Effect of carbon starvation on p-nitrophenol degradation by a Moraxella strain in buffer and river water

This study examines the effect of carbon starvation on the ability of a Moraxella sp. strain to degrade p-nitrophenol (PNP). Carbon starvation for 24 h decreased the induction time for p-nitrophenol degradation by the bacterium in a minimal salt medium from 6 to 1 h but it did not completely eliminate the induction time. Moraxella cells with 2-day carbon starvation had an induction time of 3 h and the induction time of the 3-day starved cells was 6 h. A 100% increase in density of the non-starved cells did not affect the induction time for p-nitrophenol degradation by the bacterium, indicating that the initial increase in cell density of the carbon-starved culture did not cause the faster onset of p-nitrophenol degradation. However, the initial uptake of p-nitrophenol of the 1-day carbon-starved Moraxella cells was 3-fold higher than the non-starved cells. A green fluorescent protein gene (gfp)-labelled Moraxella (M6 strain) was constructed to examine the survival of and p-nitrophenol degradation by the bacterium in non-sterile river water samples. Similar p-nitrophenol degradation behaviour was observed in the river water samples inoculated with the M6 cells. The time needed for complete degradation of p-nitrophenol by the non-starved M6 was 19-27 and 33 h in samples spiked with 80, 200 and 360 microM p-nitrophenol, respectively. However, the 1-day carbon-starved inocula required about 16 h to degrade the p-nitrophenol completely regardless of its concentration in the water samples. Survival of the carbon-starved and non-starved M6 was not significantly different from each other in the river water regardless of the p-nitrophenol concentration. In the absence of p-nitrophenol, the inoculum density decreased continuously. At 200 and 360 microM p-nitrophenol, the cell densities of M6 increased in the first two days of incubation and declined steadily afterward.     

The role of the Sphingomonas species UG30 pentachlorophenol-4-monooxygenase in p-nitrophenol degradation

Pentachlorophenol-4-monooxygenase is an aromatic flavoprotein monooxygenase which hydroxylates pentachlorophenol and a wide range of polyhalogenated phenols at their para position. The PCP-degrading Sphingomonas species UG30 was recently shown to mineralize p-nitrophenol. In this study, the UG30 pcpB gene encoding the pentachlorophenol-4-monooxygenase gene was cloned for use to study its potential role in p-nitrophenol degradation. The UG30 pcpB gene consists of 1614 bp with a predicted translational product of 538 amino acids and a molecular mass of 59,933 Da. The primary sequence of pentachlorophenol-4-monooxygenase contained a highly conserved FAD binding site at its N-terminus associated with a beta alpha beta fold. UG30 has been shown previously to convert p-nitrophenol to 4-nitrocatechol. We observed that pentachlorophenol-4-monooxygenase catalyzed the hydroxylation of 4-nitrocatechol to 1,2,4-benzenetriol. About 31.2% of the nitro substituent of 4-nitrocatechol (initial concentration of 200 microM) was cleaved to yield nitrite over 2 h, indicating that the enzyme may be involved in the second step of p-nitrophenol degradation. The enzyme also hydroxylated p-nitrophenol at the para position, but only to a very slight extent. Our results confirm that pentachlorophenol-4-monooxygenase is not the primary enzyme in the initial step of p-nitrophenol metabolism by UG30.     

对硝基苯酚冲击对UASB反应器中污泥活性和微生物种群的影响

研究了在稳定运行的上流式厌氧污泥床(Upflow Anaerobic Sludge Blanket,UASB)反应器中,对硝基苯酚(p-NP)冲击对反应器活性的影响。采用PCR-DGGE技术监测了反应器受对硝基苯酚冲击后微生物种群多样性的变化。实验结果表明,p-NP冲击对污泥的产甲烷活性和COD去除活性均有严重的抑制,污泥活性的恢复需要较长时间;高浓度冲击比低浓度冲击产生更严重的影响,20mg/L和40mg/Lp-NP冲击后污泥活性的恢复期分别为16d和27d。p-NP冲击后,真细菌和古菌的多样性均发生了显著的变化,而且p-NP冲击对真细菌的影响大于对古菌的影响。p-NP冲击后甲烷产量下降的主要与Methanosaetasp.的活性下降以及Methanomicrobia sp.丰度的下降有关。而冲击后真细菌的主要变化表现为Chloroflexisp.、Bacteroidesp.和Anaerovibrio sp.的丰度均下降;Rheinheimera sp.在受到20mg/Lp-NP冲击时丰度下降,继续受到40mg/Lp-NP冲击时该种群消失。Flavobacteria sp.是p-NP冲击后新出现的细菌种群,可能与p-NP的降解有关。     

有机磷农药在水生态系中生物净化机理研究——3.对硫磷及其降解产物对栅藻光合作用的影响和模拟藻菌系统中对硝基酚的降解

对硝基酚对栅藻光合放氧有明显抑制作用,半抑制浓度在16毫克/升左右;对硝基酚钠对栅藻光合放氧和CO2同化的抑制作用十分一致,它们的半抑制浓度分别在54毫克/升和52毫克/升左右。实验室证明对硝基酚在水体中主要是由细菌分解,藻类起供氧作用。一旦藻类的光合放氧受到抑制,细菌对对硝基酚的好气性降解能力也消失。对硫磷和二乙基硫代磷酸钾对栅藻光合放氧无明显抑制作用。       

Biotreatment of p-nitrophenol and nitrobenzene in mixed wastewater through selective bioaugmentation

This work combined selective adsorption and bioaugmentation to treat mixed wastewater of nitrobenzene and p-nitrophenol. The mixed wastewater of nitrobenzene (217 mg/L) and p-nitrophenol (500 mg/L) was adjusted its pH to 8 and then passed through the adsorption column at 100 mL/h. In effluent the nitrobenzene concentration was less than 4 mg/L. Without the toxic inhibition of nitrobenzene, p-nitrophenol in effluent could be degraded within 60 h through bioaugmentation. About 23 mg/g of nitrobenzene adsorbed the dry resin HU-05 could be desorbed and degraded through bioaugmentation. During this process the adsorption capacity of the resin HU-05 was recovered partly. The recovered extent was limited by nitrobenzene bioavailability. The performance of the resin HU-05 kept stably in the recycle experiments of 60 days.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.     

有机磷农药在水生态系中生物净化机理研究——2.CTP-02降解对硝基酚的特性和动力学

从鸭儿湖氧化塘中分离出具有分解对硝基酚能力的细菌Pseudomonas sp.,代号CTP-02。在实验室条件下,细菌培养物降解对硝基酚的速度与时间之间的动力学方程为dc/dt=-K1t-K2,细菌降解对硝基酚的最适温度为35℃,最适pH为7.5。CTP-02菌降解对硝基酚过程中首先发生脱硝基作用。       

Amperometric microbial biosensor for p-nitrophenol using Moraxella sp.-modified carbon paste electrode

An amperometric microbial biosensor for highly specific, sensitive and rapid quantitative determination of p-nitrophenol was developed. The biosensor takes advantage of the ability of Moraxella sp. to specifically degrade p-nitrophenol to hydroquinone, a more electroactive compound than p-nitrophenol. The electrochemical oxidation current of hydroquinone formed in biodegradation of p-nitrophenol was measured at Moraxella sp.-modified carbon paste electrode and correlated to p-phenol concentrations. The optimum response was realized by electrode constructed using 15 mg of dry cell weight per 1 g of carbon paste and operating at 0.3 V (versus Ag/AgCl reference) in pH 7.5, 20 mM sodium phosphate buffer. Operating at these optimum conditions the biosensor had excellent selectivity against phenol derivatives and was able to measure as low as 20 nM (2.78 ppb) p-nitrophenol with very good accuracy and reproducibility. The biosensor was stable for approximately 3 weeks when stored at 4 degrees C. The applicability of the biosensor to measure p-nitrophenol in lake water was demonstrated.     

Degradation of Parathion by Bacteria Isolated from Flooded Soil

Two bacteria, Bacillus sp. and Pseudomonas sp., were isolated from parathionamended flooded alluvial soil which exhibited parathion-hydrolyzing ability. Bacillus sp. readily liberated nitrite from the hydrolysis product, p-nitrophenol, but not from intact parathion. Pseudomonas sp. hydrolyzed parathion and then released nitrite from p-nitrophenol. These studies establish bacterial degradation of parathion past the p-nitrophenol stage to the end product, nitrite.     

Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide

The inhibitory effects of silymarin, its main constituent silibinin and the metabolite silibinin-glucuronide on UDP-glucuronosiltransferase (UGT) were evaluated in rat hepatic microsomes. Three substrates were chosen to cover both UGT1A and UGT2B family isozymes: bilirubin (substrate of UGT1A1), p-nitrophenol (UGT1A6) and ethinylestradiol (UGT2B1 and 2B3 for position C17 and UGT1A1 for position C3). The study of p-nitrophenol and bilirubin glucuronidation indicated that silymarin (SM) and silibinin glucuronide (SB-G) were enzyme inhibitors. The kinetic analysis showed that the type of inhibition was competitive in all cases and the Ki obtained were: for p-nitrophenol glucuronidation, KiSB-Gapp: 14+/-1 microg/ml and KiSMapp: 51+/-10 microg/ml and for bilirubin glucuronidation, KiSB-Gapp: 16+/-3 microg/ml. In turn, ethinylestradiol glucuronidation was not affected by any of the compounds studied suggesting that the inhibitory effect was restricted to UGT1A isozymes. Similar studies performed using human hepatic microsomes showed that SM and SB-G were also inhibitors of human UGT1A isozymes. In conclusion, administration of silymarin or its main constituent silibinin could lead to the decrease in the glucuronidation of substrates whose conjugation depends on UGT1A isozymes in a process mediated by silibinin-glucuronide, though their effect in humans needs further investigation.     

Studies of mammalian glucoside conjugation

The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.     

Estimation of Cellobiohydrolase Ⅰ Activity by Numerical Differentiation of Dynamic Ultraviolet Spectroscopy

1,4-β-D-glucan cellobiohydrolase Ⅰ （CBH Ⅰ）, p-nitrophenyl β-D-cellobioside, p-nitrophenol and cellobiose show distinct ultraviolet spectra, allowing the design of an assay to track the dynamic process of p-nitrophenyl β-D-cellobioside hydrolysis by CBH Ⅰ. Based on the linear relationship between p-nitrophenol formation in the hydrolysate and its first derivative absorption curve of AUC340-400 m （area under the curve）, a new sensitive assay for the determination of CBH Ⅰ activity was developed. The dynamic parameters of catalysis reaction, such as Vm and kcat, can all be derived from this result. The influence of β-glucosidase and endoglucanase in crude enzyme sample on the assay was discussed in detail. This approach is useful for accurate determination of the activity of CBHs.     

Sulfating-activity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A3*1 ((213)Arg) and ST1A3*2 ((213)His), both of which exist in Japanese as well as Caucasians.

We recently found single amino acid substitutions ((213)Arg/His and (223)Met/Val) in polymorphic human phenol-sulfating phenol sulfotransferase (SULT: cDNAs encoding ST1A3, P PST or HAST1/2) among Caucasians and African-Americans. In a Japanese population (n = 143), allele frequencies of (213)Arg and (213)His were 83.2 and 16. 8%, respectively, but the (223)Val allele was not found. (213)His homozygosity was reportedly associated with both very low (>7-fold) sulfating activities of p-nitrophenol (at 4 microM) and low thermostability in platelets. Sulfating-activity determinations using recombinant (213)Arg- and (213)His-forms (ST1A3*1 and ST1A3*2, respectively) did not, however, reveal appreciable deficiency in [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol (4 microM) by ST1A3*2 (7.5 vs. 10.2 nmol/min/nmol SULT for ST1A3). Kinetic parameters for p-nitrophenol for p-nitrophenol sulfation supported the slight decrease in sulfating activities at 4 microM (K(m), 0.82 vs. 1.75 microM; V(max), 13.2 vs. 13.1 nmol/min/nmol SULT, respectively, for ST1A3*1 and *2). p-Nitrophenyl sulfate-dependent 2-naphthol sulfation by ST1A3*2 was 69% of that by ST1A3*1 (p<0.05). However, ST1A3*2 was remarkably unstable at 45 and 37 degrees C as compared to ST1A3*1. The lower p-nitrophenol sulfating activity of ST1A3*2 may explain the lower platelet p-nitrophenol sulfation in ST1A3*2 homozygotes. Protein instability and ST1A3 gene regulation may be both involved in the polymorphism of p-nitrophenol sulfation in human tissues.     

Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monooxygenase

The npd gene cluster, which encodes the enzymes of a p-nitrophenol catabolic pathway from Arthrobacter sp. strain JS443, was cloned and sequenced. Three genes, npdB, npdA1, and npdA2, were independently expressed in Escherichia coli in order to confirm the identities of their gene products. NpdA2 is a p-nitrophenol monooxygenase belonging to the two-component flavin-diffusible monooxygenase family of reduced flavin-dependent monooxygenases. NpdA1 is an NADH-dependent flavin reductase, and NpdB is a hydroxyquinol 1,2-dioxygenase. The npd gene cluster also includes a putative maleylacetate reductase gene, npdC. In an in vitro assay containing NpdA2, an E. coli lysate transforms p-nitrophenol stoichiometrically to hydroquinone and hydroxyquinol. It was concluded that the p-nitrophenol catabolic pathway in JS443 most likely begins with a two-step transformation of p-nitrophenol to hydroxy-1,4-benzoquinone, catalyzed by NpdA2. Hydroxy-1,4-benzoquinone is reduced to hydroxyquinol, which is degraded through the hydroxyquinol ortho cleavage pathway. The hydroquinone detected in vitro is a dead-end product most likely resulting from chemical or enzymatic reduction of the hypothetical intermediate 1,4-benzoquinone. NpdA2 hydroxylates a broad range of chloro- and nitro-substituted phenols, resorcinols, and catechols. Only p-nitro- or p-chloro-substituted phenols are hydroxylated twice. Other substrates are hydroxylated once, always at a position para to a hydroxyl group.     

Phospholipid-dependence of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase.

Hepatic UDP-glucuronyltransferase activity was resolved into two fractions, one exhibiting oestrone glucuronyltransferase activity and the other exhibiting p-nitrophenol glucuronyltransferase activity. Hydroxyapatite-column chromatography removed greater than 95% of the phospholipids from both preparations. The partially purified delipidated enzymes were essentially devoid of catalytic activity, but activities were restored by the addition of phospholipids or phosphatidylcholine mixtures containing various saturated and unsaturated fatty acids. Both oestrone and p-nitrophenol glucuronyl-transferase activities were reconstituted to similar degrees with the phosphatidylcholine mixtures. When purified phospholipids were tested, phosphatidylcholine and lysophosphatidylcholine were most effective in restoring activity, whereas phosphatidylethanolamine was the least effective. These results further suggest that oestrone and p-nitrophenol UDP-glucuronyltransferases are dependent on phospholipids for their activity.     

Thiol-dependent changes in the properties of rat liver sulphotransferases

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The K(m) values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5mum and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0 degrees C when the K(m) and V(max.) values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0 degrees C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The K(m) value for adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.     

Stimulation of microsomal uridine diphosphate glucuronyltransferase by glucuronic acid derivatives

The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3beta-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme action.

1. In the presence of a high concentration of p-nitrophenyl beta-D-glucopyranoside (donor) the rates of production of p-nitrophenol and a transglucosylation product (1-glyceryl beta-D-glucopyranoside) increased, whereas the rate of production of glucose decreased with increasing concentration of glycerol in reactions catalysed by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. 2. When [donor] greater than Km the rate of production of p-nitrophenol was higher in the presence of glycerol than in its absence, whereas when [donor] less than Km the rate of production of p-nitrophenol was lower in the presence of glycerol than in its absence. 3. Glycerol increased both the Michaelis constant (Km) and maximum velocity (Vmax.), whereas dioxan increased Km but decreased Vmax. 4. Up to 1 mM-AgNO3 had no effect on enzyme activity. 5. A 2H-solvent-isotope-effect [Vmax. (H2O)/V max. (2H2O)] value of 1.40 +/- 0.05 was found at pH (or p2H) 5.8 6. alpha-2H-kinetic isotope-effect (kappa H/kappa 2H) values of 1.03 +/- 0.01 and 1.05 +/- 0.01 were found in the absence and presence of glycerol respectively. 7. Although maltose was a non-competitive inhibitor of beta-glucosidase activity, the ratio of velocity in the presence of glycerol to that in its absence increased, after an initial decline, with increasing concentration of maltose. 8. These results are discussed in terms of a mechanism involving a solvent-separated glucosyl cation-carboxylate ion-pair, which has greater affinity for alcoholic glucosyl acceptors, and an intimate ion-pair, which has greater affinity for water as a glucosyl acceptor and which could collapse reversibly and rapidly into a preponderance of an unreactive covalent glucosyl-enzyme.     

Pathways of microbial metabolism of parathion.

A mixed bacterial culture, consisting of a minimum of nine isolates, was adapted to growth on technical parathion (PAR) as a sole carbon and energy source. The primary oxidative pathway for PAR metabolism involved an initial hydrolysis to yield diethylthiophosphoric acid and p-nitrophenol. A secondary oxidative pathway involved the oxidation of PAR to paraoxon and then hydrolysis to yield p-nitrophenol and diethylphosphoric acid. Under low oxgen conditions PAR was reduced via a third pathway to p-aminoparathion and subsequently hydrolyzed to p-aminophenol and diethylthiophosphoric acid. PAR hydrolase, an enzyme produced by an isolate from the mixed culture, rapidly hydrolyzed PAR and paraoxon (6.0 mumol/mg per min). This enzyme was inducible and stable at room temperature and retained 100% of its activity when heated for 55 C for 10 min.     

Human neutrophil N-acetyl-beta-D-glucosaminidase: heparin inhibition

To determine N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) in human neutrophil granules separated by a method requiring heparin, the inhibition of this enzyme by heparin was studied. Neutrophils were purified from blood of five donors by modifications of the Hypaque-Ficoll and dextran separation methods resulting in a suspension which was 96% neutrophils. Neutrophil lysates were assayed for N-acetyl-beta-D-glucosaminidase by measuring the amount of p-nitrophenol released from p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The reaction showed first-order kinetics with regard to enzyme concentration. Triton X-100, 0.1% v/v, enhanced enzyme activity. Heparin was shown to reduce neutrophil lysate N-acetyl-beta-D-glucosaminidase to a specific activity of 46% at a heparin concentration of 2 units per assay and to 43% (maximal inhibition) at 17 and 50 units of heparin per assay. Substantially higher heparin concentrations partially restored the inhibited activity, the maximal restoration being a return to 80% of the original activity at 1700 units of heparin per assay. Protamine sulfate was assessed for its ability to restore N-acetyl-beta-D-glucosaminidase activity in the presence of heparin. At 1.0 mg/10 units of heparin, protamine restores enzyme activity to its heparin-free activity. These studies of human neutrophil N-acetyl-beta-D-glucosaminidase demonstrate: (1) specific enzyme activity is 28.8 +/- 7.0 nmole p-nitrophenol released per minute per milligram of protein or 1.7 +/- 0.5 nmole p-nitrophenol released per minute per 10(6) neutrophils; (2) heparin rapidly but finitely inhibits enzyme activity at very low concentrations and paradoxically restores it toward normal at high concentrations; and (3) protamine sulfate restores enzyme activity inhibited by heparin.