Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase

A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammoniumchloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [³H]Diisopropyl fluorophosphate ([³H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [³H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme.A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.Abbreviations AChE Acetylcholinesterase - BW284C51 1,5-bis(4-allyl dimethylammonium phenyl) pentan 3-one-dibromide - DFP Diisobropyl fluorophosphate - TIPP Tetra isopropyl pyrophosphoramide - TPCK N-Tosyl-L-phenylalanylchloromethyl ketone - MAP m-Aminophenyl trimethylammonium chloride - RCA₁ Ricinus communis agglutinin 120 - TEAB Tetraethylammonium bromide - DTT Dithiothreitol     

Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Localization of the peptidase activity of human serum butyrylcholinesterase in a approximately 50-kDa fragment obtained by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholinesterase (approximately 90-kDa subunit) is known to exhibit aryl acylamidase and peptidase activity. Limited alpha-chymotrypsin digestion of the purified butyrylcholinesterase gave three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa. In our earlier studies [Rao and Balasubramanian (1989) Eur. J. Biochem. 179, 639-644] we characterized the approximately 20-kDa fragment and showed that it exhibited both butyrylcholinesterase and aryl acylamidase activities. In the present studies the approximately 50-kDa fragment is characterized. This fragment, after isolation by Sephadex G-75 chromatography from a chymotryptic digest of purified butyrylcholinesterase, exhibited only peptidase activity and was devoid of cholinesterase and aryl acylamidase activities. It could bind to a column of Ricinus communis agglutinin bound to Sepharose, indicating its glycosylated nature and the presence of galactose. The peptidase activity in the approximately 50-kDa fragment could be immuno-precipitated by a polyclonal antibody raised against purified butyrylcholinesterase. SDS-gel electrophoresis of this fragment isolated by R. communis agglutinin-Sepharose and Sephadex G-75 chromatography showed a protein band of approximately 50 kDa by silver staining. Amino-terminal sequence analysis of the approximately 50-kDa fragment gave the sequence of Gly-Pro-Thr-Val-Asp which corresponded to amino acid residues 291-295 in the butyrylcholinesterase sequence [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. The combined results suggested that alpha-chymotrypsin digestion of human serum butyrylcholinesterase resulted in the formation of a approximately 20-kDa fragment exhibiting both cholinesterase and aryl acylamidase activities and a approximately 50-kDa fragment exhibiting only peptidase activity.     

Purification and biochemical characterization of a 17 kDa fibrinolytic enzyme from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.     

Purification and biochemical characterization of antioxidant peptide from horse mackerel (Magalaspis cordyla) viscera protein

In the present study, a peptide having high antioxidant properties was isolated from horse mackerel viscera protein, Magalaspis cordyla. In vitro gastrointestinal digestion was employed to obtain potential protein hydrolysate and was subjected to consecutive chromatographic methods using fast protein liquid chromatography (FPLC) connected to diethyl amino ethyl (DEAE) anion exchange column and Sephadex G-25 gel filtration column. The activity of the fractions was tested against DPPH and hydroxyl radicals and the isolated peptide showed 89.2 and 59.1 percentage of scavenging. The amino acid sequence of purified peptide was determined using ESI-MS/MS as Ala-Cys-Phe-Leu (518.5 Da), it exhibited high activity against polyunsaturated fatty acid (PUFA) peroxidation than that of natural antioxidant, α-tocopherol.     

Partial purification and some properties of a bovine brain trypsin inhibitor

Using hemoglobin modified by pyridoxal 5'-phosphate as substrate, a trypsin inhibitor from bovine brain was purified by extraction at pH 4.5, ion-exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-100 and isoelectric focusing. On a column of Sephadex G-100 the inhibitor exhibited a molecular mass of 78 kDa. The iso-electric point of the inhibitor was 4.3-4.4. The dissociation constant (Ki) for the complex of bovine trypsin and brain inhibitor was estimated to be 3.7 X 10(-10)M as tested with a protein substrate, and 2.4 X 10(-10)M when tested with a synthetic substrate. During purification two other brain trypsin inhibitors were detected.     

Purification and partial peptide sequence analysis of the boar 32 kDa sperminogen

Boar 32 kDa sperminogen was purified from acid extracts of washed epididymal spermatozoa, and partial peptide sequence was determined. Boar sperminogen was purified from the acid extracts of boar spermatozoa by gel filtration through Sephadex G-75 column, followed by preparative SDS-PAGE. Gelatin zymographic analysis of the gel-filtered fractions showed that sperminogen was composed of three separate proteolytic bands. Among the three proteolytic bands, the 32 kDa sperminogen band which showed the strongest proteolytic activities upon activation was sliced out and eluted from the gel fragments. The eluted 32 kDa sperminogen was then subjected to peptide sequencing. Since the N-terminus of the 32 kDa sperminogen was blocked for peptide sequencing by Edman degradation method, the internal amino acid sequence of the sperminogen was obtained from the CNBr-digested peptides of sperminogen. The amino acid sequence of the analyzed peptide of the 32 kDa sperminogen showed 100% identity with that of proacrosin.     

Biochemical analysis of a fibrinolytic enzyme purified from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain A1

A fibrinolytic enzyme from Bacillus subtilis strain Al was purified by chromatographic methods, including DEAE Sephadex A-50 column chromatography and Sephadex G-50 column gel filtration. The purified enzyme consisted of a monomeric subunit and was estimated to be approximately 28 kDa in size by SDS-PAGE. The specific activity of the fibrinolytic enzyme was 1632-fold higher than that of the crude enzyme extract. The fibrinolytic activity of the purified enzyme was approximately 0.62 and 1.33 U/ml in plasminogen-free and plasminogen-rich fibrin plates, respectively. Protease inhibitors PMSF, DIFP, chymostatin, and TPCK reduced the fibrinolytic activity of the enzyme to 13.7, 35.7, 15.7, and 23.3%, respectively. This result suggests that the enzyme purified from B. subtilis strain Al was a chymotrypsin-like serine protease. In addition, the optimum temperature and pH range of the fibrinolytic enzyme were 50°C and 6.0–10.0, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as Q-T-G-G-S-I-I-D-P-I-N-G-Y-N, which was highly distinguished from other known fibrinolytic enzymes. Thus, these results suggest a fibrinolytic enzyme as a novel thrombolytic agent from B. subtilis strain Al.     

Purification of a novel antibacterial short peptide in earthworm Eisenia foetida

Earthworms live in an environment with abundantpathogens. These pathogens are, firstly, bacteria living inwater or soil that are ingested during feeding or introducedinto the body following injury. Parasites, particularlylarval forms, which represent the dissemination phase,are another important group of potentially pathogenicagents. During the course of evolution, earthworms havedeveloped defense strategies against these living patho-gens [1,2]. Earthworms lack true antibodies and hence anada…     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythrocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin) kinase according to physical and site-specificity criteria. The soluble enzyme shows an Mr of about 30000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that had been phosphorylated with [gamma-32P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with 32Pi. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Purification and amino-acid sequence of a nerve growth factor from the venom of Vipera russelli russelli.

Nerve growth factor (NGF) was purified from the venom of Vipera russelli russelli by Sephadex G-50 gel filtration, S-Sepharose column chromatography and Blue-Sepharose CL-6B column chromatography. The purified NGF was found to be a glycoprotein, whose apparent molecular mass was estimated to be about 17.5 kDa by SDS-PAGE. The amino-acid sequence was determined by a combination of conventional methods. The V. r. russelli NGF was composed of 117 amino-acid residues with one residue, Asn-21, being N-linked glycosylated and the molecular mass of its protein portion was calculated to be 13,280 Da.     

Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture of medium of Diplococcus pneumoniae.

The culture medium of Diplococcus pneumoniae contains enzymic activity that cleaves Galbeta1 leads to 3GalNAc from desialized human erythrocyte membrane glycoprotein. The enzyme was purified 180-fold by ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and DEAE A-25 Sephadex chromatography. The purified enzyme liberates Galbeta1 leads to 3GalNAc from glycopeptides and glycoproteins with Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr moieties. The optimum pH of this enzyme is 6.0. Using glycopeptides obtained by trypsin digestion of human erythrocyte membrane glycoprotein as a substrate, a Km of 0.20 mM (on the basis of the amount of Galbeta1 leads to 3GalNAc residues) was obtained. So far, the enzyme appears to have a strict specificity for Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr structures, because no oligosaccharides larger than trisaccharides were liberated from porcine submaxillary mucin.     

Purification of xanthine dehydrogenase from rat liver: a rapid procedure with high enzyme yields

Xanthine dehydrogenase (EC 1.2.1.37) was purified approximately 1000-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (greater than 20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis.     

Hydrophobic binding domains of rat intestinal maltase-glucoamylase

Rat intestinal microvillus maltase-glucoamylase was isolated by detergent extraction and purification in the presence of protease inhibitors as previously described and incorporated into phospholipid vesicles. After purification of the vesicles on Sephadex G-50, maltase was labelled with 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID) by photolysis using a water-jacketed mercury vapour lamp with a saturated CuSO4 filter. The labelled enzyme was extracted with acetone, resuspended in 1% Triton X-100, reincorporated into phospholipid vesicles, and digested with activated papain to release the hydrophilic polar head of the enzyme from the vesicle bilayer. Vesicle-bound and free enzyme components were separated on Sepharose 4B. Ninety percent of the enzymatic activity was free, while a similar percentage of radioactive label remained with the vesicles in keeping with the separation of an active polar headpiece from a labelled apolar peptide in the lipid bilayer. The vesicle fractions were subjected to chromatography on Sephadex LH-60 with ethanol--formic acid (7:3) as the eluant. A single radioactive peak (14 kilodaltons (kDa)) was separated from labelled lipid. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the peak showed a radioactive doublet of 26-28 kDa, possibly representing a dimer. No other labelled peptides were found. These results suggest that detergent-solubilized maltase-glucoamylase is inserted into the phospholipid bilayer via an apolar peptide with a minimum molecular mass of 14 kDa. The peptide probably represents a terminal anchor segment of the 145-kDa subunit which is converted to 130 kDa when the membrane-bound enzyme is solubilized by papain.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythorocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin_kinase according and site-specificity criteria. The soluble enzyme shows an M_r of about 30 000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32 000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that has been phosphorylated with [γ-³²P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21 500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with ³²P_i. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8

Proteases are the hydrolytic enzymes which hydrolyzes peptide bond between proteins with paramount applications in pharmaceutical and industrial sector. Therefore production of proteases with efficient characteristics of biotechnological interest from novel strain is significant. Hence, in this study, an alkaline serine protease produced by Bacillus cereus strain S8 (MTCC NO 11901) was purified and characterized. The alkaline protease was purified by ammonium sulfate precipitation (50%), ion exchange (DEAE-Cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. As a result of this purification, a protein with specific activity of 300U/mg protein was obtained with purification fold 17.04 and recovery percentage of 34.6%. The molecular weight of the purified protease was determined using SDS-PAGE under non-reducing (71?kDa) and reducing conditions (35?kDa and 22?kDa). Zymogram analysis revealed that proteolytic activity was only associated with 22?kDa. These results indicate that existence of the enzyme as dimer in its native state. The molecular weight of the protease (22?kDa) was also determined by gel filtration (Sephadex G-200) chromatography and it was calculated as 21.8?kDa. The optimum activity of the protease was observed at pH 10.0 and temperature 70?°C with great stability towards pH and temperature with casein as a specific substrate. The enzyme was completely inhibited by PMSF and TLCK indicating that it is a serine protease of trypsin type. The enzyme exhibits a great stability towards organic solvents, oxidizing and bleaching agents and it is negatively influenced by Li²⁺ and Co²⁺ metal ions. The purified protein was further characterized by Matrix Assisted Laser Desorption Ionization/Mass Spectroscopy (MALDI/MS) analysis which reveals that total number of amino acids is 208 with isoelectric point 9.52.     

Partial purification and characterization of a gelatin-specific protease from the culture media of human pulmonary alveolar macrophages

A gelatin-specific protease from the culture media of human pulmonary alveolar macrophages has been partial purified by gel filtration and characterized. The macrophages were obtained by bronchopulmonary lavage from the lungs of disease-free smoking volunteers. The gelatin-specific protease initially requires trypsin activation. After chromatographing the culture media on a Sephadex G-200 column, trypsin is no longer required for activation. The gelatin-specific protease reported here shares many properties of previously reported gelatinases. It is inhibited by EDTA, cysteine, dithiothreitol and serum. It is unaffected by other protease inhibitors: phenylmethylsulfonyl fluoride, tosyllysine chloromethyl ketone and p-chloromercuribenzoate. Of all substrates tested activity was observed only with gelatin. It was inactive toward collagen, elastin and methemoglobin. This enzyme may have a role in the digestion of collagen which has been cleaved by a mammalian collagenase.     

Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.