Enzymatic properties of a neutral endo-1,3(4)-beta-xylanase Xyl II from Bacillus subtilis

A Bacillus sp. CCMI 966, characterised as Bacillus subtilis, has a duplication time of about 24 min. It produces at least two extracellular xylanases, Xyl I and Xyl II. The extracellular xylanase activity seems to be strongly correlated with the biomass growth profile. The Xyl II isoenzyme was purified by ammonium sulphate precipitation and anionic exchange chromatography, with a purification factor of 8.3. The molecular weight of the isoenzyme was estimated by SDS-PAGE revealing that Xyl II is a multimeric enzyme with a catalytic subunit of about 20 kDa. Under non-denaturing conditions, a molecular weight of about 340 kDa was obtained by native PAGE gel and of 20 kDa by gel filtration chromatography. The enzyme showed an optimum pH and temperature of 6.0 at 60 degrees C. Xyl II was stable at 40 degrees C for 180 min at pH 6.0. The specificity of Xyl II for different substrates was evaluated. Xyl II presents a higher affinity towards OSX, with a K(m) of 1.56 g l(-1) and showed the ability to hydrolyse laminarin, with a K(m) of 1.02 g l(-1). Xylotetraose is the main product of xylan degradation. The Xyl II ability for binding to cellulose and/or xylan was also studied.     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutase from Nicotiana plumbaginifolia leaves.

The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites.     

Cyclic AMP modulates electrical signaling in a weakly electric fish

Many species of electric fish show diurnal or socially elicited variation in electric organ discharge amplitude. In Sternopygus macrurus, activation of protein kinase A by 8-bromo-cAMP increases electrocyte sodium current magnitude. To determine whether the behavioral plasticity in electric organ discharge amplitude is controlled by electrocyte biophysical properties, we examined whether the effects of phosphorylation on ion currents in the electric organ translate directly into electric organ discharge changes. We injected the electric organ of restrained fish with 8-bromo-cAMP and monitored the electric organ discharge. The effect of protein kinase A activation on electrocyte action potentials was examined in isolated electric organ using two-electrode current clamp. Electric organ discharge and action potential amplitude and pulse duration increased in response to 8-bromo-cAMP. Pulse and action potential duration both increased by about 25%. However, the increase in electric organ discharge amplitude (approximately 400%) was several-fold greater than the action potential amplitude increase (approximately 40%). Resting membrane resistance decreased in electrocytes exposed to 8-bromo-cAMP. We propose that in the Thevenin equivalent circuit of the electric organ a moderate increase in action potential amplitude combined with a decrease in internal resistance produces a greater voltage drop across the external resistance (the water around the fish), accounting for the large increase in the externally recorded electric organ discharge.     

Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization,developmental change,in-vitro translation,and processing

The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of Pisum sativum L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa).Abbreviations IgG immunoglobulin G - kDa kilodalton - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehüser for help in the immunocytochemical studies.     

Oltipraz-induced decrease in the activity of cytosolic glutathione S-transferase in Schistosoma mansoni.

The activity of glutathione S-transferase (GST) decreased progressively in Schistosoma mansoni from mice treated with oltipraz (OPZ). However, the peroxidase activity of GST (selenium-independent) and selenium-dependent glutathione peroxidase was not affected by OPZ treatment. Purification and quantification of GST from worms after OPZ treatment indicated that the decrease in enzyme activity was greater than could be accounted for by the decrease in GST protein content. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with GST isoenzyme specific antisera revealed a slight decrease in the quantity of both 26 and 28 kDa GSTs. Fractionation of cytosolic GSTs from male S. mansoni by chromatofocusing resolved three major isoenzymes (SmI, II and III) and a minor form which eluted first from the column. SmI, II and III all had a molecular weight of about 28 kDa on SDS-polyacrylamide gel electrophoresis. However, on electrophoresis in the absence of SDS, the three GST forms exhibited different mobilities. The pattern of SmI, II and III was similar in untreated and OPZ-treated worms, but the activities of the isoenzymes from treated worms were lower. The results suggest that OPZ interacts with the GST isoenzymes SmI, II and III in a similar manner; thus, the effects are not isoenzyme specific. Taken together, these results suggest that OPZ and/or its metabolites interact directly with GST resulting in inhibition of activity and reduction in total enzyme protein. This mechanism may be important in the antischistosomal action of OPZ.     

Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp

We isolated cytoplasmic glycocyamine kinase (GK) and creatine kinase (CK) from the tropical marine worm Namalycastis sp. by ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-5PW chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the isolated GK is highly purified and appears to be a heterodimer of two distinct subunits, alpha and beta, with molecular masses of approximately 40 kDa. The complete nucleotide sequences of the cDNAs for Namalycastis GKalpha and GKbeta were 1527 (encoding 374 amino acids) and 1579 bp (encoding 390 amino acids), respectively. The predicted amino acid sequences differ only in the N-terminal 50 residues. This is consistent with the characteristics of Neanthes GKalpha and GKbeta chains, which we have previously shown to be generated by alternative splicing. The recombinant enzymes GKalpha, GKbeta, and CK from Namalycastis were successfully expressed in Escherichia coli as maltose-binding protein fusion proteins. In contrast to the stable GKbeta enzyme, GKalpha was quite unstable, and its activity decreased remarkably with time. Thus, the N-terminal 50 residues appear to play a key role in enzyme stability. The kinetic parameters for the native GK heterodimer were similar to GKbeta, suggesting that GKalpha would have an activity similar to GKbeta if part of a heterodimer. This is the first report of precise kinetic parameters for GK. Finally, based on our results, we present a model for pluriphosphagen function in Namalycastis wherein cytoplasmic GK and CK and mitochondrial CK function together with phosphocreatine and phosphoglycocyamine to enable cells to respond quickly to a sudden large energy requirement.     

Purification and characterization of polygalacturonase from banana fruit

Polygalacturonase isoenzyme 3 (PG-3) was purified to homogeneity with a specific activity of 0.7 mu katal mg-1 protein from banana fruit pulp. The purified enzyme was a glycoprotein with ca. 8% carbohydrate. The molecular weight of the native enzyme was found to be 90 +/- 10 kDa with a subunit molecular weight of 29 +/- 2 kDa. The enzyme exhibited optimum activity at pH 4.3 and temperature 40 degrees C with activation energy 35.4 kJ mol-1. A unique property of the enzyme was the requirement of -SH groups for the enzyme activity. The enzyme was inhibited by p-CMB and activated by 2-ME and DTT. The inhibition of p-CMB could be reversed by DTT. The enzyme contained eight free -SH groups. The Km of the enzyme was 0.15% for polygalacturonic acid.     

Alpha-actinin in the electric organ of Electrophorus electricus, L

We have identified Alpha-actinin from the electric organ of the Electrophorus electricus, L. It was analysed by polyacrylamide gel electrophoresis, and identified by immunoblotting. This protein was also found in a membrane fraction of the electric organ enriched with components of the cytoskeleton. Our results suggest that this protein might play a role either in the organization of the microfilaments or its interactions with the membrane to maintain a polarized electrocyte.     

Localization of phosphatidylinositol 4-kinase isoenzymes in rat liver plasma membrane domains

The presence of different isoenzymes of phosphatidylinositol 4-kinase in isolated rat liver plasma membranes and their further distribution in plasma membrane domains was examined. Both wortmannin-sensitive and -insensitive PtdIns 4-kinase activities were detected in highly purified plasma membranes obtained by aqueous two-phase affinity partitioning. The wortmannin-sensitive enzyme was identified as the 230 kDa isoform by Western blotting, whereas the 92 kDa isoform was not detected in plasma membranes. The apparent molecular weights of these isoforms were 205 and 105 kDa on SDS polyacrylamide gel electrophoresis, but approximately 500 and 230 kDa respectively on gel filtration, suggesting that both enzymes either are dimers or composed of heterologous subunits. Approximately 25% of the total 230 kDa isoenzyme present in liver, and only ca 5% of the wortmannin-insensitive one, was associated with the plasma membrane fraction. Plasma membrane domains were isolated by a combination of sucrose and Nycodenz gradient centrifugations. The 230 kDa isoform was identified in the blood sinusoidal domain, but not in the bile canalicular one, and was also found in lateral plasma membranes. The wortmannin-insensitive isoenzyme was present only in this latter material. The functional implications of this distribution of PtdIns 4-kinase isoenzymes in plasma membrane regions are discussed.     

Polarized distribution of Na+, K(+)-ATPase alpha-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na+, K(+)-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the alpha subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of alpha isoforms (alpha1 and alpha2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K(+)-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0+/-0.1 microM) than the activity of innervated membranes (I50=2.6+/-0.2 microM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K(+)-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of alpha1 and alpha2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K(+)-ATPase alpha-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Growth-rate-related and hydroxysterol-induced changes in membrane fluidity of cultured hepatoma cells: correlation with 3-hydroxy-3-methyl glutaryl CoA reductase activity

Creatine kinase (CK) activity in plasma obtained non-invasively from adult healthy, Sprague-Dawley, male rats was found to be 528 +/- 270 U/L (N = 17), a value which was 7 times that obtained in human specimens. Agarose gel electrophoresis revealed that the only detectable CK isoenzyme present was CK-BB, in contrast to the human serum isoenzyme which was CK-MM. Furthermore, it was found that the rat CK-BB could be detected using an RIA technique designed to quantitate human CK-BB occasionally present in blood after brain injury (rat CK-BB = 84.5 +/- 55.2 micrograms/L, N = 17, human CK-BB: Not detectable). It was thus possible to calculate the CK-BB specific activity (SA) in rat plasma using total CK assay and RIA (rat CK-BB SA = 6.25 +/- 3.87 U/micrograms, N = 17). When six rats (156 +/- 23 g) were treated with lead acetate in the drinking water (26 mM) for 3 weeks, the CK-BB SA rose to 18 +/- 5.8 U/micrograms (P less than .02). At this point the electrophoresis pattern of the CK-BB showed a transient change from a single band to a doublet. The dose was then increased to 52 mM for 6 weeks, during which time the CK-BB SA declined steadily to 1.6 +/- 0.6, a level significantly less than that of the untreated animals (p less than .02). The results suggest that chronic lead treatment evokes a biphasic response in CK-BB SA with the initial release of enzyme of high SA from tissues. Further treatment apparently results in an inactivation of the enzyme within lead sensitive tissues.     

Purification and characterization of alpha-amylase from the infective juveniles of the nematode Heterorhabditis bacteriophora

Glycogen content and alpha-amylase activity were estimated in the infective juveniles (IJs) of Heterorhabditis bacteriophora at different times of storage. The glycogen content declined from 5.8 to 2.5 ng/IJ during storage for 40 days at 27 degrees C. The change in glycogen content coincided with the change of alpha-amylase activity during storage. alpha-Amylase was purified from IJs at zero time of storage by ion exchange chromatography and gel filtration. Ion exchange chromatography resolved alpha-amylase into three isoenzymes. The major isoenzyme alpha-amylase I had the highest specific activity and was purified to homogeneity. A molecular mass of 46-47 kDa was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. The Km values were 6.5 and 9.6 mg/ml using starch and glycogen as substrates, respectively. alpha-Amylase I showed optimum activity at pH 7.0 and had an optimum temperature of 40 degrees C. The enzyme was unstable at temperatures above 40 degrees C. The enzyme activity was severely inhibited by EDTA, p-CMB and iodoacetic acid, but potentiated by CaCl2 and NaCl. These results are discussed and compared with previously reported alpha-amylases in the insect hosts of the parasite.     

Electric organ morphology of Sternopygus macrurus, a wave-type, weakly electric fish with a sexually dimorphic EOD.

In several species of electric fish with a sex difference in their pulse-type electric organ discharge (EOD), the action potential-generating cells of the electric organ (electrocytes) of males are larger and more invaginated compared to females. Androgen treatment of females and juveniles produces a longer-duration EOD pulse that mimics the mature male EOD, with a concurrent increase in electrocyte size and/or membrane infolding. In Sternopygus macrurus, which generates a wave-type EOD, androgen also increases EOD pulse duration. To investigate possible morphological correlates of hormone-dependent changes in EOD in Sternopygus, we examined electric organs from both fish collected in the field, and untreated and androgen-treated specimens in the laboratory. The electrocytes are cigar shaped, with prominent papillae on the posterior, innervated end. Electrocytes of field-caught specimens were significantly larger in all parameters than were electrocytes of specimens maintained in the laboratory. EOD pulse duration and frequency were highly correlated, and were significantly different between the sexes in sexually mature fish. Nevertheless, no sex difference in electrocyte morphology was observed, nor did any parameters of electrocyte morphology correlate with EOD pulse duration or frequency. Further, whereas androgen treatment significantly lowered EOD frequency and broadened EOD pulse duration, there was no difference in electrocyte morphology between hormone-treated and control groups. Thus, in contrast to results from studies on both mormyrid and gymnotiform pulse fish, electrocyte morphology is not correlated with EOD waveform characteristics in the gymnotiform wave-type fish Sternopygus. The data, therefore, suggest that sex differences in EOD are dependent on changes in active electrical properties of electrocyte membranes.     

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

Purification and characterization of an NADH oxidase from Eubacterium ramulus

An NADH oxidase from the strictly anaerobic Eubacterium ramuluswas purified to homogeneity. The enzyme is composed of two types of subunits with molecular masses of 40 and 30 kDa. The molecular mass of the native enzyme is 450 kDa according to gel filtration and PAGE analysis. Six to eight mol of FAD were found per mol of native enzyme. The NADH-specific enzyme was inhibited by N-bromosuccinimide and sulfhydryl reagents such as N-ethylmaleimide, CuCl(2) or ZnCl(2). The physiological function of the purified enzyme is unclear, but the demonstration of NADH-dependent O(2)-consumption suggests that it plays a role in the scavenging of oxygen.     

Evaluation of a new radioimmunological assay for the determination of the B subunit of creatine kinase enzyme

We used a new radioimmunological (RIA) kit for the assay of B subunit of creatine kinase enzyme (CK). This RIA system uses a specific antisera against the B subunit as ligant, human CK-BB labelled with 125I as tracer, and purified human CK-BB isoenzyme as standard. The mean (+/- SD) sensitivity obtained was 0.25 +/- 0.16 ng/tube and the between assay variability was about 9-10%. Serum levels of 113 normal subjects was not normally distributed. The 95% of values was found below 5 ng/ml. This new RIA is usefull in clinical practice when serum levels of CK-BB isoenzyme must be determined. This method is quickly and it is characterized by a good degree of precision, but the CK-MB isoenzyme cross-reacts for about 40% in this RIA system. Therefore, for the clinical diagnosis by means of this RIA it is necessary to rule out the concomitant elevations of serum CK-MB values.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.