cecropia silkmoth during the first 7 days after transfer from cold conditions to room temperature was compared to that of similar animals whose development was arrested with aminophylline. The major difference observed was the failure of the intrinsic secretory cells of the corpus cardiacum to degenerate in the arrested animals. This failure to degenerate coincides with the expected period of brain hormone release. After long periods of arrest, the medial neurosecretory cells and their axons became distended with neurosecretory granules. The significance of these observations in the initiation of adult development is discussed.     

Mouse transcobalamin II: Biosynthesis and uptake by L-929 cells

Mouse fibroblast (L-929) cells, in culture, synthesized and secreted into the growth medium a vitamin B₁₂-binding substance which was identical to mouse transcobalamin II (TC II) as judged by the following criteria: (i) gel filtration on Sephadex G-200, (ii) ion-exchange chromatography on DEAE-cellulose and CM-cellulose, and (iii) the ability to facilitate cellular B₁₂ uptake by L-929 cells. The secretion of mouse fibroblast binder was blocked by cycloheximide and puromycin; and in both cases the cells' ability to secrete this binder was partially restored when the inhibitor was removed. Within 30 h after the cells were exposed to [⁵⁷Co]B₁₂ bound to mouse serum TC II (M_r ~ 38,000) the [⁵⁷Co]B₁₂ was bound to a large molecular weight intracellular binder (M_r ~ 120,000) which was not released into the culture medium. During this same incubation period, the cells released free [⁵⁷Co]B₁₂ and [⁵⁷Co]B₁₂ bound to a protein which had the same elution volume as mouse serum TC II on Sephadex G-200.     

Skin banking methodology: An evaluation of package format,cooling and warming rates,and storage efficiency

The two major skin packaging formats for transplantable human skin, flat — folded and rolled — cylindrical, were evaluated with respect to the control of cooling rate, warming rate, and storage efficiency. Experiments were performed with six amounts of skin ranging from 7.6 × 20 cm (0.17 ft²) up to 7.6 × 120 cm (1.00 ft²).Contrary to previously published statements, when skin packaged in either of the two formats is cooled at an uncontrolled rate in a low temperature (?70 °C) mechanical refrigerator or dry-ice chest, the smaller skin dimensions cool too rapidly (up to ?24 °C min^?1), while the packets containing larger skin dimensions exhibit prolonged exothermic temperature plateaus (8–44 min), allowing the possibility of significant crystallization damage to the cells. On the other hand, controlled-rate cooling of ?1 °C min^?1 can be obtained using a temperature-feedback controlled-rate freezer along with a flat skin packet geometry. Much less control is obtained if a cylindrical skin packet geometry is used with a controlled-rate freezer.Skin processed in the flat format is capable of being warmed by water immersion about 10 times more quickly than equivalent amounts of skin processed in the rolled format. The longer warming times associated with the cylindrical package format (3.5–25 min, depending upon the amount of skin per packet) result from extended endothermic temperature plateaus in the subzero region, which have been shown to damage skin cells and reduce their subsequent viability. The short warming times (0.25–3.5 min) associated with the flat skin package format are devoid of such complications, since they are within the needed warming rate of 50 °C–70 °C min^?1.Package geometry affects the storage requirements of transplantable skin. The flat format possesses a two- to threefold advantage in storage efficiency. Capital equipment and liquid nitrogen usage for storage is drastically decreased if a flat package format is chosen.     

Further characterization of the effects of 3-methylcholanthrene administration upon hepatic ribonucleic acid polymerase activities

The administration of 3-methylcholanthrene (MC) to rats results in a marked increase in the specific activities of hepatic RNA polymerases I and II. In the present study, we were able to show that this increase was not caused by a shift in the ratio of ‘free’ to ‘template-engaged’ RNA polymerase. By means of binding studies with [³H]amatoxin, we were unable to demonstrate any increase in the number of RNA polymerase II molecules in liver after MC administration to the rats. RNA polymerase I was purified in excess of 3000-fold from hepatic nuclei isolated both from control and MC-treated rats. The stimulation in activity was demonstrated at each step in the purification scheme until glycerol sedimentation analysis. Results from cation-exchange chromatography on phosphocellulose indicated that the polycyclic hydrocarbon increased the enzyme activity of RNA polymerase I_b somewhat specifically. Subsequent to glycerol gradient centrifugation, this stimulatory advantage was no longer evident. Reconstitution experiments revealed the presence of a stimulatory component, which was demonstrated in low molecular weight fractions from both control and experimental preparations.     

Purification and properties of d-galactose-binding lectins from some erythrina species: Comparison of properties of lectins from E. indica,E. arborescens,E. suberosa, and E. lithosperma

The lectins of the seeds of four species of the genus Erythrina, namely E. indica, E. arborescens, E. lithosperma, and E. suberosa were isolated by affinity chromatography on acid-treated ECD-Sepharose 6B. The lectins were found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. In SDS-gel electrophoresis, E. indica and E. lithosperma lectins each gave two bands with subunit molecular weights of 30,000 and 33,000 in the case of the former and 26,000 and 28,000 in the case of the latter. E. arborescens and E. suberosa gave single bands corresponding to polypetide chain molecular weight of 28,000. The lectins were found to be glycoproteins with their neutral sugar contents ranging from 4–9%. In carbohydrate specificity all the lectins were d-galactose specific. Their close similarity was also demonstrated by their homologous cross-reaction against the antiserum to E. indica lectin. In hemagglutinating activity toward human erythrocytes, E. indica and E. suberosa lectins showed higher activity toward the O group and E. arborescens toward the B group. The results show the similarity of the lectins derived from different species of the same genus in respect of immunochemical properties and carbohydrate specificity. In studies on E. indica lectin, the protein was found homogeneous by electrophoretic, immunochemical, and sedimentation experiments. Its molecular weight of 68,000 determined from sedimentation and diffusion data indicated that the molecule was a dimer of two noncovalently bound unequal subunits whose SDS-gel electrophoretic molecular weights are noted above. The lectin was devoid of cysteine and methionine and contained valine as its N-terminal amino acid. It had 9% neutral sugars and 1.5% glucosamine. Equilibrium dialysis studies with lactose showed that the values of the association constant K at different temperatures were of similar orders of magnitude to other lectins and the dimeric molecule possessed two noninteracting binding sites.     

Relationship between epitope density and immunoprecipitation of multivalent antigens by bivalent antibody: immunoprecipitation of adenylylated glutamine synthetase by anti-AMP antibodies

Escherichia coli glutamine synthetase (GS) preparations composed of 12 adenylylated subunits (GS₁₂^?) are almost completely precipitated by sheep Anti-AMP immunoglobulin G (IgG), whereas glutamine synthetase preparations containing 6 adenylylated subunits (GS₆^?) are only partially precipitated by the antibodies (R.J. Hohman, S.G. Rhee, and E.R. Stadtman, 1980, Proc. Nat. Acad. Sci. USA77, 7410–7414). By means of ¹²⁵I-labeled anti-AMP antibodies and double immunoprecipitation techniques, in which rabbit antiserum to sheep IgG or anti-GS antibodies were used to precipitate soluble immune complexes, it was demonstrated that under optimal conditions, both the soluble and insoluble immune complexes obtained with either GS₆^? or GS₁₂^? contain 0.5 mol antibody/mol adenylylated subunit. In agreement with the lattice theory of immuno-precipitation, soluble immune complexes are formed in antibody excess. Scatchard plots of binding data indicate that under conditions of antibody excess, one antibody molecule is bound to each AMP moiety of GS₁₂^?, whereas GS₆^? binds a maximum of only 0.68 antibody molecule/adenylylated subunit. We propose that with some species of GS₆^?, the distribution of adenylylated subunits favors monogamous interactions of the bivalent antibody with two subunits within the same GS molecule and thereby leads to the formation of small, soluble, immune complexes. Other explanations are considered. Only 30% of the antibody population that recognizes unconjugated 5′-AMP binds to the AMP moiety of adenylylated GS. Anti-AMP antiserum can be fractionated on a GS₁₂^?-Sepharose matrix into two subpopulations of antibody with strikingly different immunoprecipitation characteristics. Conversely, species of GS with various states of adenylylation ranging from 0 to 8 were separated from a GS₆^? preparation by means of affinity chromatography on an anti-AMP antibody-Sepharose matrix. Under optimal conditions, antibodies purified by affinity chromatography precipitated a smaller fraction of a GS₆^? preparation than did unfractionated antiserum. Competence of the purified antibody was nearly restored to that of the unfractionated serum by the addition of an enhancement factor present in the IgG fraction of nonimmune serum. The enhancement factor was not required for complete precipitation of GS^?₁₂ by purified antibodies. Contrary to most antibody-antigen reactions, immunoprecipitation of GS₆^? with anti-AMP antibodies is greater at 30 °C than at 4 °C.     

Limited proteolysis of liver and muscle aldolases: Effects of subtilisin,cathepsin B,and Staphylococcus aureus protease

Limited proteolysis of rabbit liver and muscle aldolases by subtilisin and cathepsin B results in decreased catalytic activity, associated with the release of acid-soluble peptides from the COOH terminus. Analysis of the sequence of these peptides confirms the COOH-terminal sequence of the muscle enzyme and provides new information on the COOH-terminal sequence of the liver enzyme. As previously reported for muscle aldolase, cathepsin B releases mainly dipeptides from the COOH terminus of liver aldolase. The COOH-terminal sequence of rabbit liver aldolase is SerThrGlnSerLeuPheThrAla SerTyrThrTyr. The Gln-Ser bond is resistant to Staphylococcus aureus protease, which hydrolyzes a GluSer bond at the corresponding positions in the muscle enzyme.     

Effects of lysolecithin on the surface properties of human erythrocytes

Human red blood cells (RBCs), transformed by incubation with the amphiphatic compound lysolecithin from their normal discocyte shape into echinocytes, have increased rates of agglutination in the presence of either poly- -lysine (PLL) or soybean agglutinin (SBA). Removal of lysolecithin by washing caused a reversal of shape back to the discocyte configuration and a lowering of agglutination rates. Methochlorpromazine, another amphiphatic echinocytogenic substance produced a similar increase in agglutination rates, suggesting that increased agglutinability may be a general property of echinocytes. Lysolecithin treatment of RBCs caused a decrease in the binding of cationized ferritin (CF) particles/μm² of RBC surface. The decrease in CF binding is due to a rearrangement of negative charge bearing molecules on the RBC surface rather than shedding of charged groups. These observations support the hypothesis that integral membrane proteins which bear negative charges and receptors are associated with a cytoskeleton within the red cell. Alterations in cell shape which result in distortion of the cytoskeleton may cause a redistribution of integral membrane proteins which bear charged groups at the RBC surface.     

pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The V_max for the demethylation reaction exhibited a pH optimum at approximately 4.5. The K_m for N,N-dimethylaniline increased with decreasing pH, while the K_m for ethyl hydroperoxide varied in a manner paralleling V_max. Comparison of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the V_max versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.     

Membrane (Na+ + K+)-ATPase of canine brain,heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures

Effects of commonly used purification procedures on the yield and specific activity of (Na⁺ + K⁺)-ATPase (Mg²⁺-dependent, Na⁺ + K⁺-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP-dependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na⁺ + K⁺)-ATPase and the ratio between (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na⁺ + K⁺)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na⁺ + K⁺)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzyme preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purification procedure. These results indicate that the presently used purification procedures may alter.     

Effects of density-dependent inhibition of growth on phospholipid metabolism in monolayer cultures of animal cells.

The onset of density-dependent inhibition of growth is correlated with a change in the synthesis of the major phospholipids of either BHK-21 (hamster) and 3T3 (mouse) cells. The change consists of inhibition of phosphatidylcholine synthesis while the synthesis of phosphatidylethanolamine remains constant or increases. The incorporation of radioactive precursors reflects turnover rates, since no significant differences were observed in phospholipid composition. No major differences in fatty acyl chains of these same phospholipids were observed as a function of growth rate despite considerable individuality of relative patterns of polar lipids in both cell lines. No other density-dependent differences in phospholipid metabolism were observed which were common to both cell lines. These results suggest that major aspects of growth regulation may be metabolic rather than compositional and that regulation of the enzymes of lipid metabolism may be useful probes of effector mechanisms in growth control.     

Enzymic hydration of benzene oxide: Assay and properties

A radiometric assay for epoxide hydratase using [¹⁴C]benzene oxide as substrate has been developed. The reaction product trans-1,2-[¹⁴C]dihydroxy-1,2-dihydrobenzene (benzene dihydrodiol) was separated from the other components by simple extraction of the unreacted substrate and phenol (a rearrangement product) into a mixture of light petroleum and diethyl ether followed by extraction of the benzene dihydrodiol into ethyl acetate. The product was then estimated by scintillation counting. Using this assay the enzymic hydration of benzene oxide and the possible existence of a microsomal epoxide hydratase with a greater specificity toward benzene oxide were reinvestigated. The sequence of activities of microsomes from various organs was liver > kidney > lung > skin, the pH optimum of enzymic benzene oxide hydration was about pH 9.0, which is similar to that of styrene oxide hydration and both activities were equally stable when liver microsomal fractions were stored. The effect of low molecular weight inhibitors upon the hydration of styrene and benzene oxide by liver microsomes was similar in some cases and dissimilar in others. However, all the dissimilarities could be explained without recourse to the hypothesis of the existence of a separate benzene oxide hydratase. During enzyme purification studies the activity toward benzene oxide was inhibited by the detergent used (cutscum) but was recovered when the detergent was removed. Solubilization without significant loss of activity was successful using sodium cholate. This allowed immunoprecipitation studies, which were performed using monospecific antiserum raised against homogeneous epoxide hydratase. The dose-response curves of the extent of precipitation of activity with increasing amounts of added antiserum were indistinguishable for benzene oxide and styrene oxide as substrate. At high antiserum concentrations precipitation was complete with both substrates. The findings, taken together, indicate the presence in rat liver microsomes of a single epoxide hydratase catalyzing the hydration of both styrene and benzene oxide or the presence of enzymes so closely related that these cannot be distinguished by any of the criteria tested.