193
Clinica Chin&a Act4 196 (1991) 193-206 8 1991 Elsevier Science Publishers B.V. ~9-8981/91/$03.50 ~898191~7lM
ADONIS
CCA 04930
Purification and properties of human serum carnosinase Mel C. Jackson, Pharmacololly Beparrment,
Christine .Sehooi
M. Kucera
and James F. Lenney
of ~edjc;~e, Univer~it.v of Hawaii, Honv~~~u~Hawaii (USA)
(Received 30 July 1990; revision received 13 October 1990; accepted 16 October 1990) Key words; Serum carnosinase; Dipeptidase
Summaty
Carnosinase from human plasma was purified 18 OOO-foldto apparent homogeneity in a four step procedure. The dipeptidase was partially inactivated during DEAE-cellulose chromatography; however, it reactivated slowly when concentrated and stored at 4*C. In the second purification step, hydroxylapatite column chromatography, two forms of the enzyme were separated from one another. Human serum camosinase was found to be a glycoprotein with a p1 of 4.4 and a subunit n/r, of 75 000; the active enzyme was a dimer, the two subunits being connected by one or more disulfide bonds. The enzyme was especially active in hydrolyzing carnosine and anserine, preferring dipeptides with histidine in the C-terminal position. In most human tissues, the concentration of serum carnosinase was proportional to the percentage of trapped blood in the sample. However, the brain contained about 9 times more enzyme than expected, based on the amount of trapped blood present. The physiological function of this enzyme seems to be the hydrolysis of homocamosine in the brain and the splitting of carnosine and anserine in the blood stream. Six higher primates were found to have serum carnosinase. Twelve nonprimate mammals were tested; all were lacking the serum enzyme except for the Golden hamster, which had very high concentrations of a camosinase having somewhat different properties than the higher primate enzyme, Introduction
The human body contains two dipeptidases which hydrolyze carnosine (palanylhistidine). One of these is present in tissues, especially kidney, liver and brain, Correspondence to: J.F. Lenney, Pharmacology Department, School of Medicine, University of Hawaii, 1960 East West Road, Honolulu, Hawaii 96822, USA.
194
and should be referred to as ‘non-specific cytosolic dipept~dase’ [I]. The other is serum camosinase [Z], a unique enzyme which is the subject of this commu~cation. These two dipeptidases are very different from one another with regard to specificity, metal ion activation, M,, groups essential for activity, etc. Perry et al. [3] were the first to note that human serum hydrolyzes carnosine and anserine (fi-alanyl-1-methylhistidine). These authors also reported that two young children with hypercamosinuria on a meat-free diet were deficient in serum carnosinase. van Munster et al. [4] developed a radiometric assay for serum carnosinase and reported that normal children less than one year old had only a trace of this enzyme; thereafter the concentration of camosinase in serum increased with age up to about 15 years. Murphey et al. [5] developed a simplified and more sensitive ~uo~rnet~c assay for carnosinase. Lenney et al. [2] increased the sensitivity of the fluorimetric serum camosinase assay about 10-fold. They partially characterized the enzyme and showed that it hydrolyzed homocamosine (GABA-histidine) and was different from human tissue camosinase. Lenney et al. [6] found that patients with homocamosinosis are lacking serum carnosinase and this appears to be the reason for greatly elevated levels of brain and CSF homocarnosine in these individuals. They also reported that CSF contains relatively high concentrations of serum camosinase. Duane and Peters [7] studied patients with chronic alcoholic myopathy and found that the activity of serum carnosinase was inversely correlated to the degree of type II muscle fiber atrophy and was not correlated to the severity of liver disease. In patients with myopathy who abstained from alcohol, enzyme activity increased sig~ficantly with improvement in the myopathy. In this coruscation we report the pu~fication of human serum camosinase to apparent homogeneity. The relatively high concentrations of serum camosinase in human brain and CSF indicate that it may be synthesized in the brain and secreted into CSF. Six higher primates were found to have serum carnosinase; most other mammals, with the exception of the Golden hamster, did not have detectable carnosinase activity in the blood stream. Experimental Materials
Human blood plasma was supplied by the Blood Bank of Hawaii. Human tissues were obtained at autopsy 12 h after death, from an l&yr-old female (traffic accident victim). Great ape serum samples were from the Yerkes Regional Primate Research Center (Atlanta, GA). Hydroxylapatite (Bio-Gel HT) and a Protean vertical gel electrophoresis cell were from Bio-Rad Laboratories (Richmond, CA). ‘Ampholine’ ampholytes were from Pharmacia LKB Biotechnology (Piscataway, NJ). Carnosine, other dipeptides, tripeptides and all other reagents were from Sigma Chemical Co. (St. Louis, MO). Column chromatography was conducted using a Perkin-Elmer Isopure series 410 LC pump (Norwalk, CT). Ultrafiltration cells and Diaflo YM30 membranes were from Amicon Corp. (Danvers, MA). Collodion dialysis sacs and a dialyzing concentrator apparatus were from Schleicher and Schuell (Keene, NH).
195
Enzyme assays Serum camosinase activity against camosine and homocamosine was measured using the procedures described by Lenney et al. [2]. The free histidine was quantitated by reacting it with o-phthaldialdehyde to form a fluorescent adduct under conditions making the reaction specific for histidine. In studying the specificity of serum camosinase, the activity against a variety of peptide substrates was measured using the Butterworth and Priestman [8] modification of the method of Roth [9], as further modified by Lenney [l]. Tissue camosinase was assayed using the method of Lenney et al. [lo], activating the enzyme with MnCl, and dithiothreitol. Assay of tissues for serum carnosinase To 1 g of tissue 5 ml of 10 mmol/l Tris/HCl buffer, pH 7.8, containing 0.1 mmol/l MnCl, and 0.02% NaN, were added and the tissue was ground in a glass mortar with fine glass beads. The homogenate was centrifuged for 30 min at 40000 x g. A portion of the supematant was assayed for serum carnosinase; homocamosine was used as substrate because serum carnosinase is the only human enzyme hydrolyzing this dipeptide [6]. Another portion of the supernatant was centrifuged at 105 000 x g for 60 min and assayed for blood content using the method of Dahlberg [ll]. Gel electrophoresis Vertical slab SDS-PAGE (14 X 16 X 0.2 cm) was performed using the stock solutions and running buffers described by Laemmli [12]. A 7-17% polyacrylamide linear gradient analytical gel pH 8.8, with a 3% stacking gel pH 6.8 was used throughout. Electrophoresis was conducted for 12 h at 50 V and 15 mA at 22°C. SDS dissociation buffer was made according to Laemmli [12], substituting dithiothreitol for fi-mercaptoethanol. Molecular weight was estimated using carbonic anhydrase, ovalbumin, bovine serum albumin, phosphorylase b, fi-galactosidase and myosin as calibrating proteins. Vertical slab non-denaturing PAGE with 7.5% running gels and 4% stacking gels was run according to Davis [13]. Electrophoresis was for 3.5 h at 210 V and 20 mA at 10°C. Gels were stained with 0.2% Coomassie Blue G250 dye using the method of Neuhoff et al. [14]. Visualization of protein bands after SDS-PAGE, prior to excising the serum camosinase band, was performed using 4 mol/l sodium acetate according to Higgins and Dahmus [15]. Isoelectric focusing was carried out in a vertical slab gel 2 mm thick containing 5% polyacrylamide and 3% carrier ampholytes pH 4-6. Focusing was at 8°C for 12 h at 250 V. Marker proteins were glucose oxidase from A. niger (~1 4.2), soybean trypsin inhibitor (PI 4.55), P-lactoglobulin A from bovine milk (~1 5.13) and carbonic anhydrase II from bovine erythrocytes (PI 5.95).
196
Enzyne purification
Column chromatographic procedures were conducted at 22°C. Unused DEAEcellulose (settled volume of 240 ml) was pretreated with 2 mol/l NaCl, washed with water, then equilibrated with 25 mmol/l N-ethylmorpholine buffer, pH 7.0, containing 0.1 mmol/l MnCl, and 0.02% NaN, (buffer A). The settled matrix was mixed with 215 ml of human plasma and incubated for 1 h at 22’C with occasional stirring. The mixture was poured into a column (4 cm diameter) and washed with buffer A until the absorption at 280 nm fell below 0.05. Then a 540 ml linear gradient of NaCI (O-O.5 mol/l in buffer A) was pumped through the column at a rate of 1.5 ml/min. Fractions (7.5 ml) were collected and those containing serum carnosinase were pooIed (68 ml) and concentrated to 9.2 ml on a Diaflo YM30 ultrafiltration membrane. This concentrate was applied to a 2.5 x 25 cm column bed of hydroxylapatite which had been equilibrated with 10 mmol/l sodium phosphate buffer pH 6.8 containing 0.1 mmol/l M&l, and 0.02% NaN, (buffer B). Unbound material was eluted using 50 ml of buffer B (0.5 ml/min), collecting 3 ml fractions in tubes containing 0.5 ml of 0.5 mol/l NH,OH/HCl buffer pH 8.5 and 0.3 mI glycerol. Bound materiai was efuted using a linear gradient (50 ml) of sodium phosphate buffer, pH 6.8 (lo-500 mmol/l), containing 0.1 mmol/l MnCl, and 0.02% NaN,, and an additional 50 ml of 0.5 moI/l buffer was pumped through the column to ensure complete eiution of bound solute. Fractions containing serum camosinase (peak I, Fig. 1) were pooled (41 ml) and concentrated by ultrafiltration to 3.1 ml.
r 50
FRACTION No. (X3.8 ml)
Fig. 1. Chromato~aphy of a human plasma DEAE-~~~~_I~oss eluate on a hydroxylapatjt~ column. Fractions were assayed for activity against camosine (0) and for absorbance at 280 nm (m). ~bosphate buffer gradient (- - - - - -).
197
The hydroxylapatite column was reused 8 or 10 times before its performance began to deteriorate. This concentrate was applied to a 0.8 X 10 cm column bed of Cibacron Blue 3000 CL-L-agarose which had been equilibrated with buffer A. Non-adsorbed proteins were eluted using 20 ml buffer A at a flow rate of 0.7 ml/mm, collecting 2 ml fractions. Bound materials were eluted using 20 ml of 0.5 mol/l NaCl in buffer A. The serum carnosinase did not adsorb on the matrix; fractions containing the enzyme were pooled (10 ml) and dialyzed against 5 mmol/l Tris/HCl buffer, pH 7.4, while vacuum concentrating to a volume of 0.48 ml in a collodian sac (M, cutoff 25 000). This enzyme concentrate was applied across the width of a vertical gel slab and subjected to SDS-PAGE as described above. Protein bands were visualized using the procedure of Higgins and Dahmus [15]. The gel strip containing the serum carnosinase band was excised and placed in a dialysis bag with 0.5 ml of 10 mmol/l sodium phosphate buffer pH 7.4 containing 0.9% NaCl; dialysis was for 90 min against 1.5 1 of this buffer at 22°C. The gel slice was then fragmented and the suspension (1.9 ml) stored at - 70°C.
Identification
of enzyme
band on SDS-gel
Two ml of the peak 1 enzyme concentrate obtained after hydroxylapatite chromatography (0.4 mg protein/ml, 600 pmol/ml per h enzyme activity) was electrophoresed in a non-denaturing polyacrylamide gel. The region of the gel known from earlier experiments to contain the enzyme was cut into 2 mm slices and each slice was soaked in a solution containing 2 ml of 125 mmol/l NH,OH/HCl buffer pH 8.5 and 1.5 ml of 2.5 mmol/l CdCl, in 0.5% sodium citrate. Each slice was macerated using a handheld Potter-Elvehjem homogenizer and the resulting gel suspension was allowed to settle for 1 h at 4°C; a portion of the supernatant was then assayed for serum carnosinase. Portions of consecutive gel slice fragments containing enzyme were then electrophoresed on an SDS-gel and the resulting protein bands were visualized by staining with Coomassie Blue G-250. In the majority of the active gel slice suspensions, two protein bands were present. However in the slice from the anode end of the series, only one band was observed. It was concluded that this band represented serum carnosinase; its location in the gel slab was used to identify the enzyme band in subsequent SDS-PAGE experiments.
Preparation
of enzyme for substrate specificity
studies
Highly purified serum carnosinase was prepared by electrophoresing a hydroxylapatite column enzyme concentrate in a non-denaturing gel slab as described in the preceding paragraph. The gel slice extract having the highest activity per ml was tested for activity against 34 peptides.
198
Protein determinations
Protein concentrations in whole plasma, DEAE-cellulose concentrates and hydroxylapatite concentrates were determined using the method of Lowry et al. 1161. For Cibacron Blue-agarose concentrates the micro bicinchoninic acid method of Smith et al. [17] was used. Total protein in the gel suspension obtained after the final step was estimated as follows: To separate lanes of an SDS-gel slab known amounts of bovine serum albumin and ovalbumin (OS-20 pg) were applied, as were measured volumes of the gel suspension containing apparently homogeneous enzyme. After electrophoresis, the gel was stained using Coomassie Blue G-250 1141. The intensity of staining of the standards and the serum carnosinase bands was determined by a computerized gel scanning procedure using the ICC version 2.07 scanning program and the Dage-MTI series 68 camera. A calibration curve was constructed for the protein standards (mean integral of light transmission versus pg protein) and the amount of protein in the serum carnosinase bands was obtained by reference to this curve. Glycosylation of the enzyme
To determine the presence or absence of serum carnosinase glycosylation, 0.5 ml of hydroxylapatite peak 1 concentrate was applied to a 0.8 x 8 cm column of Concanavalin A-Sepharose. After washing with 2.5 column volumes of 20 mmol/l Tris/HCl buffer pH 7.5 containing 0.1 mmol/l MnCl, and 0.02% NaN,, bound material was eluted with either a-D-glucose, tu-D-mannose or methyl-a-D-glucopyranoside in this buffer, increasing the concentration of sugar in 50 mmol/l steps every 2.5 column volumes from 50 to 500 mmol/l. Fractions (2.5 ml) were collected and assayed for serum camosinase activity. Results
Purification
The purification of human serum camosinase is summarized in Table I. The DEAE-cellulose chromato~aphy step was very effective because the enzyme was TABLE I Purification of human serum carnosinase Step
Volume (ml)
U/ml
Protein (m&ml)
U/mg
Recovery @I
Purification (-fold)
Whole plasma DEAE cont. Hydroxylapatite Cibacron Blue cont. SDS-PAGE
215 9.2 3.1 9.0 1.9
61 725 740 100 474
66 15.8 0.45 0.022 0.029
0.92 46 1640 4550 16300
100 51 I8 6.9 6.9
I 50 1780 4950 17700
One unit of enzyme was the amount which hydrolyzed 1 pmol carnosinefh. Recovery of activity in the final step was assumed to be 100%. The final preparation represented one band on SDS-PAGE under reducing conditions.
199
eluted late in the NaCl gradient, after most (ca. 99%) of the other proteins had eluted. In the hydroxylapatite column step, two carnosinase peaks were eluted, as shown in Fig. 1. The majority of the activity was in peak 1, which did not bind to the column. Peak 2 eluted at approximately 0.2 mol/l phosphate, just before the major protein peak. Both peaks were analyzed for hydrolytic activity against homocarnosine. The ratio of activity against carnosine to activity against homocarnosine was 8.8 for peak 1 and 7.8 for peak 2, indicating that the two peaks probably represent different forms of the same enzyme. The pooled and concentrated peak 1 fractions were used for further purification; SDS-gel electrophoresis followed by Coomassie Blue G250 staining showed that this preparation contained six proteins. One of these was identified as serum carnosinase, using a procedure described in the Experimental section. When peak 1 concentrate was chromatographed on a Cibacron Blue-agarose column, the enzyme did not bind to the matrix. The active fractions were pooled, concentrated and analyzed by SDS-PAGE and non-denaturing PAGE. Both procedures showed the presence of three protein bands. On the SDS gel, the carnosinase band was well separated from the other two proteins; this was not true of the non-denaturing gel. Therefore, SDS-PAGE was used for the final purification step; although the pure enzyme was inactive, it was suitable for injection into rabbits to produce a specific antiserum. As shown in Table I, serum carnosinase was purified 17700-fold in four steps. Plasma contains about 4 mg of this enzyme per liter, which is less than 0.01% of the total plasma protein. Reactivation
of serum carnosinase
The purification procedure was repeated many times. In several cases, the recovery of enzyme activity after DEAE-cellulose chromatography was only lo-40%. In these cases, when the concentrated preparations were stored for 6 days at 4’C, a 25-400% increase in activity was observed. Substrate
specificity
Twenty nine dipeptides and 3 tripeptides were tested as potential substrates, using a highly purified serum carnosinase preparation. As shown in Table II, 15 dipeptides and 2 tripeptides were hydrolyzed. Carnosine and anserine were the two most rapidly hydrolyzed substrates; the four most rapidly hydrolyzed contained histidine at the carboxyl end of the dipeptide. Two tripeptides were slowly hydrolyzed, suggesting that serum carnosinase is a dipeptidase with weak aminopeptidase activity. The following compounds were not hydrolyzed: P-ala-try, P-ala-gly, P-alalys, ala-phe, ala-val, his-ala, his-gly, his-ser, pro-ala, pro-leu, y-glu-his, leu-arg, gly-gly-gly, N-acetyl-carnosine, N-acetyl-met, N-acetyl-his, leu$-naphthylamide and fl-ala-fl-naphthylamide.
200 TABLE
II
Hydrolysis
of peptides
by human
serum camosinase
Substrate (0.8 mmol/l)
Fluorescence to camosine
P-ala-his (camosine) P-ala-1Mehis (anserine) ala-his gly-his gly-leu P-ala-phe gly-his-gly ala-leu ala-ala GABA-his (homocarnosine) p-ala-ala phe-ala ala-try ser-his leu-leu
100 88~23 38+10 31+24 21+ 6 18i 5 13* 4 13* 4 11* 4 11+ 3 lo+ 1 8+ 2 8k 5 8+ 1 8+ 5 7-i 2 77t 6
gly-glY gly-his-lys Enzyme preparation and assay procedure are described the average +SD for 6 assays, and are not corrected different amino acid reaction products.
relative = 100
in Experimental section. The numbers shown are for variations in the fluorescence obtained from
M,, pZ and test for glycoprotein Highly purified human serum carnosinase peak 1 was run on a SDS-gel slab in lanes adjacent to six calibrating proteins. The enzyme had an apparent IV, of 75 000 in the presence of dithiothreitol and 155 000 in the absence of reducing agents. Apparently homogeneous enzyme was subjected to isoelectric focusing in a vertical slab gel in a lane adjacent to isoelectric point marker proteins. A pZ value of 4.4 & 0.1 was obtained. When human serum carnosinase peak 1 was applied to a column of concanavalin A-Sepharose, the enzyme was bound to the matrix. Mannose (0.05 mol/l) or methyl a-D-glucopyranoside (0.05 mol/l) eluted the enzyme, whereas glucose (0.05-0.5 mol/l) did not. These results indicate that the enzyme is a glycoprotein. Serum carnosinase
in various tissues
Samples from nine human tissues were analyzed for serum carnosinase concentration and for trapped blood content. Homocarnosine was used as the substrate in the enzyme assay because serum carnosinase appears to be the only human enzyme splitting this dipeptide [6]. As shown in Table III, enzyme activity was roughly proportional to blood content for all of the tissues except brain, indicating that serum carnosinase was present only in the trapped blood of these tissues.
201 TABLE
III
Distribution
of serum camosinase
in human
tissues
Tissue
% Trapped blood
Activity (pmol homocamosine.g-‘.h-‘)
Ratio Activity/ ‘%blood
Pancreas Stomach Brain Adrenal gland Skeletal muscle Heart muscle Kidney Liver Blood
1.3 1.3 1.4 1.5 2.5 3.4 4.0 4.8 100
0.02 0.045 0.30 0.07 0.055 0.09 0.11 0.13 2.5
0.015 0.035 0.214 0.047 0.022 0.026 0.028 0.027 0.025
Each extract was assayed for activity against blood. All tissues were from one individual.
4 mmoI/l
homocamosine
and for the amount
of trapped
The brain sample contained approximately 9 times as much serum carnosinase expected from its trapped blood content. This finding indicates that almost 90% the serum carnosinase in brain is extravascular in localization. The enzyme samples of human brain which hydrolyzes homocarnosine has been identified serum camosinase [6]. Serum carnosinase
as of in as
in great apes and the Golden hamster
Six higher primate serum samples were analyzed for camosinase activity. As shown in Table IV, serum carnosinase activity ranged from 8 U/ml in the orangutan to 58 U/ml in the pygmy chimpanzee, as compared to an average value of 50
TABLE
IV
Comparison Species
of serum camosinases Activity vs. camosine
from six higher primates
Relative rate of hydrolysis (camosine = 100)
Effect of inhibitors: % inhibition activity against camosine
of
Anserine
Homocarnosine
Bestatin (30 pmol/l)
DTT (2 mmol/l)
(D/ml) Human Chimpanzee Gibbon Gorilla Orangutan
50 48 23 37 8
88 61 69 109 271
10.7 0.8 2.1 2.1 12.5
39 26 48 60 65
Pygmy chimpanzee
58
75
0.9
59
pHMB (0.2 mmol/l) 8 9 0 4
94 96 94 92 82
16
92
-21
Relative rates of hydrolysis of substrates by human serum are from Table II. Other primate data represented an average of two determinations using a single serum sample from each species. Inhibitor concentrations are final concentrations in 0.5 ml digest.
202
U/ml in adult human serum. All samples hydrolyzed anserine and homocarnosine and responded similarly to the three inhibitors, bestatin, p-hydroxymercuribenzoate and dithiothreitol. The data in Table IV indicate that the six higher primates have similar serum carnosinases. Serum samples from three monkey species (cynomologus, owl and rhesus) contained low concentrations of carnosinase. No carnosinase was detected in serum from dog, horse, calf, hog, rat, mouse, rabbit, baboon, guinea pig, armadillo or Chinese hamster. However, Golden hamster serum samples hydrolyzed carnosine at an average rate of 570 ~mol/rnl per h, approximately 11 times more rapidly than the average adult human sample. These hamster samples were activated more effectively by CdC12 than by MnCl,; they hydrolyzed anserine and had weak activity against homocarnosine. In contrast to the inhibition data for higher primates in Table IV, the Golden hamster enzyme was inhibited 94% by p-hydroxymercuribenzoate, 74% by dithiothreitol and was unaffected by bestatin. Furthermore, the pH optimum for the Golden hamster enzyme was 7.4 whereas human serum carnosinase had optimum activity at pH 8.3. Therefore, the Golden hamster serum carnosine-hydrolyzing enzyme appears to be somewhat different from that present in the serum of higher primates.
Discussion Human serum carnosinase was, purified 17700-fold; apparent homogeneity was achieved using a four step procedure. The first step, DEAE-cellulose chromatography, was very effective, probably because of the relatively low pl (4.4) of this enzyme. In the second step, hydroxylapatite chromatography (Fig. l), two forms of serum camosinase were separated from one another. These enzymes had similar activities in the hydrolysis of carnosine and homocarnosine and are probably two forms of the same enzyme. SDS-gel electrophoresis showed that the purified serum carnosinase had a molecular weight of 155 000 in the absence of reducing agents and 75000 in the presence of dithiothreitol. The active enzyme had an apparent M, of 160000 (by s&e-exclusion chromatography) and was inhibited by dithiothreitol [2]. Thus, a homodimeric structure, with the two subunits held together by disulfide bonds, appears to be essential for activity. In some of the DEAE-cellulose column runs, abnormally low yields were encountered. In these cases, after the eluted enzyme was concentrated by ultrafiltration, a marked reactivation occurred. One possible explanation for this phenomenon is that the enzyme lost activity by reduction of intra-chain -S-S- bonds and regained it by oxidation of the resulting -SH groups to form active dimers. Human serum camosinase had a unique specificity (Table II). Of 34 substrates tested, carnosine and anserine were hydrolyzed much more rapidly than any of the others. The four best substrates were dipeptides containing histidine at the Cterminus. Serum camosinase split anserine and homocarnosine, but not pro-leu or pro-ala, whereas human tissue carnosinase hydrolyzed prolinase substrates (e.g. pro-leu), but not anserine or homocarnosine [l]. Although serum carnosinase split a
203
variety of dipeptides, its specificity was not very broad, since 12 of the dipeptides tested were not attacked. Another unique feature of serum camosinase is that it is present in higher primates (man and the great apes) but not in most other mammals. Of 12 non-primate mammals tested, only the Golden hamster had serum carnosinase activity; this serum was extremely active, but the properties of this enzyme were different from those of the higher primate enzyme. Curiously, Chinese hamster serum was inactive against carnosine. Table III shows that the activity of serum carnosinase in eight human tissues was proportional to the level of trapped blood. However, in brain only about 10% of the serum carnosinase was in the trapped blood. The brain contains about 12% interstitial fluid, which is the equivalent of CSF, and the concentration of serum carnosinase in CSF is about 10% of that in serum [6]. Thus, about 17% of the serum carnosinase in a brain sample is in the interstitial fluid. The remainder of the serum enzyme (about 70%) appears to be cellular in localization. Kish et al. [18] analyzed eight regions of human brain for homocarnosine-hydrolyzing activity (serum carnosinase, in retrospect) and found an uneven distribution of the enzyme. The highest activity was in the dentate nucleus and the lowest in the cerebellar cortex. All activities were much higher than would be expected if the enzyme were in trapped blood and CSF only. Human serum proteins are present in CSF, the concentrations in serum being approximately 300 times higher than those in CSF. Since the concentration of serum carnosinase is only 10 times higher in serum than in CSF, the concentration of this enzyme in CSF is 30 times higher than expected, as compared with the levels of other serum proteins. Evidently serum camosinase is selectively secreted by brain cells into CSF. It seems unlikely that the enzyme is selectively transported across the blood-brain barrier into brain cells. Therefore it can be speculated that serum camosinase may be synthesized in brain cells. Several reports have shown that the level of camosinase in the blood stream increases with age. In the first 10 months of life, no activity is detectable; thereafter the concentration rises gradually, reaching the adult level at age 12-15 years [2]. Roesel et al. [19] have shown that the urinary excretion of camosine (expressed as pmol/g creatinine) decreases with age during the first four years of life; older children and adults excrete less than 4% of the amounts excreted by infants. These workers attributed the drop in carnosinuria to the rising concentration of serum carnosinase. Homocarnosinosis is a rare familial metabolic disorder. In patients with this disease, CSF homocarnosine concentrations are elevated 20-fold above normal [20] and brain homocarnosine in one of the patients was four times normal [21]. The elevated concentrations of homocarnosine are probably attributable to the fact that these patients lack serum carnosinase [6]. On a meat-free diet, normal individuals excrete very small amounts of carnosine, whereas the homocamosinosis patients excreted more than 20 times this amount [22]. When normal individuals eat chicken meat, which contains carnosine and anserine, they hydrolyze these compounds and excrete a reaction product from anserine, 1-methylhistidine. When the homocarno-
204
sinosis patients consumed chicken, they excreted relatively large amounts of the unhydrolyzed dipeptides and very little 1-methylhistidine [22]. Thus it appears that serum carnosinase has two functions: the hydrolysis of homocarnosine in the brain, and the hydrolysis of carnosine and anserine in the blood stream. Since the homocarnosinosis patients have hypercarnosinuria on a meat-free diet, carnosine must be ‘leaking’ from their skeletal muscles. These patients probably have normal concentrations of tissue carnosinase [lo], since a brain biopsy of one of the patients showed a normal level of this enzyme (Lenney et al., unpubl. results). However, tissue carnosinase probably has little or no activity against carnosine in vivo because of its high pH optimum (9.5) and its high K, value (20 mmol/l carnosine). This enzyme is much more active against other dipeptides such as gly-leu and pro-leu [l]. Homocarnosine and GABA are present only in the central nervous system. Human brain contains much more homocarnosine than hog or rat brains [23]. Kish et al. [18] showed that homocarnosine was unevenly distributed in 13 regions of human brain; little or no camosine was detected except in the olfactory bulb, where the carnosine concentration was l/5 of the homocarnosine concentration. Homocarnosine may serve as a storage depot for GABA in higher primates, where serum carnosinase is present to release the GABA. The hog and rat have no serum carnosinase, and their brains do not hydrolyze homocarnosine [24,25]. Acknowledgements We thank George R. Lenney and Richard W.M. Child for financial support, Alan Komeya for excellent technical assistance, the Blood Bank of Hawaii for human plasma, Dr. Robert V. Cooney for performing the gel scanning measurements, Dr. Charles Odom for autopsy tissues and the Yerkes Regional Primate Research Center for great ape serum samples. References Lenney JF. Human cytosolic camosinase: evidence for identity with prolinase, a non-specific dipeptidase. Biol Chem Hoppe-Seyler 1990;371:167-171. Lenney JF, George RP, Weiss AM, Kucera CM, Chan PWH, Rinzler GS. Human serum carnosinase: characterization, distinction from cellular carnosinase, and activation by cadmium. Clin Chim Acta 1982;123:221-231. Perry TL, Hansen S, Love DL. Serum carnosinase deficiency in carnosinemia. Lancet 1968;June 8:1229-1230. van Munster PJJ, Trijbels JMF, van Heeswijk PJ, Schut-Jansen B, Moerkerk C. A new sensitive method for the determination of serum camosinase activity using L-carnosine-[l-‘4C]fl-alanyl as substrate. Clin Chim Acta 1970;29:243-248. Murphey WH, Patchen L, Lindmark DG. Carnosinase: a fluorometric assay and demonstration of two electrophoretic forms in human tissue extracts. Clin Chim Acta 1972;42:309-314. Lenney JF, Peppers SC, Kucera CM, Sjaastad 0. Homocarnosinosis: lack of serum carnosinase is the defect probably responsible for elevated brain and CSF homocarnosine. Clin Chim Acta 1983:132:157-165.
205 7 Duane P, Peters T3. Serum camosinase activities in patients with aicohohc chronic skeletal muscle myopathy. Clin Sci 1988;75:185-190. 8 Butterworth J, Priestman D. Fluorimetric assay for prohnase and partial characterization in cultured skin fibroblasts. Clin Chim Acta 1982;122:51-60. 9 Roth M. Fluorescence reaction for amino acids. Anal Chem 1971;43:880-882. 10 Lenney JF, Peppers SC, Kucera-Orallo CM, George RP. Characterization of human tissue camosinase. B&hem J 1985;228:653-660. 11 Dahlberg E. Estimation of the blood contamination of tissue extracts. Anal B&hem 19S3;130:108113. I2 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685, 13 Davis BJ. Disc electrophoresis-IL Method and application to human serum proteins. Ann NY Acad Sci 1964:121:404-427. 14 Neuhoff V, Amid N, Taube D, Ehrhardt W. Improved staining of proteins in po~yac~~amide gels inctuding isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. El~trophor 1988;9:255-262. 15 Higgins RC, Dahmus ME. Rapid visualization of protein bands OR SDS-gels. Anal Biochem 1979;93:25?-260. 16 Lowry OH, Rosebrougb NJ, Farr AL, Randall RI. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 17 Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal B&hem 1985;150:76-85. 18 Kish SJ, Perry TL, Hansen S. Regional distribution of homocamosine, homocamosine-carnosine synthetase and homocamosinase in human brain. J Neurochem 1979;32:1629-1636. 19 Roesel RA, Kearse EC, Blankenship PR. Camosine excretion in infants and children. Fed Proc 1986;45:470 (abstract). 20 Sjaastad 0, Berstad J, Gjesdahf P. Gjessing L. Homocamosinosis. 2. A familial metabolic disorder associated with spastic paraplegia, progressive mental deficiency, and retinal pigmentation. Acta Nemo1 Stand 1976;53:275-290. 21 Perry TL, Kish SJ, Sjaastad 0, et al. Homocamosinosis: increased content of homocarnosine and deficiency of homocamosinase in brain. J Neurochem 1979;32:1637-1640. 22 Lunde H. Sjaastad 0, Gjessing L. Homoeamosinosis: hypercamosinuria. J Neurochem 1982;38:242245. 23 Abraham D, Pisano JJ, Udenfriend S. The distribution of homocamosine in mammals. Arch Biochem Biophys 1962;99:210-213, 24 Ziesler 0, Hole K, Haugan I, Borresen AL, Gjessing LR, Sjaastad 0. Transport and distribution of homocamosine after intracerebroventricular and intravenous injection in the rat. Neurochem Res 1984;9:637-648. 25 Lenney JF. Separation and characterization of two camosine-sphtting cytosolic dipeptidases from hog kidney (camosinase and non-specific dipeptidase). Biol Chem Hoppe-Seyler 1990,371:433-440.
Clinica Chin&a Act4 196 (1991) 193-206 8 1991 Elsevier Science Publishers B.V. ~9-8981/91/$03.50 ~898191~7lM
ADONIS
CCA 04930
Purification and properties of human serum carnosinase Mel C. Jackson, Pharmacololly Beparrment,
Christine .Sehooi
M. Kucera
and James F. Lenney
of ~edjc;~e, Univer~it.v of Hawaii, Honv~~~u~Hawaii (USA)
(Received 30 July 1990; revision received 13 October 1990; accepted 16 October 1990) Key words; Serum carnosinase; Dipeptidase
Summaty
Carnosinase from human plasma was purified 18 OOO-foldto apparent homogeneity in a four step procedure. The dipeptidase was partially inactivated during DEAE-cellulose chromatography; however, it reactivated slowly when concentrated and stored at 4*C. In the second purification step, hydroxylapatite column chromatography, two forms of the enzyme were separated from one another. Human serum camosinase was found to be a glycoprotein with a p1 of 4.4 and a subunit n/r, of 75 000; the active enzyme was a dimer, the two subunits being connected by one or more disulfide bonds. The enzyme was especially active in hydrolyzing carnosine and anserine, preferring dipeptides with histidine in the C-terminal position. In most human tissues, the concentration of serum carnosinase was proportional to the percentage of trapped blood in the sample. However, the brain contained about 9 times more enzyme than expected, based on the amount of trapped blood present. The physiological function of this enzyme seems to be the hydrolysis of homocamosine in the brain and the splitting of carnosine and anserine in the blood stream. Six higher primates were found to have serum carnosinase. Twelve nonprimate mammals were tested; all were lacking the serum enzyme except for the Golden hamster, which had very high concentrations of a camosinase having somewhat different properties than the higher primate enzyme, Introduction
The human body contains two dipeptidases which hydrolyze carnosine (palanylhistidine). One of these is present in tissues, especially kidney, liver and brain, Correspondence to: J.F. Lenney, Pharmacology Department, School of Medicine, University of Hawaii, 1960 East West Road, Honolulu, Hawaii 96822, USA.
194
and should be referred to as ‘non-specific cytosolic dipept~dase’ [I]. The other is serum camosinase [Z], a unique enzyme which is the subject of this commu~cation. These two dipeptidases are very different from one another with regard to specificity, metal ion activation, M,, groups essential for activity, etc. Perry et al. [3] were the first to note that human serum hydrolyzes carnosine and anserine (fi-alanyl-1-methylhistidine). These authors also reported that two young children with hypercamosinuria on a meat-free diet were deficient in serum carnosinase. van Munster et al. [4] developed a radiometric assay for serum carnosinase and reported that normal children less than one year old had only a trace of this enzyme; thereafter the concentration of camosinase in serum increased with age up to about 15 years. Murphey et al. [5] developed a simplified and more sensitive ~uo~rnet~c assay for carnosinase. Lenney et al. [2] increased the sensitivity of the fluorimetric serum camosinase assay about 10-fold. They partially characterized the enzyme and showed that it hydrolyzed homocamosine (GABA-histidine) and was different from human tissue camosinase. Lenney et al. [6] found that patients with homocamosinosis are lacking serum carnosinase and this appears to be the reason for greatly elevated levels of brain and CSF homocarnosine in these individuals. They also reported that CSF contains relatively high concentrations of serum camosinase. Duane and Peters [7] studied patients with chronic alcoholic myopathy and found that the activity of serum carnosinase was inversely correlated to the degree of type II muscle fiber atrophy and was not correlated to the severity of liver disease. In patients with myopathy who abstained from alcohol, enzyme activity increased sig~ficantly with improvement in the myopathy. In this coruscation we report the pu~fication of human serum camosinase to apparent homogeneity. The relatively high concentrations of serum camosinase in human brain and CSF indicate that it may be synthesized in the brain and secreted into CSF. Six higher primates were found to have serum carnosinase; most other mammals, with the exception of the Golden hamster, did not have detectable carnosinase activity in the blood stream. Experimental Materials
Human blood plasma was supplied by the Blood Bank of Hawaii. Human tissues were obtained at autopsy 12 h after death, from an l&yr-old female (traffic accident victim). Great ape serum samples were from the Yerkes Regional Primate Research Center (Atlanta, GA). Hydroxylapatite (Bio-Gel HT) and a Protean vertical gel electrophoresis cell were from Bio-Rad Laboratories (Richmond, CA). ‘Ampholine’ ampholytes were from Pharmacia LKB Biotechnology (Piscataway, NJ). Carnosine, other dipeptides, tripeptides and all other reagents were from Sigma Chemical Co. (St. Louis, MO). Column chromatography was conducted using a Perkin-Elmer Isopure series 410 LC pump (Norwalk, CT). Ultrafiltration cells and Diaflo YM30 membranes were from Amicon Corp. (Danvers, MA). Collodion dialysis sacs and a dialyzing concentrator apparatus were from Schleicher and Schuell (Keene, NH).
195
Enzyme assays Serum camosinase activity against camosine and homocamosine was measured using the procedures described by Lenney et al. [2]. The free histidine was quantitated by reacting it with o-phthaldialdehyde to form a fluorescent adduct under conditions making the reaction specific for histidine. In studying the specificity of serum camosinase, the activity against a variety of peptide substrates was measured using the Butterworth and Priestman [8] modification of the method of Roth [9], as further modified by Lenney [l]. Tissue camosinase was assayed using the method of Lenney et al. [lo], activating the enzyme with MnCl, and dithiothreitol. Assay of tissues for serum carnosinase To 1 g of tissue 5 ml of 10 mmol/l Tris/HCl buffer, pH 7.8, containing 0.1 mmol/l MnCl, and 0.02% NaN, were added and the tissue was ground in a glass mortar with fine glass beads. The homogenate was centrifuged for 30 min at 40000 x g. A portion of the supematant was assayed for serum carnosinase; homocamosine was used as substrate because serum carnosinase is the only human enzyme hydrolyzing this dipeptide [6]. Another portion of the supernatant was centrifuged at 105 000 x g for 60 min and assayed for blood content using the method of Dahlberg [ll]. Gel electrophoresis Vertical slab SDS-PAGE (14 X 16 X 0.2 cm) was performed using the stock solutions and running buffers described by Laemmli [12]. A 7-17% polyacrylamide linear gradient analytical gel pH 8.8, with a 3% stacking gel pH 6.8 was used throughout. Electrophoresis was conducted for 12 h at 50 V and 15 mA at 22°C. SDS dissociation buffer was made according to Laemmli [12], substituting dithiothreitol for fi-mercaptoethanol. Molecular weight was estimated using carbonic anhydrase, ovalbumin, bovine serum albumin, phosphorylase b, fi-galactosidase and myosin as calibrating proteins. Vertical slab non-denaturing PAGE with 7.5% running gels and 4% stacking gels was run according to Davis [13]. Electrophoresis was for 3.5 h at 210 V and 20 mA at 10°C. Gels were stained with 0.2% Coomassie Blue G250 dye using the method of Neuhoff et al. [14]. Visualization of protein bands after SDS-PAGE, prior to excising the serum camosinase band, was performed using 4 mol/l sodium acetate according to Higgins and Dahmus [15]. Isoelectric focusing was carried out in a vertical slab gel 2 mm thick containing 5% polyacrylamide and 3% carrier ampholytes pH 4-6. Focusing was at 8°C for 12 h at 250 V. Marker proteins were glucose oxidase from A. niger (~1 4.2), soybean trypsin inhibitor (PI 4.55), P-lactoglobulin A from bovine milk (~1 5.13) and carbonic anhydrase II from bovine erythrocytes (PI 5.95).
196
Enzyne purification
Column chromatographic procedures were conducted at 22°C. Unused DEAEcellulose (settled volume of 240 ml) was pretreated with 2 mol/l NaCl, washed with water, then equilibrated with 25 mmol/l N-ethylmorpholine buffer, pH 7.0, containing 0.1 mmol/l MnCl, and 0.02% NaN, (buffer A). The settled matrix was mixed with 215 ml of human plasma and incubated for 1 h at 22’C with occasional stirring. The mixture was poured into a column (4 cm diameter) and washed with buffer A until the absorption at 280 nm fell below 0.05. Then a 540 ml linear gradient of NaCI (O-O.5 mol/l in buffer A) was pumped through the column at a rate of 1.5 ml/min. Fractions (7.5 ml) were collected and those containing serum carnosinase were pooIed (68 ml) and concentrated to 9.2 ml on a Diaflo YM30 ultrafiltration membrane. This concentrate was applied to a 2.5 x 25 cm column bed of hydroxylapatite which had been equilibrated with 10 mmol/l sodium phosphate buffer pH 6.8 containing 0.1 mmol/l M&l, and 0.02% NaN, (buffer B). Unbound material was eluted using 50 ml of buffer B (0.5 ml/min), collecting 3 ml fractions in tubes containing 0.5 ml of 0.5 mol/l NH,OH/HCl buffer pH 8.5 and 0.3 mI glycerol. Bound materiai was efuted using a linear gradient (50 ml) of sodium phosphate buffer, pH 6.8 (lo-500 mmol/l), containing 0.1 mmol/l MnCl, and 0.02% NaN,, and an additional 50 ml of 0.5 moI/l buffer was pumped through the column to ensure complete eiution of bound solute. Fractions containing serum camosinase (peak I, Fig. 1) were pooled (41 ml) and concentrated by ultrafiltration to 3.1 ml.
r 50
FRACTION No. (X3.8 ml)
Fig. 1. Chromato~aphy of a human plasma DEAE-~~~~_I~oss eluate on a hydroxylapatjt~ column. Fractions were assayed for activity against camosine (0) and for absorbance at 280 nm (m). ~bosphate buffer gradient (- - - - - -).
197
The hydroxylapatite column was reused 8 or 10 times before its performance began to deteriorate. This concentrate was applied to a 0.8 X 10 cm column bed of Cibacron Blue 3000 CL-L-agarose which had been equilibrated with buffer A. Non-adsorbed proteins were eluted using 20 ml buffer A at a flow rate of 0.7 ml/mm, collecting 2 ml fractions. Bound materials were eluted using 20 ml of 0.5 mol/l NaCl in buffer A. The serum carnosinase did not adsorb on the matrix; fractions containing the enzyme were pooled (10 ml) and dialyzed against 5 mmol/l Tris/HCl buffer, pH 7.4, while vacuum concentrating to a volume of 0.48 ml in a collodian sac (M, cutoff 25 000). This enzyme concentrate was applied across the width of a vertical gel slab and subjected to SDS-PAGE as described above. Protein bands were visualized using the procedure of Higgins and Dahmus [15]. The gel strip containing the serum carnosinase band was excised and placed in a dialysis bag with 0.5 ml of 10 mmol/l sodium phosphate buffer pH 7.4 containing 0.9% NaCl; dialysis was for 90 min against 1.5 1 of this buffer at 22°C. The gel slice was then fragmented and the suspension (1.9 ml) stored at - 70°C.
Identification
of enzyme
band on SDS-gel
Two ml of the peak 1 enzyme concentrate obtained after hydroxylapatite chromatography (0.4 mg protein/ml, 600 pmol/ml per h enzyme activity) was electrophoresed in a non-denaturing polyacrylamide gel. The region of the gel known from earlier experiments to contain the enzyme was cut into 2 mm slices and each slice was soaked in a solution containing 2 ml of 125 mmol/l NH,OH/HCl buffer pH 8.5 and 1.5 ml of 2.5 mmol/l CdCl, in 0.5% sodium citrate. Each slice was macerated using a handheld Potter-Elvehjem homogenizer and the resulting gel suspension was allowed to settle for 1 h at 4°C; a portion of the supernatant was then assayed for serum carnosinase. Portions of consecutive gel slice fragments containing enzyme were then electrophoresed on an SDS-gel and the resulting protein bands were visualized by staining with Coomassie Blue G-250. In the majority of the active gel slice suspensions, two protein bands were present. However in the slice from the anode end of the series, only one band was observed. It was concluded that this band represented serum carnosinase; its location in the gel slab was used to identify the enzyme band in subsequent SDS-PAGE experiments.
Preparation
of enzyme for substrate specificity
studies
Highly purified serum carnosinase was prepared by electrophoresing a hydroxylapatite column enzyme concentrate in a non-denaturing gel slab as described in the preceding paragraph. The gel slice extract having the highest activity per ml was tested for activity against 34 peptides.
198
Protein determinations
Protein concentrations in whole plasma, DEAE-cellulose concentrates and hydroxylapatite concentrates were determined using the method of Lowry et al. 1161. For Cibacron Blue-agarose concentrates the micro bicinchoninic acid method of Smith et al. [17] was used. Total protein in the gel suspension obtained after the final step was estimated as follows: To separate lanes of an SDS-gel slab known amounts of bovine serum albumin and ovalbumin (OS-20 pg) were applied, as were measured volumes of the gel suspension containing apparently homogeneous enzyme. After electrophoresis, the gel was stained using Coomassie Blue G-250 1141. The intensity of staining of the standards and the serum carnosinase bands was determined by a computerized gel scanning procedure using the ICC version 2.07 scanning program and the Dage-MTI series 68 camera. A calibration curve was constructed for the protein standards (mean integral of light transmission versus pg protein) and the amount of protein in the serum carnosinase bands was obtained by reference to this curve. Glycosylation of the enzyme
To determine the presence or absence of serum carnosinase glycosylation, 0.5 ml of hydroxylapatite peak 1 concentrate was applied to a 0.8 x 8 cm column of Concanavalin A-Sepharose. After washing with 2.5 column volumes of 20 mmol/l Tris/HCl buffer pH 7.5 containing 0.1 mmol/l MnCl, and 0.02% NaN,, bound material was eluted with either a-D-glucose, tu-D-mannose or methyl-a-D-glucopyranoside in this buffer, increasing the concentration of sugar in 50 mmol/l steps every 2.5 column volumes from 50 to 500 mmol/l. Fractions (2.5 ml) were collected and assayed for serum camosinase activity. Results
Purification
The purification of human serum camosinase is summarized in Table I. The DEAE-cellulose chromato~aphy step was very effective because the enzyme was TABLE I Purification of human serum carnosinase Step
Volume (ml)
U/ml
Protein (m&ml)
U/mg
Recovery @I
Purification (-fold)
Whole plasma DEAE cont. Hydroxylapatite Cibacron Blue cont. SDS-PAGE
215 9.2 3.1 9.0 1.9
61 725 740 100 474
66 15.8 0.45 0.022 0.029
0.92 46 1640 4550 16300
100 51 I8 6.9 6.9
I 50 1780 4950 17700
One unit of enzyme was the amount which hydrolyzed 1 pmol carnosinefh. Recovery of activity in the final step was assumed to be 100%. The final preparation represented one band on SDS-PAGE under reducing conditions.
199
eluted late in the NaCl gradient, after most (ca. 99%) of the other proteins had eluted. In the hydroxylapatite column step, two carnosinase peaks were eluted, as shown in Fig. 1. The majority of the activity was in peak 1, which did not bind to the column. Peak 2 eluted at approximately 0.2 mol/l phosphate, just before the major protein peak. Both peaks were analyzed for hydrolytic activity against homocarnosine. The ratio of activity against carnosine to activity against homocarnosine was 8.8 for peak 1 and 7.8 for peak 2, indicating that the two peaks probably represent different forms of the same enzyme. The pooled and concentrated peak 1 fractions were used for further purification; SDS-gel electrophoresis followed by Coomassie Blue G250 staining showed that this preparation contained six proteins. One of these was identified as serum carnosinase, using a procedure described in the Experimental section. When peak 1 concentrate was chromatographed on a Cibacron Blue-agarose column, the enzyme did not bind to the matrix. The active fractions were pooled, concentrated and analyzed by SDS-PAGE and non-denaturing PAGE. Both procedures showed the presence of three protein bands. On the SDS gel, the carnosinase band was well separated from the other two proteins; this was not true of the non-denaturing gel. Therefore, SDS-PAGE was used for the final purification step; although the pure enzyme was inactive, it was suitable for injection into rabbits to produce a specific antiserum. As shown in Table I, serum carnosinase was purified 17700-fold in four steps. Plasma contains about 4 mg of this enzyme per liter, which is less than 0.01% of the total plasma protein. Reactivation
of serum carnosinase
The purification procedure was repeated many times. In several cases, the recovery of enzyme activity after DEAE-cellulose chromatography was only lo-40%. In these cases, when the concentrated preparations were stored for 6 days at 4’C, a 25-400% increase in activity was observed. Substrate
specificity
Twenty nine dipeptides and 3 tripeptides were tested as potential substrates, using a highly purified serum carnosinase preparation. As shown in Table II, 15 dipeptides and 2 tripeptides were hydrolyzed. Carnosine and anserine were the two most rapidly hydrolyzed substrates; the four most rapidly hydrolyzed contained histidine at the carboxyl end of the dipeptide. Two tripeptides were slowly hydrolyzed, suggesting that serum carnosinase is a dipeptidase with weak aminopeptidase activity. The following compounds were not hydrolyzed: P-ala-try, P-ala-gly, P-alalys, ala-phe, ala-val, his-ala, his-gly, his-ser, pro-ala, pro-leu, y-glu-his, leu-arg, gly-gly-gly, N-acetyl-carnosine, N-acetyl-met, N-acetyl-his, leu$-naphthylamide and fl-ala-fl-naphthylamide.
200 TABLE
II
Hydrolysis
of peptides
by human
serum camosinase
Substrate (0.8 mmol/l)
Fluorescence to camosine
P-ala-his (camosine) P-ala-1Mehis (anserine) ala-his gly-his gly-leu P-ala-phe gly-his-gly ala-leu ala-ala GABA-his (homocarnosine) p-ala-ala phe-ala ala-try ser-his leu-leu
100 88~23 38+10 31+24 21+ 6 18i 5 13* 4 13* 4 11* 4 11+ 3 lo+ 1 8+ 2 8k 5 8+ 1 8+ 5 7-i 2 77t 6
gly-glY gly-his-lys Enzyme preparation and assay procedure are described the average +SD for 6 assays, and are not corrected different amino acid reaction products.
relative = 100
in Experimental section. The numbers shown are for variations in the fluorescence obtained from
M,, pZ and test for glycoprotein Highly purified human serum carnosinase peak 1 was run on a SDS-gel slab in lanes adjacent to six calibrating proteins. The enzyme had an apparent IV, of 75 000 in the presence of dithiothreitol and 155 000 in the absence of reducing agents. Apparently homogeneous enzyme was subjected to isoelectric focusing in a vertical slab gel in a lane adjacent to isoelectric point marker proteins. A pZ value of 4.4 & 0.1 was obtained. When human serum carnosinase peak 1 was applied to a column of concanavalin A-Sepharose, the enzyme was bound to the matrix. Mannose (0.05 mol/l) or methyl a-D-glucopyranoside (0.05 mol/l) eluted the enzyme, whereas glucose (0.05-0.5 mol/l) did not. These results indicate that the enzyme is a glycoprotein. Serum carnosinase
in various tissues
Samples from nine human tissues were analyzed for serum carnosinase concentration and for trapped blood content. Homocarnosine was used as the substrate in the enzyme assay because serum carnosinase appears to be the only human enzyme splitting this dipeptide [6]. As shown in Table III, enzyme activity was roughly proportional to blood content for all of the tissues except brain, indicating that serum carnosinase was present only in the trapped blood of these tissues.
201 TABLE
III
Distribution
of serum camosinase
in human
tissues
Tissue
% Trapped blood
Activity (pmol homocamosine.g-‘.h-‘)
Ratio Activity/ ‘%blood
Pancreas Stomach Brain Adrenal gland Skeletal muscle Heart muscle Kidney Liver Blood
1.3 1.3 1.4 1.5 2.5 3.4 4.0 4.8 100
0.02 0.045 0.30 0.07 0.055 0.09 0.11 0.13 2.5
0.015 0.035 0.214 0.047 0.022 0.026 0.028 0.027 0.025
Each extract was assayed for activity against blood. All tissues were from one individual.
4 mmoI/l
homocamosine
and for the amount
of trapped
The brain sample contained approximately 9 times as much serum carnosinase expected from its trapped blood content. This finding indicates that almost 90% the serum carnosinase in brain is extravascular in localization. The enzyme samples of human brain which hydrolyzes homocarnosine has been identified serum camosinase [6]. Serum carnosinase
as of in as
in great apes and the Golden hamster
Six higher primate serum samples were analyzed for camosinase activity. As shown in Table IV, serum carnosinase activity ranged from 8 U/ml in the orangutan to 58 U/ml in the pygmy chimpanzee, as compared to an average value of 50
TABLE
IV
Comparison Species
of serum camosinases Activity vs. camosine
from six higher primates
Relative rate of hydrolysis (camosine = 100)
Effect of inhibitors: % inhibition activity against camosine
of
Anserine
Homocarnosine
Bestatin (30 pmol/l)
DTT (2 mmol/l)
(D/ml) Human Chimpanzee Gibbon Gorilla Orangutan
50 48 23 37 8
88 61 69 109 271
10.7 0.8 2.1 2.1 12.5
39 26 48 60 65
Pygmy chimpanzee
58
75
0.9
59
pHMB (0.2 mmol/l) 8 9 0 4
94 96 94 92 82
16
92
-21
Relative rates of hydrolysis of substrates by human serum are from Table II. Other primate data represented an average of two determinations using a single serum sample from each species. Inhibitor concentrations are final concentrations in 0.5 ml digest.
202
U/ml in adult human serum. All samples hydrolyzed anserine and homocarnosine and responded similarly to the three inhibitors, bestatin, p-hydroxymercuribenzoate and dithiothreitol. The data in Table IV indicate that the six higher primates have similar serum carnosinases. Serum samples from three monkey species (cynomologus, owl and rhesus) contained low concentrations of carnosinase. No carnosinase was detected in serum from dog, horse, calf, hog, rat, mouse, rabbit, baboon, guinea pig, armadillo or Chinese hamster. However, Golden hamster serum samples hydrolyzed carnosine at an average rate of 570 ~mol/rnl per h, approximately 11 times more rapidly than the average adult human sample. These hamster samples were activated more effectively by CdC12 than by MnCl,; they hydrolyzed anserine and had weak activity against homocarnosine. In contrast to the inhibition data for higher primates in Table IV, the Golden hamster enzyme was inhibited 94% by p-hydroxymercuribenzoate, 74% by dithiothreitol and was unaffected by bestatin. Furthermore, the pH optimum for the Golden hamster enzyme was 7.4 whereas human serum carnosinase had optimum activity at pH 8.3. Therefore, the Golden hamster serum carnosine-hydrolyzing enzyme appears to be somewhat different from that present in the serum of higher primates.
Discussion Human serum carnosinase was, purified 17700-fold; apparent homogeneity was achieved using a four step procedure. The first step, DEAE-cellulose chromatography, was very effective, probably because of the relatively low pl (4.4) of this enzyme. In the second step, hydroxylapatite chromatography (Fig. l), two forms of serum camosinase were separated from one another. These enzymes had similar activities in the hydrolysis of carnosine and homocarnosine and are probably two forms of the same enzyme. SDS-gel electrophoresis showed that the purified serum carnosinase had a molecular weight of 155 000 in the absence of reducing agents and 75000 in the presence of dithiothreitol. The active enzyme had an apparent M, of 160000 (by s&e-exclusion chromatography) and was inhibited by dithiothreitol [2]. Thus, a homodimeric structure, with the two subunits held together by disulfide bonds, appears to be essential for activity. In some of the DEAE-cellulose column runs, abnormally low yields were encountered. In these cases, after the eluted enzyme was concentrated by ultrafiltration, a marked reactivation occurred. One possible explanation for this phenomenon is that the enzyme lost activity by reduction of intra-chain -S-S- bonds and regained it by oxidation of the resulting -SH groups to form active dimers. Human serum camosinase had a unique specificity (Table II). Of 34 substrates tested, carnosine and anserine were hydrolyzed much more rapidly than any of the others. The four best substrates were dipeptides containing histidine at the Cterminus. Serum camosinase split anserine and homocarnosine, but not pro-leu or pro-ala, whereas human tissue carnosinase hydrolyzed prolinase substrates (e.g. pro-leu), but not anserine or homocarnosine [l]. Although serum carnosinase split a
203
variety of dipeptides, its specificity was not very broad, since 12 of the dipeptides tested were not attacked. Another unique feature of serum camosinase is that it is present in higher primates (man and the great apes) but not in most other mammals. Of 12 non-primate mammals tested, only the Golden hamster had serum carnosinase activity; this serum was extremely active, but the properties of this enzyme were different from those of the higher primate enzyme. Curiously, Chinese hamster serum was inactive against carnosine. Table III shows that the activity of serum carnosinase in eight human tissues was proportional to the level of trapped blood. However, in brain only about 10% of the serum carnosinase was in the trapped blood. The brain contains about 12% interstitial fluid, which is the equivalent of CSF, and the concentration of serum carnosinase in CSF is about 10% of that in serum [6]. Thus, about 17% of the serum carnosinase in a brain sample is in the interstitial fluid. The remainder of the serum enzyme (about 70%) appears to be cellular in localization. Kish et al. [18] analyzed eight regions of human brain for homocarnosine-hydrolyzing activity (serum carnosinase, in retrospect) and found an uneven distribution of the enzyme. The highest activity was in the dentate nucleus and the lowest in the cerebellar cortex. All activities were much higher than would be expected if the enzyme were in trapped blood and CSF only. Human serum proteins are present in CSF, the concentrations in serum being approximately 300 times higher than those in CSF. Since the concentration of serum carnosinase is only 10 times higher in serum than in CSF, the concentration of this enzyme in CSF is 30 times higher than expected, as compared with the levels of other serum proteins. Evidently serum camosinase is selectively secreted by brain cells into CSF. It seems unlikely that the enzyme is selectively transported across the blood-brain barrier into brain cells. Therefore it can be speculated that serum camosinase may be synthesized in brain cells. Several reports have shown that the level of camosinase in the blood stream increases with age. In the first 10 months of life, no activity is detectable; thereafter the concentration rises gradually, reaching the adult level at age 12-15 years [2]. Roesel et al. [19] have shown that the urinary excretion of camosine (expressed as pmol/g creatinine) decreases with age during the first four years of life; older children and adults excrete less than 4% of the amounts excreted by infants. These workers attributed the drop in carnosinuria to the rising concentration of serum carnosinase. Homocarnosinosis is a rare familial metabolic disorder. In patients with this disease, CSF homocarnosine concentrations are elevated 20-fold above normal [20] and brain homocarnosine in one of the patients was four times normal [21]. The elevated concentrations of homocarnosine are probably attributable to the fact that these patients lack serum carnosinase [6]. On a meat-free diet, normal individuals excrete very small amounts of carnosine, whereas the homocamosinosis patients excreted more than 20 times this amount [22]. When normal individuals eat chicken meat, which contains carnosine and anserine, they hydrolyze these compounds and excrete a reaction product from anserine, 1-methylhistidine. When the homocarno-
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sinosis patients consumed chicken, they excreted relatively large amounts of the unhydrolyzed dipeptides and very little 1-methylhistidine [22]. Thus it appears that serum carnosinase has two functions: the hydrolysis of homocarnosine in the brain, and the hydrolysis of carnosine and anserine in the blood stream. Since the homocarnosinosis patients have hypercarnosinuria on a meat-free diet, carnosine must be ‘leaking’ from their skeletal muscles. These patients probably have normal concentrations of tissue carnosinase [lo], since a brain biopsy of one of the patients showed a normal level of this enzyme (Lenney et al., unpubl. results). However, tissue carnosinase probably has little or no activity against carnosine in vivo because of its high pH optimum (9.5) and its high K, value (20 mmol/l carnosine). This enzyme is much more active against other dipeptides such as gly-leu and pro-leu [l]. Homocarnosine and GABA are present only in the central nervous system. Human brain contains much more homocarnosine than hog or rat brains [23]. Kish et al. [18] showed that homocarnosine was unevenly distributed in 13 regions of human brain; little or no camosine was detected except in the olfactory bulb, where the carnosine concentration was l/5 of the homocarnosine concentration. Homocarnosine may serve as a storage depot for GABA in higher primates, where serum carnosinase is present to release the GABA. The hog and rat have no serum carnosinase, and their brains do not hydrolyze homocarnosine [24,25]. Acknowledgements We thank George R. Lenney and Richard W.M. Child for financial support, Alan Komeya for excellent technical assistance, the Blood Bank of Hawaii for human plasma, Dr. Robert V. Cooney for performing the gel scanning measurements, Dr. Charles Odom for autopsy tissues and the Yerkes Regional Primate Research Center for great ape serum samples. References Lenney JF. Human cytosolic camosinase: evidence for identity with prolinase, a non-specific dipeptidase. Biol Chem Hoppe-Seyler 1990;371:167-171. Lenney JF, George RP, Weiss AM, Kucera CM, Chan PWH, Rinzler GS. Human serum carnosinase: characterization, distinction from cellular carnosinase, and activation by cadmium. Clin Chim Acta 1982;123:221-231. Perry TL, Hansen S, Love DL. Serum carnosinase deficiency in carnosinemia. Lancet 1968;June 8:1229-1230. van Munster PJJ, Trijbels JMF, van Heeswijk PJ, Schut-Jansen B, Moerkerk C. A new sensitive method for the determination of serum camosinase activity using L-carnosine-[l-‘4C]fl-alanyl as substrate. Clin Chim Acta 1970;29:243-248. Murphey WH, Patchen L, Lindmark DG. Carnosinase: a fluorometric assay and demonstration of two electrophoretic forms in human tissue extracts. Clin Chim Acta 1972;42:309-314. Lenney JF, Peppers SC, Kucera CM, Sjaastad 0. Homocarnosinosis: lack of serum carnosinase is the defect probably responsible for elevated brain and CSF homocarnosine. Clin Chim Acta 1983:132:157-165.
205 7 Duane P, Peters T3. Serum camosinase activities in patients with aicohohc chronic skeletal muscle myopathy. Clin Sci 1988;75:185-190. 8 Butterworth J, Priestman D. Fluorimetric assay for prohnase and partial characterization in cultured skin fibroblasts. Clin Chim Acta 1982;122:51-60. 9 Roth M. Fluorescence reaction for amino acids. Anal Chem 1971;43:880-882. 10 Lenney JF, Peppers SC, Kucera-Orallo CM, George RP. Characterization of human tissue camosinase. B&hem J 1985;228:653-660. 11 Dahlberg E. Estimation of the blood contamination of tissue extracts. Anal B&hem 19S3;130:108113. I2 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685, 13 Davis BJ. Disc electrophoresis-IL Method and application to human serum proteins. Ann NY Acad Sci 1964:121:404-427. 14 Neuhoff V, Amid N, Taube D, Ehrhardt W. Improved staining of proteins in po~yac~~amide gels inctuding isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. El~trophor 1988;9:255-262. 15 Higgins RC, Dahmus ME. Rapid visualization of protein bands OR SDS-gels. Anal Biochem 1979;93:25?-260. 16 Lowry OH, Rosebrougb NJ, Farr AL, Randall RI. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 17 Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal B&hem 1985;150:76-85. 18 Kish SJ, Perry TL, Hansen S. Regional distribution of homocamosine, homocamosine-carnosine synthetase and homocamosinase in human brain. J Neurochem 1979;32:1629-1636. 19 Roesel RA, Kearse EC, Blankenship PR. Camosine excretion in infants and children. Fed Proc 1986;45:470 (abstract). 20 Sjaastad 0, Berstad J, Gjesdahf P. Gjessing L. Homocamosinosis. 2. A familial metabolic disorder associated with spastic paraplegia, progressive mental deficiency, and retinal pigmentation. Acta Nemo1 Stand 1976;53:275-290. 21 Perry TL, Kish SJ, Sjaastad 0, et al. Homocamosinosis: increased content of homocarnosine and deficiency of homocamosinase in brain. J Neurochem 1979;32:1637-1640. 22 Lunde H. Sjaastad 0, Gjessing L. Homoeamosinosis: hypercamosinuria. J Neurochem 1982;38:242245. 23 Abraham D, Pisano JJ, Udenfriend S. The distribution of homocamosine in mammals. Arch Biochem Biophys 1962;99:210-213, 24 Ziesler 0, Hole K, Haugan I, Borresen AL, Gjessing LR, Sjaastad 0. Transport and distribution of homocamosine after intracerebroventricular and intravenous injection in the rat. Neurochem Res 1984;9:637-648. 25 Lenney JF. Separation and characterization of two camosine-sphtting cytosolic dipeptidases from hog kidney (camosinase and non-specific dipeptidase). Biol Chem Hoppe-Seyler 1990,371:433-440.
