Protease activities present in wheat germ and rabbit reticulocyte lysates

Rabbit reticulocyte lysates and wheat germ lysates were found to contain significant neutral protease activity when assayed against the highly sensitive 7-amino-4-methylcoumarin (AMC) peptide substrates Phe-AMC, succinyl-Ala-Ala-Phe-AMC and t-boc-Ala-Ala-Pro-Ala-AMC (substrates for aminopeptidase, chymotrypsin and elastase-like enzymes, respectively). Additionally, wheat germ lysates contain a trypsin-like activity when assayed against CBZ-Gly-Gly-Arg-AMC and a post-proline cleaving activity which hydrolyzed the Pro-Ala bond of t-boc-Ala-Ala-Pro-Ala-AMC.     

Cell-bound peptidase activities of Treponema denticola ATCC 33520 in continuous culture.

The oral spirochaete Treponema denticola ATCC 33520 was grown at a mean generation time of 10 h in anaerobic continuous culture in a serum- and carbohydrate-free medium at pH 7.0. The extracellular proteolytic activities of this spirochaete were then investigated by incubating washed cells with 68 2-naphthylamide derivatives of the Extended API System. Chymotrypsin-like, trypsin-like, elastase-like and iminopeptidase activities were demonstrated. The phenylalanine peptidase or chymotrypsin-like activity of T. denticola ATCC 33520, estimated with N-succinyl-L-phenylalanyl-L-leucyl-L-phenylalanine-thiobenzyl ester (SPLP) had a pH optimum at pH 8.5, a specific activity of 36.6 nmol min-1 (mg dry wt)-1 and was inhibited only slightly by HgCl2. The trypsin-like activity, estimated with benzoyl-DL-arginine-7-amido-4-methylcoumarin (BAMC), had a pH optimum at pH9, and a specific activity of 0.3 nmol min-1 (mg dry wt)-1; inhibition by HgCl2 indicated the involvement of active thiol groups. The activity should preferably be termed arginine peptidase activity, according to the carboxy-terminal amino acid of the test substrate. The extracellular proline peptidase activity, estimated with L-proline-7-amido-4-methylcoumarin. HBr (PRAMC), had an activity of 1.5 nmol min-1 (mg dry wt)-1, an optimum at pH 8.5 and the properties of a thiol protease. The main cell-bound and extracellular active peptidase activities of fast-growing cells of T. denticola ATCC 33520 are phenylalanine peptidase, proline peptidase, arginine peptidase and an oligopeptide-dependent alanine peptidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A preliminary study of the use of Colilert for water quality monitoring

Substrate specificities of proteases produced by two putrefactive marine bacteria, Shewanella putrefaciens and Alteromonas haloplanktis , were surveyed by using peptidyl-7-amino-4-methylcoumarin (MCA-substrates). Shewanella putrefaciens produced trypsin-like enzyme(s) showing broad spectrum specificity and chymotrypsin-like enzyme specifically hydrolysing Glt-Gly-Gly-Phe-MCA. Alteromonas haloplanktis produced high activity of ammopeptidase and trypsin-like enzyme(s) preferring Z-Phe-Arg-MCA, Bz-Arg-MCA and Boc-Leu-Ser-Thr-Arg-MCA. The two organisms would be able to utilize different proteins for their growth.     

An elastase-like enzyme in the oocytes of Arbacia Punctulata.

An elastase-like enzyme was demonstrated in the unfertilized eggs of Arbacia punctulata. The enzyme was discovered in the fertilization product of A. punctulata, but was also found to be present in the seawater surrounding unfertilized eggs. Isolation of the elastase-like enzyme was accomplished by preparation of a 100,000g supernatant from a homogenization of unfertilized eggs. Its presence and specificity were determined by assay using a synthetic peptide substrate. The elastase-like activity was nondialyzable, heat labile, and unstable at pH 4. The enzyme was inhibited by antipain, elastatinal, and DFP, but not by leupeptin or soybean trypsin inhibitor. Eggs were fertilized and developed normally in the presence of 1.0 mM elastatinal. Trypsin-like and chymotrypsin-like enzymes were also found in the seawater surrounding fertilized eggs. The trypsin-like enzyme was isolated from this source and characterized by inhibitor profile.     

Comparison of the peptidase activity in the oncosphere excretory/secretory products of Taenia solium and Taenia saginata

We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.     

Proteasome inactivation upon aging and on oxidation-effect of HSP 90

Increases of oxidatively modified protein in the cell have been associated with the aging process. Such an accumulation of damaged protein may be the result of increase in the rate of protein oxidation and/or decrease in the rate of degradation of oxidized protein. The multicatalytic proteinase or proteasome is known to be the major proteolytic system involved in the removal of oxidized protein. We have reported that, after isolation of the 20S proteasome from the liver of young and old male Fischer 344 rat, out of the three peptidase activities (chymotrypsin-like, trypsin-like and peptidyl-glutamyl peptide hydrolase) we assayed with fluorogenic peptides, the peptidyl-glutamyl peptide hydrolase activity was declining with age to a value approximately 50% of that observed for protease purified from young rats. The proteasome was subjected to metal catalyzed oxidation to determine the susceptibility of the different peptidase activities to oxidative inactivation. Both trypsin-like and peptidyl-glutamyl peptide hydrolase activities were found sensitive to oxidation. Treatment of the proteasome with 4-hydroxy-2-nonenal, a major lipid peroxidation product, was also found to inactivate the trypsin-like activity. However, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation in proteasome preparations contaminated with HSP 90, a protein that often copurifies with the proteasome. Upon addition of HSP 90 to pure 20S active proteasome, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation and from inactivation by treatment with 4-hydroxy-2-nonenal. These results suggest a possible intervention of HSP 90 in response to oxidative stress in preventing the inactivation of the proteasome by oxidative damage. Abbreviations: AAF-amc – Ala-Ala-Phe-7-amido-4-methylcoumarin; LSTR-amc – N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin; LLE-na – Leu-Leu-Glu-b-naphthylamide; HSP 90: heat shock protein 90, MCP – multicatalytic proteinase or 20S proteasome.     

Identification and characterization of a Drosophila nuclear proteasome regulator. A homolog of human 11 S REGgamma (PA28gamma )

We report the cloning and characterization of a Drosophila proteasome 11 S REGgamma (PA28) homolog. The 28-kDa protein shows 47% identity to the human REGgamma and strongly enhances the trypsin-like activities of both Drosophila and mammalian 20 S proteasomes. Surprisingly, the Drosophila REG was found to inhibit the proteasome's chymotrypsin-like activity against the fluorogenic peptide succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological analysis reveals that the Drosophila REG is localized to the nucleus but is distributed throughout the cell when nuclear envelope breakdown occurs during mitosis. Through site-directed mutagenesis studies, we have identified a functional nuclear localization signal present in the homolog-specific insert region. The Drosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REGgamma proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that gamma class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the alpha and beta REG classes.     

Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. I. Subcellular localization and functional role.

Our investigations indicate that a variety of neutral serine proteases exist in highly purified, IL-2-activated rat NK (A-NK) cells. These enzymatic activities are not restricted to only cytolysin-containing granules and are not defined by only the assay of N-alpha-benzyloxycarbonyl-L-lysine thiobenzylesterase activity. These activities, which we term A-NKP 1, A-NKP 2, A-NKP 3, and A-NKP 4, cleave, respectively, the following fluorogenic peptide substrates: Boc-Phe-Ser-Arg-7-amino-4-methylcoumarin (AMC, trypsin-like); Suc-Ala-Ala-Phe AMC (chymotrypsin-like); Suc-Gly-Pro-Leu-Gly-Pro AMC (collagenase-like), and Z-Phe-Arg AMC (another trypsin-like enzyme). The proteases A-NKP 1, A-NKP 2, and A-NKP 3 are not cell surface-associated and appear to be cytosolic as defined by isopycnic sucrose density gradient centrifugation. In contrast, A-NKP 4 appears to be located in lysosomes. Treatment of rat A-NK cells with protease inhibitors that inhibit A-NKP 2 and A-NKP 3 also substantially inhibit A-NK cell-mediated cytotoxicity against both NK-sensitive and -resistant targets (YAC-1 and P815, respectively). These results indicate that A-NKP2 and A-NKP 3 may play a role in IL-2-activated NK cell-mediated cytotoxicity. A variety of proteolytic enzymes, in addition to granzymes, therefore exist in A-NK cells. Our studies indicate that a prerequisite to a thorough understanding of the role of proteases in killer cell function is the investigation of several classes of enzymes in addition to granzymes contained in lytic granules.     

Characterisation of rhodococci using peptide hydrolase substrates based on 7-amino-4-methylcoumarin

Enzyme activity profiles of 105 rhodococci and related actinomycetes were obtained using peptide hydrolase substrates based on 7-amino-4-methylcoumarin. All of the test strains produced l-alanyl-, l-lysyl-, l-methionyl, l-seryl-, l-tyrosyl, S-Bz-l-cysteinyl and l-alanyl-l-alanyl-l-phenyl-alanyl-peptidases but the distribution of the remaining enzymes gave results of differential value. Fluorogenic probes prepared from 7-amino-4-methylcoumarin provide a simple and rapid means of detecting specific exo- and endopeptidases in small amounts of whole rhodococci.     

Cytochemistry of proteolytic activity and pH status of vacuoles in Medicago truncatula root nodules

The vacuole development in root nodules of Medicago truncatula was analyzed by light and electron microscopy. Histochemistry of protease activity in root nodules was studied using fluorogenic substrates for proteolytic enzymes, 7-amino-4-methylcoumarin, CBZ-L-phenylalanyl-L-arginine amide, hydrochloride (AMC), and rhodamine 110, bis-(CBZ-L-phenylalanyl-L-arginine amide) dihydrochloride (RPA). Furthermore, the topology of acidification of the central vacuoles in infected and noninfected cells in root nodules of Medicago truncatula was analyzed with the fluorescent pH-sensitive acidotropic dye Neutral Red. It was shown that vacuoles were acidic, lytic organelles in noninfected cells and young infected cells of the nodule where they displayed protease activity. Mature vacuoles of infected cells had high pH and did not show any substantial protease activity. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 31–38. The text was submitted by the authors in English.     

Cell associated elastase activities of rat mammary tumour cells

As part of our studies into the role of tumour cell proteinases in cancer invasion, we have adapted a fluorogenic assay to measure the elastase activities of intact rat mammary adenocarcinoma cells using the elastase specific substrates Cbz-Ala-Ala-Pro-Val-6-aminoquinoline and Ac-Ala-Ala-Pro-Ala-7-amino-4-methylcoumarin. This is a sensitive assay which enables rapid (30-120 min) measurement of enzyme activities under conditions of physiological pH and ionic strength and can differentiate between cell-associated and secreted enzyme activities. As the substrates are non-toxic and the method is non-invasive, cells can be reclaimed for further studies. This method thus provides a useful means for screening intact cells for elastase activity. Cell-surface elastase extracts were inhibited by phenylmethylsulphonyl fluoride but not by EDTA, indicating that they are serine proteinases. Extracts also degraded insoluble elastin confirming that these rat mammary adenocarcinoma cells produce elastase.     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.     

Crude and purified proteasome activity assays are affected by type of microplate

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate.     

Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability

Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by pchloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2–3 g/10⁶ cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins produced in heterologous systems may vary depending on the host cells.Abbreviations ABTS 2,2-azino-di-(3-ethylbenzothiazoline) sulfonic acid diammonium salt - AMC 7-amino-4-methylcoumarin - CHO Chinese hamster ovary - DFP diisopropylfluorophosphate - DHFR dihydrofolate reductase - ELISA enzyme-linked immunosorbent assay - MCA 4-methylcoumaryl-7-amide - MTX methotrexate - NEAA non essential amino acid - NEM N-ethylmaleimide - PCMB p-chloromercuribenzonate - PMSF phenylmethanesulfonyl fluoride - pro-UK pro-urokinase     

Novel thiocoumarins as inhibitors of TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs) and microsomal lipid peroxidation

Different coumarin/thiocoumarin derivatives, that is, 7-hydroxy-4-methylcoumarin, 7,8-dihydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 7,8-diacetoxy-4-methylcoumarin, 7-hydroxy-4-methylthiocoumarin, 7,8-dihydroxy-4-methylthiocoumarin, 7-acetoxy-4-methylthiocoumarin and 7,8-diacetoxy-4-methylthiocoumarin were synthesized and evaluated for their effects on TNF-alpha induced expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and on NADPH-catalyzed rat liver microsomal lipid peroxidation with a view to identify modulators for expression of cell adhesion molecules and to establish structure-activity relationship. We found that dihydroxy and diacetoxy derivatives of thiocoumarin were more potent in comparison to the corresponding coumarin derivatives in inhibiting TNF-alpha-induced expression of ICAM-1. However, coumarin derivatives were found to be more potent in comparison to the corresponding thiocoumarins in inhibiting microsomal lipid peroxidation. We have also tested the intermediate compounds 7,8-dibenzyloxy-4-methylcoumarin and 7,8-dibenzyloxy-4-methylthiocoumarin for their inhibitory activity on TNF-alpha-induced ICMA-1 expression. We found that dibenzyloxy-4-methylthiocoumarin is better than dibenzyloxy-4-methylcoumarin. The mechanisms underlying the observed activities of coumarins and thiocoumarins have been discussed with reference to their structures. Such structure-function relationship studies may help in developing molecules with better anti-inflammatory and anti-oxidant activities.     

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.     

Aprotinin derivatives with chromophoric leaving groups can be used as highly selective active-site titrants for serine proteinases and permit the determination of kinetic constants of enzyme-inhibitor complexes

This paper reports a novel and valuable approach to active-site titration. The starting substance for the preparation of the active-site titrants is aprotinin (bovine pancreatic trypsin inhibitor) in which the reactive-site peptide bond, Lys15-Ala16, is split. Two cystine disulfide bonds hold together the two peptide chains. The Lys15 of the reactive site is substituted by arginine-, phenylalanine- and valine-4-nitroanilide or by valine-7-amido-4-methylcoumarin. The different incorporated amino acid residues correspond to different specificities against serine proteinases. Serine proteinases with suitable specificity are able to remove 4-nitroaniline or 7-amino-4-methylcoumarin from these aprotinin derivatives while at the same time resynthesis of the reactive-site peptide bond occurs. The proteinase is then trapped in a stable enzyme-inhibitor complex, which prevents the proteinase from releasing further leaving groups. The quantity of 4-nitroaniline or 7-amino-4-methylcoumarin, which can be assayed spectrophotometrically or fluorometrically is equimolar to the quantity of proteinase used and trapped. The aprotinin derivatives with an incorporated Phe15 or Val15 residue are highly specific for chymotrypsin or for elastase from human leukocytes, respectively. The kinetic constants kon and koff of the enzyme-inhibitor complexes, and hence the equilibrium dissociation constants, can be calculated from the respective titration curves.     

New chromogenic and fluorogenic substrates for pyrrolidonyl peptidase.

L-Pyroglutamyl derivatives of p-nitroaniline and 7-amino-4-methylcoumarin were synthesized as new sensitive substrates for pyrrolidonyl peptidase (pyrrolidonecarboxylyl peptidase) from Bacillus amyloliquefaciens. Their hydrolyses could be followed by conventional colorimetric and fluorometric procedures; i.e., in terms of the increase in absorbance at 410 nm caused by the liberation of p-nitroaniline and the emission at 440 nm after excitation at 370 nm depending on the liberation of 7-amino-4-methylcoumarin. Values of Km were estimated to be 0.69 mM for anilide substrate and 0.33 mM for methylcoumarin substrate in the pyrrolidonyl peptidase reaction at pH 8.0. The methylcoumarin compound was about one thousand fold more sensitive than the anilide substrate.     

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.     

Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.