Regulation of Glutamine Synthetase X. Effect of Growth Conditions on the Susceptibility of Escherichia coli Glutamine Synthetase to Feedback Inhibition

The kinetic properties of Escherichia coli glutamine synthetase are markedly influenced by the manner in which the organism is grown. Enzyme obtained from stationary-phase cells grown on glycerol and glutamate is strongely inhibited by each of the eight feedback effectors known to influence this enzyme; however, the enzyme from log-phase cells grown on glucose and growth-limiting concentrations of NH(4)Cl is stimulated by some of these effectors. Of the growth variables examined, nitrogen source and time of harvest were the most important; carbon source and aeration seemed to have no effect. Two purified enzyme preparations have been obtained from cells grown under two different conditions, designated enzymes I and II for convenience. Enzyme I is stimulated by adenosine 5'-monophosphate, histidine, and tryptophan in the transfer assay, whereas enzyme II is strongly inhibited by all effectors tested. Enzyme I has a higher specific activity in the forward assay in the presence of Mg(++) or Co(++), whereas enzyme II is more active in the presence of Mn(++).     

Coagglutination and enzyme capture tests for detection of Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.     

Coagglutination and enzyme capture tests for detection of Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.     

Sugar transport by the bacterial phosphotransferase system. Characterization of the sulfhydryl groups and site-specific labeling of enzyme I

Enzyme I is the first protein of the phospho transfer sequence in the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This protein exhibits a temperature-dependent monomer/dimer equilibrium. The nucleotide sequence of Escherichia coli ptsI indicates four -SH residues per subunit (Saffen, D. W., Presper, K. A., Doering, T. L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253). In the present experiments, the sulfhydryl groups of the E. coli enzyme were studied with various -SH-specific reagents. Titration of Enzyme I with 5,5'-dithiobis-2-nitrobenzoic acid also revealed four reacting -SH groups. The kinetics of the 5,5'-dithiobis-2-nitrobenzoic acid reaction with Enzyme I exhibit biphasic character, with pseudo-first order rate constants of 2.3 x 10(-2)/s and 2.3 x 10(-3)/s at pH 7.5, at room temperature. Fractional amplitudes associated with the rate constants were 25 +/- 5% for the fast and 75 +/- 5% for the slow rate. The "slow" rate was influenced by ligands that react with Enzyme I (the protein HPr, Mg2+, Mg2+ plus P-enolpyruvate), and also by temperature (at the temperature range where the monomer/dimer association occurs). The fractional ratio of the two rates remained at 1:3 under these conditions. Thus, under all conditions tested, two classes of -SH groups were detected, one reacting more rapidly than the other three -SH groups. Modification of the "fast" -SH group results in an active enzyme capable of forming dimer, whereas modification of the slow -SH groups results in inactive and monomeric Enzyme I. The enzyme was labeled with pyrene maleimide under conditions where only the more reactive sulfhydryl group was derivatized. Hydrolysis by trypsin followed by reverse-phase high performance liquid chromatography analysis of the peptide mixture resulted in only one fluorescent peak. This peak was not observed when the more reactive sulfhydryl residue was protected prior to pyrene maleimide labeling. Amino acid sequencing of the fluorescent peak indicated that the more reactive residue is the C-terminal amino acid residue, cysteine 575. The results provide a means for selectively labeling Enzyme I with a fluorophore at a single site while retaining full catalytic activity.     

Isolation,partial purification,biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis,Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37?°C for 48?h with a constant agitation of 120?rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1?M ice cold CaCl₂ and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60?s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.     

Application of rapid enzyme assay techniques for monitoring of microbial water quality

Rapid enzyme assay techniques based on direct measurement of beta-d-galactosidase (GALase) or beta-d-glucuronidase (GLUase) activity without selective cultivation are used for rapid estimation of the level of coliform bacteria and Escherichia coli in water samples. Reported detection limits using fluorogenic substrates correspond to culturable target bacteria concentrations that can be appropriate within present guidelines for recreational waters. The rapidity, that is detection within one hour, compromises the specificity of the assay; enzyme activity contributions from other than target bacteria need to be considered, particularly at low levels of target bacteria. Enzyme activities are more persistent than the culturability of target bacteria to environmental and disinfection stress, thus water samples may express enzyme activities of both culturable and viable non-culturable cells.     

Effect of seawater on Escherichia coli β-galactosidase activity

An investigation of β-galactosidase activity of Escherichia coli strain H10407, under different physiological and environmental conditions, e.g. induced and uninduced osmotic stress, light, etc., was undertaken. In this study E. coli was employed as a model for faecal coliforms in waste water. β-Galactosidase activity was induced by isopropyl-β-D-thiogalactoside (IPTG). Enzyme activity (U cell^-1)/cell for sewage bacteria and for induced E. coli was similar, i.e. log U cell^-1= -8.5 whereas uninduced E. coli yielded log U cell^-1= -12.1. Initial enzyme activity was not dependent on phase of growth of the cell (exponential vs stationary phase) or whether marine or fresh water at the time of initial dilution. However, osmotic change resulted in a decrease in culturable cells, even though enzyme activity remained constant. A significant decrease in the number of culturable bacteria, followed by a decrease in β-galactosidase activity, was observed after exposure of cells to visible light radiation. It is concluded that β-galactosidase enzyme is retained in viable but non-culturable E. coli. Furthermore, β-galactosidase appears to offer a useful and rapid (25 min) measure of the viability of faecal coliforms, and therefore, of the water quality of bathing and shellfishing areas.     

Measurement of folylpolyglutamate synthetase in mammalian tissues

Previous methods for the measurement of folylpolyglutamate synthetase have been modified and combined to facilitate assay of this enzyme at the levels found in mammalian tissues. Batch adsorption of product onto charcoal allowed the rapid analysis of multiple samples of partially purified enzyme, e.g., column fractions. This technique, however, was unsuitable for the assay of folylpolyglutamate synthetase in crude cytosols due to the presence of interfering enzyme activities. On the other hand, the sequential use of charcoal adsorption and batch elution from DEAE-cellulose permitted isolation of the folate product from assay mixtures containing crude enzyme fractions. Under these conditions, interference from other enzyme activities and background values were low enough for the quantitation of 10 pmol of oligoglutamyl folate product. Folylpolyglutamate synthetase was measured in a series of mouse tissues and tumors. Enzyme activity was quite low in all cases. Mouse liver and kidney and some of the tumors studied had the highest levels (50-100 pmol product/h/mg protein); other tumors and spleen had lower levels. Enzyme activity was at the limit of detection in intestine and lung and was below detection in brain, heart, and skeletal muscle.     

A rapid TLC bioautographic method for the detection of acetylcholinesterase and butyrylcholinesterase inhibitors in plants

A simple and rapid bioautographic enzyme assay on TLC plates has been developed for the screening of acetylcholinesterase and butyrylcholinesterase inhibition by plant extracts. Enzyme activity was detected by the conversion of naphthyl acetate into naphthol and the formation of the corresponding purple-coloured diazonium dye with Fast Blue B salt. Inhibitors of cholinesterases produced white spots on the dye-coloured background of the TLC plates. The alkaloids galanthamine and physostigmine, which are known inhibitors of acetylcholinesterase, were used to determine the sensitivity of the assay. Various plant extracts were tested using the bioassay.     

Plant and algal interference in bacterial beta-D-galactosidase and beta-D-glucuronidase assays.

Several commonly occurring freshwater and marine plants and algae were screened for beta-D-galactosidase and beta-D-glucuronidase activities by using a 60-min enzyme assay based on the hydrolysis by these enzymes of 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl- beta-glucuronide, respectively. All freshwater plant extracts tested showed beta-D-galactosidase activity several at relatively high levels, and a number also showed beta-D-glucuronidase activity. A number of the macroalgae showed no activity of either enzyme, but those showing beta-D-galactosidase activity also showed beta-D-glucuronidase activity. The majority of microalgae showed some beta-D-galactosidase activity, but few showed beta-D-glucuronidase activity. Further studies, using the commercial Colilert test and the marine water formulation of Colilert, revealed that 2 of 11 of the microalgal species and several of the plant extracts tested caused positive reactions. It was concluded that several plant extracts and algae could significantly interfere with the detection of coliform bacteria and Escherichia coli with the use of rapid assays, on the basis of their production of beta-D-galactosidase and beta-D-glucuronidase, respectively. The significance of the plant and algal interferences in tests such as Colilert is dependent on the levels of enzymes released under natural conditions, the dilution which they may undergo, and the numbers of algal cells present. This also applies to interferences in rapid enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Enzyme Regulation during Growth of Maize: I. Progressive Desensitization of Homoserine Dehydrogenase during Seedling Growth

The sensitivity of homoserine dehydrogenase (EC 1.1.1.3) to inhibition by the feed-back modifier, l-threonine, was examined in preparations derived from etiolated shoots, roots, and lightgrown tissues of Zea mays L. var. earliking. A progressive decrease in enzyme sensitivity was observed during seedling growth. Enzyme derived from internode tissue retained a greater sensitivity to the effector than enzyme derived from apical portions of etiolated shoots, whereas enzyme from root tips was characteristically more sensitive than that prepared from mature cells of the root. Enzyme desensitization occurred rapidly during culture of excised shoots and the activities of both homoserine dehydrogenase and aspartokinase (EC 2.7.2.4) declined during shoot culture under a variety of conditions. The initial enzyme levels and the characteristic sensitivity of homoserine dehydrogenase were preserved during culture at 5 to 7 C, but desensitization was not prevented by inclusion of cycloheximide in the culture medium.Results of control experiments provide evidence that desensitization occurs in vivo. No alteration of the enzyme properties was detected during extraction or concentration of sensitive or insensitive enzyme or during coextraction of enzyme from mixed populations of different age shoots; nor was a differential distribution of inhibitors or activators indicated during assay of mixed preparations. The change in enzyme sensitivity was apparent under a variety of assay conditions and was not accompanied by changes in the apparent affinity of the enzyme for the substrate, homoserine. It is suggested that systematic changes in the regulatory characteristics of certain enzymes could be an important level of metabolic regulation during cellular differentiation.Three forms of maize homoserine dehydrogenaase were detected after acrylamide gel electrophoresis of samples derived from 72-hr shoots. Similar analysis of samples from older shoots revealed a broad asymmetric band of enzyme activity, suggesting that changes in the relative distribution of specific forms of the enzyme could be related to the growth-dependent changes in the sensitivity of maize homoserine dehydrogenase.     

The Escherichia coli adenylate cyclase complex: Activation by phosphoenolpyruvate

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

Biosynthesis of biotin: synthesis of 7,8-diaminopelargonic acid in cell-free extracts of Escherichia coli

Cell-free extracts prepared from a biotin auxotroph of Escherichia coli were active in catalyzing the synthesis of 7,8-diaminopelargonic acid, an intermediate of the biotin pathway, from 7-oxo-8-aminopelargonic acid. The product was identified on the basis of its chromatographic characteristics and its biotin activities for biotin auxotrophs of E. coli. Enzyme activity was determined in a reaction coupled with the desthiobiotin synthetase system, which is required for the conversion of 7,8-diaminopelargonic acid to desthiobiotin, and by measuring the amount of desthiobiotin formed by microbiological assay. The reaction was stimulated by l-methionine and pyridoxal-5'-phosphate. l-Methionine could not be replaced by any other amino acids tested. Pyridoxamine and pyridoxamine-5'-phosphate were as active as pyridoxal phosphate. The enzyme, presumably an aminotransferase, was demonstrable in the parent strain of E. coli and all mutant strains tested with the exception of a strain which is able to grow on diaminopelargonic acid but not on 7-oxo-8-aminopelargonic acid. Furthermore, the enzyme was repressible by biotin. The results were consistent with the hypothesis that the biosynthesis of 7,8-diaminopelargonic acid from 7-oxo-8-aminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.     

“Enzyme Test Bench,” a high‐throughput enzyme characterization technique including the long‐term stability

A new high throughput technique for enzyme characterization with specific attention to the long term stability, called “Enzyme Test Bench,” is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests in 96‐well microtiter plates under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimal non‐linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The engineered instrumentation provides for simultaneous automated assaying by fluorescent measurements, mixing and homogenous temperature control in the range of 10–85 ± 0.5°C. A universal fluorescent assay for online acquisition of ester hydrolysis reactions by pH‐shift is developed and established. The developed instrumentation and assay are applied to characterize two esterases. The results of the characterization, carried out in microtiter plates applying short term experiments of hours, are in good agreement with the results of long term experiments at different temperatures in 1 L stirred tank reactors of a week. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature regarding such process parameters as turn over number, space time yield or optimal process duration. The comparison of the temperature dependent behavior of both characterized enzymes clearly demonstrates that the frequently applied estimation of long term stability at moderate temperatures by simple activity measurements after exposing the enzymes to elevated temperatures may lead to suboptimal enzyme selection. Thus, temperature dependent enzyme characterization is essential in primary screening to predict its long term behavior. Biotechnol. Bioeng. 2009;103: 305–322. © 2008 Wiley Periodicals, Inc.     

Microbial metabolism of amino alcohols. Control of formation and stability of partially purified ethanolamine ammonia-lyase in Escherichia coli.

Induction of ethanolamine ammonia-lyase formation in Escherichia coli required both the ethanolamine and vitamin B12, and was gratuitous during growth on glycerol. Ethanolamine analogues inhibited enzyme activity and inhibited growth with ethanolamine as the the nitrogen source, but did not act as inducers. Enzyme formation was more rapid when ethanolamine was added to cultures containing vitamin B12 rather than the reverse. Enzyme formation was subject to catabolic repression, glucose and acetate being particularly effective. Chloramphenicol, I-aminopropan 2-01 and 1,3-diaminopropan-2-01 prevented enzyme induction. Ethanolamine ammonia-lyase, resolved from its cobamide coenzyme, was purified 35-fold. The apoenzyme was stable for several days in the presence of ethanolamine, dithiothreitol, glycerol and K+ ions. Enzyme formation therefore requires both substrate and cobamide coenzyme to be present simultaneously as inducers.     

Residual activity of human porphobilinogen deaminase with R167Q or R167W mutations: an explanation for survival of homozygous and compound heterozygous acute intermittent porphyrics.

To find an explanation for survival of homozygous or compound heterozygous variants of acute intermittent porphyria, we studied the three mutant forms of porphobilinogen deaminase (PBG-d) described in the four reported patients with homozygous acute intermittent porphyria. Wild-type human PBG-d and the PBG-d R167W, R167Q and R173Q mutants were expressed in Escherichia coli and the recombinant mutant human enzyme were examined for enzyme activity. Specific antibodies against human PBG-d detected the three human PBG-d mutants. All three had less than 2% of wild-type enzyme activity when examined under customary assay conditions (pH 8.0), but the R167W and R167Q mutants were found to have about 25% of normal activity when assayed at pH 7.0. This residual activity at a more physiological pH provides an explanation for survival when these mutations are inherited in a homozygous or compound heterozygous fashion.     

Production of a recombinant carotenoid cleavage dioxygenase from grape and enzyme assay in water-miscible organic solvents

A recombinant carotenoid cleavage dioxygenase from Vitis vinifera L. was produced by Escherichia coli as a fusion with the glutathione-S-transferase (GST) protein under different bacterial growth conditions. The enzyme production was monitored by a GST assay. Addition of Triton X-100 prior to bacterial cell disruption doubled the release of soluble protein. A simple spectrophotometric enzyme assay was developed to measure carotenoid cleavage activity using lutein as substrate. Enzyme activity showed a 26-fold increase with the addition of 10% (v/v) acetone in the reaction mixture.     

Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface.

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.     

The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system.

The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene.     

Comparative immunological studies on arylsulfatase in bacteria of the family Enterobacteriaceae: occurrence of latent arylsulfatase protein regulated by sulfur compounds and tyramine.

The arylsulfatases of 21 strains of the family Enterobacteriaceae were compared by measuring their enzymatic activities and immunological reactivities. Enzyme formation under repressing, nonrepressing, and derepressing conditions was tested. Antiserum prepared against pure arylsulfatase from Klebsiella aerobgenes W70 was tested against the enzyme extracts from the strains using double diffusion, quantitative precipitation, and immunoelectrophoresis. No close relationship was found between arylsulfatase activity and immunological cross-reactionship was found between arylsulfatase activity and immunological cross-reactivity. The strains in the family Enterobacteriaceae could be divided into two groups on the basis of the immunological properties of their enzyme. Antisera formed a precipitin band with both active and inactive enzyme proteins from Escherichia, Citrobacter, Salmonella, Klebsiella, and Enterobacter, but not with the proteins from Serratia, Proteus, and Erwinia, even though some strains of these species had enzyme activity. It was also found that the formation of arylsulfatase proteins, irrespective of whether they had enzyme activity, were under regulation by sulfur compounds and tyramine.