Production of pyridoxal phosphate by a mutant strain of Schizosaccharomyces pombe

Conditions for extracellular production of vitamin B6 compounds (B6), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leul strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B6 and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B6 and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H202 increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and, in contrast, increased pyridoxine.     

Further Characterization of Glycolaldehyde Dehydrogenase Isozymes from Escherichia coli B Involvement in Vitamin B6 Biosynthesis

Kinetic study of glycolaldehyde dehydrogenase was performed with isozymes of Escherichia coli B. The reaction equilibrium of isozyme A was estimated to lie in glycolate formation, while those of isozymes B and C were in glycolaldehyde formation. Isozyme A was released from cells with osmotic shock, while the others were not. Isozymes B and C were found in cytoplasmic fraction. Some reversal mutants derived from WG3 strain (one of vitamin B₆ auxotrophs) acquired ability to produce isozyme C. Based on these results, the non-involvement of isozyme A in vitamin B₆ biosynthesis was discussed.     

Mechanism of thiamine-induced respiratory deficiency in Saccharomyces carlsbergensis.

Cells of Saccharomyces carlsbergensis 4228 grown aerobically with added thiamine (1 microgram . ml-1) in a vitamin B6-free medium contained no detectable heme precursors, such as delta-aminolevulinate, coproporphyrin III, or protoporphyrin IX. The deficiency in heme precursors in the thiamine-grown cells was accompanied by previously reported phenomena, i.e., growth depression, vitamin B6 deficiency, and respiratory deficiency due to a marked decrease in the activities of heme-containing enzymes and cytochrome level (I. Nakamura et al., FEBS Lett. 62: 354-358, 1976). It has been reported that all of the effects of thiamine are abolished by adding pyridoxine to the medium. delta-Aminolevulinate was found to have quite similar effects to those of pyridoxine, except that growth was partially improved by delta-aminolevulinate, whereas it was fully restored by pyridoxine. Incubation of the thiamine-grown cells with delta-aminolevulinate resulted in the appearance of the heme precursors and the heme-containing enzymes. Consistent with the lowered amount of vitamin B6, the thiamine-grown cells had a lowered activity of delta-aminolevulinate synthase, a pyridoxal phosphate-dependent enzyme. Not only the holoenzyme activity but also the apoenzyme activity was very low in these cells. These results indicate that the thiamine-induced vitamin B6 deficiency brings about the decrease in delta-aminolevulinate synthase activity, which leads to heme deficiency and therefore to respiratory deficiency.     

Effect of the extracellular matrix on plasminogen activator isozyme activities of human mammary epithelial cells in culture.

Multiple molecular forms of plasminogen activator were detected in normal human mammary epithelial cells in culture. Cells derived from (normal) breast mammoplasty specimens and grown on the surface of collagen gels exhibited three major classes of plasminogen activator isozymes (Mr = 100,000 [100K], 75,000 [75K], and 55,000 [55K]). The activity of the 100K and 75K isozymes was greatly reduced when the cells were grown on conventional tissue-culture-grade plastic surfaces. MCF-7, a human mammary carcinoma cell line, exhibited predominantly or exclusively the 55K isozyme, irrespective of the cell growth substratum. The activity of the 55K isozyme was more than twofold higher for MCF-7 cells grown on collagen gels than for cells grown on plastic. Progesterone, diethylstilbestrol, and estrogen stimulated the activity of the 55K isozyme of MCF-7 cells, but only when the cells were grown on a plastic surface. The plasminogen activator activities of the normal human mammary epithelial cells were not stimulated by these hormones, irrespective of the growth substratum. These results show that the expression of plasminogen activator isozymes by human mammary epithelial cells is subject to modulation by the extracellular matrix. Normal and malignant cells may differ in their responsiveness to these effects.     

Secretion of hexosaminidase isozymes by Tetrahymena.

Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A1 and B1, and 2 minor forms, A2 and B2, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B1 has a molecular weight of approximately 93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase B1 has a molecular weight of approximately 93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A1 has a molecular weight of approximately 170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-beta-D-galactosaminide than the B forms. Neither hexosaminidases A1 or B1 has any endo-beta-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete approximately 20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxybenzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.     

Interconversions of different forms of vitamin B6 in tobacco plants

There are six different vitamin B₆ (VB₆) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP), and pyridoxine 5′-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB₆ in the human diet, and VB₆ plays an important role in plants, the mechanisms underlying the interconversions of different VB₆ forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B₆ vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB₆ was about 3.9 μg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB₆ by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B₆ vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.     

Disruption of the plr1+ gene encoding pyridoxal reductase of Schizosaccharomyces pombe

Pyridoxal (PL) reductase encoded by the plr1(+) gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN). The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B(6), or the excretion of PL as PN from yeast cells. In this study, a PL reductase-disrupted (plr1 Delta) strain was constructed and its phenotype was examined. The plr1 Delta cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media. In EMM, the plr1 Delta strain became flocculent at the late stationary phase for an unknown reason. The plr1 Delta cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1(+) gene, which maintained the flow of "PL --> PN --> PNP --> PLP" in the salvage synthesis of PLP. The total vitamin B(6) and pyridoxamine 5'-phosphate contents in the plr1 Delta cells were significantly lower than those in the wild-type ones. The percentages of the PLP amount as to the other vitamin B(6) compounds were similar in the two cell types. The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type. In contrast, PN was much higher in the latter than the former. The plr1 Delta cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL. The results showed that PL reductase encoded by the plr1(+ )gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis.     

Antitumor effect of vitamin B6 and its mechanisms

Epidemiological studies have reported an inverse association between vitamin B(6) intake and colon cancer risk. Our recent study has been conducted to examine the effect of dietary vitamin B(6) on colon tumorigenesis in mice. Mice were fed diets containing 1, 7, 14 or 36 mg/kg pyridoxine for 22 weeks, and given a weekly injection of azoxymethane (AOM) for the initial 10 weeks. Compared with the 1 mg/kg pyridoxine diet, 7, 14 and 35 mg/kg pyridoxine diets significantly suppressed the incidence and number of colon tumors, colon cell proliferation and expressions of c-myc and c-fos proteins. Supplemental vitamin B(6) lowered the levels of colonic 8-hydroxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE, oxidative stress markers) and inducible nitric oxide (NO) synthase protein. In an ex vivo serum-free matrix culture model using rat aortic ring, supplemental pyridoxine and pyridoxal 5'-phosphate (PLP) had antiangiogenic effect. The results suggest that dietary vitamin B(6) suppresses colon tumorigenesis by reducing cell proliferation, oxidative stress, NO production and angiogenesis.     

Impairments in pyridoxine-dependent sulphur amino acid metabolism are highly sensitive to the degree of vitamin B6 deficiency and repletion in the pig

The objectives of the current study included the characterization of the temporal changes in indices of sulphur amino acid metabolism in piglets in response to vitamin B6 deficiency and repletion with graded levels of pyridoxine hydrochloride. In Experiment 1, 12 piglets (average initial weight = 5.3 kg; n = 6 per group) were fed a semi-purified diet containing either 0 (deficiency group) or 3 mg (control group) pyridoxine·HCl/kg diet, using a pair-feeding design, for 6 weeks. Piglets consuming vitamin B6-deficient diets exhibited decreased average daily gains on the 4th week and feed conversion efficiency from the 4th week until the end of the trial (P < 0.05). Plasma pyridoxal-5'-phosphate (PLP), in pigs consuming vitamin B6-deficient diets, was significantly lower than controls throughout the experiment (P < 0.01), reaching a nadir of 14% of the control animals' value by the end of the trial. Indices of sulphur amino acid metabolism, including activities of hepatic cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CGL) and serine hydroxymethyltransferase, as well as hepatic-free cysteine concentrations were markedly decreased after 6 weeks of B6 deficiency (P < 0.05). Total hepatic mRNA expressions for CBS and CGL were not affected. Concurrently, hepatic-free homocysteine concentrations increased by more than eight-fold (P < 0.01) at the end of the trial. An examination of plasma total homocysteine and cysteine concentrations revealed significant (P < 0.05) differences between treatments, with evidence of an abrupt shift in concentrations at 3 weeks post-initiation of dietary treatments (>25-fold increase in homocysteine; halving of cysteine values). At the end of Experiment 1, vitamin B6 deficiency significantly increased plasma methionine and serine levels, but decreased plasma glycine concentrations (P < 0.05). In Experiment 2, 20 pigs of 14 days old (initial BW = 5.0 kg) were subjected to a 4-week vitamin B6 depletion protocol, based on results obtained in Experiment 1. After the depletion period and assessment of baseline status (four pigs), remaining pigs were allocated to one of four dietary vitamin B6 repletion treatments: 0.75, 1.5, 2.25 and 3 mg/kg diet as pyridoxine·HCl (n = 4 per level) for 14 days. Significant dose-dependent increases in plasma PLP and cysteine, and decreases in homocysteine were observed, and these were sensitive to the duration of repletion. In conclusion, data from the current studies support the use of both plasma PLP and homocysteine as sensitive indices of vitamin B6 status in the pig. Additionally, the observed patterns of responses in vitamin B6-sensitive metabolites are supportive of an inclusion level of 2.25 mg/kg diet, as pyridoxine·HCl, in diets for young pigs.     

Inhibition of cathepsin activities by vitamin B6 derivatives

We found that pridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibited the activity of cathepsins B, K, S and L in vitro. Cathepsins activities in cultured splenocytes were suppressed by the addition of pridoxal (PL) or pridoxine (PI) in to the culture medium. A newly synthesized artificial vitamin B6 derivative, a pridoxal propionate derivative, CLIK-164, showed selective inhibition of cathepsin O/K.     

Human brain pyridoxal-5'-phosphate phosphatase (PLPP):protein transduction of PEP-1-PLPP into PC12 cells

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).     

Hydrocellulase of Aspergillus aculeatus

Conditions for extracellular production of vitamin B₆ compounds (B₆), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leu1 strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B₆ and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B₆ and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H₂O₂ increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and in contrast, increased pyridoxine.     

Hypertension,calcium channel and pyridoxine (vitamin B6)

The moderately pyridoxine (vitamin B₆)-deficient male rat was introduced by us as an animal model (B6DHT) for the study of hypertension. Hypertension in this rat is associated with increased sympathetic stimulation. Arterial segments from B6DHT rats maintained a higher resting tone. The influx of ⁴⁵calcium into intracellular compartment of the vascular smooth muscle of the caudate artery of B6DHT rats was also enhanced. Administration of pyridoxine attenuated the hypertension in B6DHT rats as well as in genetic or dietary-induced moderately hypertensive conditions such as in the Zucker obese rat and sucrose or low calcium-fed rats. However, pyridoxine did not have any effect or the spontaneously hypertensive rat. All classes of calcium channel blockers were effective in lowering the systolic blood pressure of B6DHT rats. The increased in vitro influx of⁴⁵ calcium into intracellular compartment of artery segments of B6DHT rats as well as the BAY K 8644-induced influx of⁴⁵ calcium into artery segments from normal rats were blocked by pyridoxal phosphate as well as by dihydropyridine-sensitive calcium channel blockers (DHP). Pyridoxal phosphate (PLP) in vitro enhances the binding of calcium channel antagonists to membrane preparations from vascular tissue. PLP corrects the membrane abnormality in responsive hypertensive conditions and thus, could be an endogenous modulator of DHP - sensitive calcium channels.     

Vitamin B6 metabolism in microbes and approaches for fermentative production

Vitamin B6 is a designation for the six vitamers pyridoxal, pyridoxine, pyridoxamine, pyridoxal 5′-phosphate (PLP), pyridoxine 5′-phosphate, and pyridoxamine. PLP, being the most important B6 vitamer, serves as a cofactor for many proteins and enzymes. In contrast to other organisms, animals and humans have to ingest vitamin B6 with their food. Several disorders are associated with vitamin B6 deficiency. Moreover, pharmaceuticals interfere with metabolism of the cofactor, which also results in vitamin B6 deficiency. Therefore, vitamin B6 is a valuable compound for the pharmaceutical and the food industry. Although vitamin B6 is currently chemically synthesized, there is considerable interest on the industrial side to shift from chemical processes to sustainable fermentation technologies. Here, we review recent findings regarding biosynthesis and homeostasis of vitamin B6 and describe the approaches that have been made in the past to develop microbial production processes. Moreover, we will describe novel routes for vitamin B6 biosynthesis and discuss their potential for engineering bacteria that overproduce the commercially valuable substance. We also highlight bottlenecks of the vitamin B6 biosynthetic pathways and propose strategies to circumvent these limitations.     

Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay

Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all other compounds in plasma in an analytical run of 46 min. The imprecisions and mean values (presented in parenthesis in nmol/L) were (unspiked and spiked sample) 9-8% (41-65) for PLP, 12-7% (18-40) for 4-PA, 67-28% (4-19) for PL, 15% (21) for PN, 10% (27) for PM, and 27% (17) for PMP. All three B6 vitamers (PLP, 4-PA, and PL) present in unspiked plasma showed an excellent linearity within the range of (nM) 8-60 (4-PA), 1-19 (PL), and 11-99 (PLP). In conclusion, we report a HPLC-based method that separates and detects nanomolar quantities of six B6 vitamers and demonstrate that the method will be suitable for routine quantitation of PLP and 4-PA in human plasma.     

Vitamin B(6) salvage enzymes: mechanism, structure and regulation

Vitamin B(6) is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B(6), and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5'-phosphate is recycled from food and from degraded B(6)-enzymes in a "salvage pathway", which essentially involves two ubiquitous enzymes: an ATP-dependent pyridoxal kinase and an FMN-dependent pyridoxine 5'-phosphate oxidase. Once it is made, pyridoxal 5'-phosphate is targeted to the dozens of different apo-B(6) enzymes that are being synthesized in the cell. The mechanism and regulation of the salvage pathway and the mechanism of addition of pyridoxal 5'-phosphate to the apo-B(6)-enzymes are poorly understood and represent a very challenging research field. Pyridoxal kinase and pyridoxine 5'-phosphate oxidase play kinetic roles in regulating the level of pyridoxal 5'-phosphate formation. Deficiency of pyridoxal 5'-phosphate due to inborn defects of these enzymes seems to be involved in several neurological pathologies. In addition, inhibition of pyridoxal kinase activity by several pharmaceutical and natural compounds is known to lead to pyridoxal 5'-phosphate deficiency. Understanding the exact role of vitamin B(6) in these pathologies requires a better knowledge on the metabolism and homeostasis of the vitamin. This article summarizes the current knowledge on structural, kinetic and regulation features of the two enzymes involved in the PLP salvage pathway. We also discuss the proposal that newly formed PLP may be transferred from either enzyme to apo-B(6)-enzymes by direct channeling, an efficient, exclusive, and protected means of delivery of the highly reactive PLP. This new perspective may lead to novel and interesting findings, as well as serve as a model system for the study of macromolecular channeling. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.     

Secretion of Hexosaminidase Isozymes by Tetrahymena*

SYNOPSIS. Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of β-N-acetylhexosaminidase (2-acetamido-2-deoxy-β-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A₁ and B₁, and 2 minor forms, A₂ and B₂, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B₁ has a molecular weight of ?93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-β-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A₁ has a molecular weight of ?170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-β-D-galactosaminide than the B forms. Neither hexosaminidases A₁ or B₁ has any endo-β-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete ?20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxy-benzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.