Journal of Neuroscience Research 4: 365-370 (1979)
Hypothalamic Cathepsin D: Assay and lsoenzyme Cornposition T. N. Akopyan, N. A. Barchudaryan, L. V. Karabashyan, A. A. Arutunyan, A. Lajtha, and A. A. Galoyan Institute of Biochemistry, Yerevan, USSR; and Center for Neurochemistry, WardS Island, New York 12
sensitive and convenient method of endopeptidase assay using as substrate
glohin modified with pyridoxal-5-phosphate was used for determination of acid proteinases in bovine hypothalamus separated by isoelectric focusing. The soluble protein fraction of hypothalamus upon elution from Sephadex gave five peaks of proteinase activity at p H 3.2. 'The properties indicate that these peaks of endopeptidase activity are isoenzyme forms of cathepsin D. Key words: cathepsin D, endopeptidasc assay, acid proteinases
I NTROD UCTlON
Cathepsin D, one of the main intr-acellular proteinases. plays an important role in the breakdown of proteins in normal cells [ l , 21 and also in thc pathologic processes of tissue breakdown [ 3,4]. Although the successful purification of this enzyme has been reported from different sources [5-9] , data concerning the isoenzyme composition of this enzyme are lacking. Previously, we reported thc purification and some properties of acid proteinase from bovine hypothalamus, including its action o n somatostatin and substance P [ l o ] . This enzyme was identified b y us a s cathepsin D (EC 3.4.2.3). During purification of calhepsin D from hypothalamus we observed multiple peaks of enzyme activity. This observation was extended t o study the isoenzyme composition of cathepsin D in hypothalamus. For resolution of enzymes having similar activities, isoelectric focusing is a suitable and powerful method. A difficulty of this method is that fractions from focusing columns contain ampholines and sucrose, which interfere with those methods of measuring proteinase activity that utilize amino group and tyrosinc-detecting reagents. In this communication, we report the development of a simple and sensitive method for endopeptidase assay and the use of this method for determination of acid proteinases of bovine hypothalamus separated by an isoelectric focusing column.
Address reprint requests to T.N. Akopyan, Institute of Biochemistry, Baruyr Sevag sir 5/1, Yerevan 375044, Armenia, LJSSR. 0360-4012/79/040S/6-036~$01.40 0 1979 Alan R . Liss, Inc.
366
Akopyan et a1
M A T E R I A L S AND METHODS
Sephadex G- 100 (fine grade) and anipholine were obtained from Pharmacia; PMSF (phenylmethyl sulfonylfluoride), TPCK (L-1 -tosylamide-2-phenylethyl chlormethyl ketone), and 0-phenylpyruvate were secured from Sigma. Pepstatin, leupeptin. and antipain were kindly supplied by Dr. H. Uinezawa (Institute of Microbial Chemistry, Tokyo). Pepsin and chymotrypsin were from Worthington; pyridoxal-5'-phosphate, from Reanal (Budapest). Preparation of Pyridoxal-Globin
Globin powder (4 gm) was dissolved in water (300 ml) containing 3 M guanidine C1, with the pH adjusted to 9.8 with 2 N NaOH. The pII was maintained at 9.8 while 0.8 gm pyridoxal-5-phospbate was added. After two hours Na BH4 was added t o the solution until the brilliant yellow color disappeared. After prolonged dialysis. the pyridoxal-globin obtained was stored at -10°C. Enzyme Assay
Endopeptidase assay was performed in 0.6 nil of reaction mixture containing 0.05 M citrate buffer, pH 3.5 (for pepsin and hypothalamus acid proteinases). or 0.1 M phosphate buffer, pH 7.6 (for chyrnotrypsin), and 100 pg pyridoxal-globin. After incubation at 37°C for one hour the reaction was stopped by addition of 0.15 ml of 30% (w/v) trichloroacetic acid. After centrifugation the pH of the supernatant was adjusted to 5.5 with 1 M sodium citrate, and the fluorescence was measured at 41 0 nm (excitation at 330 nm) on a Farrand Optical Co., Inc. fluorescence spectrophotometer. Controls were the substrate and enzymes incubated separately as above. Electrofocusing Procedure
Bovine hypothalamus was homogenized for 90 seconds in 2 volumes of cold 0.025 M phosphate buffer (pH 7.6) containing 0.3 M sucrose and 0.276 Triton X-100. The crude extract was centrifuged for one hour at 100,OOOg. The obtained supernatant was desalted by passage through a column of Sephadex G-25 (3 X 2.5 cm). Isoelectric focusing was performed in a 110-in1 column with cooling jacket (LKB) using ampholines mixtures (1%) producing pH gradients of 3-10 or 4-8 and sucrose-density gradient (40-0%). The protein sample (40-60 mg) was applied on the column as the component of light solution. The electrophoresis was carried out for 20 -24 hours at 4°C and a potential of 500 V. The experiments were stopped when the current through the column reached a constant value. Molecular Weight Determination
The method used was based on that described by Andrew [ I 11. The hypothalamicsoluble proteins (100,OOOg) were precipitated by (NI14)2SO4 (0-80%) and added to a column of Sephadex G-100 ( 3 X 90 cm) equilibrated previously with 0.025 M phosphate buffer (pH 7.6). The column was calibrated with Blue Dextran, bovine serum albumin, ovalbumin, chymotrppsinogen A, and myoglobin (proteins from Serva). RESULTS AND DISCUSSION
Pyridoxal-5'-phosphate reacts at basic pH with the €-amino group of lysine residues of proteins, resulting in the formation of Schiff bases. We used globin because this protein is a substrate for a large number of endopeptidases and contains a relatively high amount of
Hypothalamic Cathepsin D
367
1.5 W
v
$
1-0
m p:
~.0 - 5
m Q
k*-”nm 300
8
_1-I-
w
10
LL
U
5
15
25
35
02
45
04
PYRIDOXAL-GLOB1N
U
L
ENZYME
U
0.8
06
1.0
(m)
Fig. 1. a: Absorbance spectra of pyridoxal-globin in 0.1 M citmte-phosphate buffer pH (5.5). b: pII-dependence of absorbance of ppridoxal-globin at 330 nin; 0.1 M citrate-phosphate buffers (pH 3-9) were used. c: The concentration dependence of fluorescence of pyridoxal-globin. d: Dependence of the cleavage of pyridoxal-glohin on pepsin (0-3) and chymotrypsin ( 0 - * ) concentrations.
I1,7 = i a
15
I
30
40
50
60
J 70
F R A C T I O N NUMBER Fig. 2. Resolution o f acid protease activity of the hypothalamus by isoelectric focusing.
lysine residues. The modified globin obtained had maximum absorbance at 330 nm in the pH region 5 6 (Fig. la, b). Under our conditions about 10 mol pyridoxal-5’-phosphate coupled with 1 in01 globin. It was possible to detect the presence of 1 pmol of protein (Fig. Ic). Pyridoxal-globin was tested as substrate with pepsin and chymotrypsin. A linear relationship was found between the fluorescence of acid-soluble pyridoxal peptide products and enzyme concentration in the regions tested (Fig. Id). Hypothalamic acid protease was investigated by isoelectric focusing. In preliminary experiments pH 3--10 was used, but proteinase activity was detected only in the region of
368
Akopyan e t a1
* -> c
I-
U
*z N
Z 04 h
0
m N
4
50
100
150
200
250
ml
w n 20
40
60
80
FRACTION NUMBER Fig. 3. Molecular weight estimation of hypothalamus acid protease. Soluble proteins (1 00,000g) of hypothalamus were precipitated by (NH4)2S04 and t h e dissolved proteins (100 mg) were applied to a column of Sephadex G-100; the volume of each fraction was 5 ml.
pH 4.5-7. Therefore the resolution of the enzyme was carried out using carrier ampholine, pH 4-8 (Fig. 2). Five distinct peaks of endopeptidase activity were found with isoelectric points of 4.8, 5.2, 5.7, 6.2, and 6.7. Although five peaks were obtained in all the experiments, the isoelectric points varied by f 0.1 pH units. We performed several experiments to test for possible experimental artifacts. Five peaks of activity were obtained if electrofocusing was run so that enzymes were focused in different regions of the sucrose gradient. The medium in which hypothalamus was homogenized did not affect the elution pattcrn of enzymes. There was a similar picture of resolution whether distilled water, 0.025 M phosphate, or tris-HCI buffer (pH 7.6) was used. Hypothalamic acid protease activity upon gel filtration on a column with Sephadex G-100 before isoelectric focusing was eluted in one sharp peak corresponding to a molecular weight of about 50,000 (Fig. 3). After gel filtration the resolution by isoelectric focusing into five peaks was not altered. To detect any possible differences in the properties of the five enzyme peaks, we tested the effect of some inhibitors. The pattern of inhibition was similar in each of t h e five peaks analyzed. Little or no effect was observed with ethylene diaminetetracetic acid (EDTA), iodoacetate, parachlormercuribenzoate, or TPCK. Only small inhibition was found with antipain and PMSF, but more significant inhibition with leupeptin and phenylpyruvate (Table I). Pepstatin completely inhibited activity at very low concentrations (ID5,, equal to 6-30 ngiml). The high affinity of pepstatin to the hypothalamic acid protease is parallel to its inhibition of pepsin; cathepsin D from liver, uterus. and whole brain; gastricin; and renin [ 12-1 51 . Three endopeptidases are believed to initiate proteolysis at acid pH: cathepsin B l ? cathepsin D, and cathepsin E [16]. All five peaks of activity obtained by isoelectric focusing in our work were identified as endopeptidases having properties similar to those of
Hypothalamic Cathepsin D
3 69
TABLE I. Inhibition of the Five Peaks of Hypothalamus Acid Proteinwe Activity Per cent ag c inhibition
Pepstatin Leupeptin .4ntipain
PMSF TPCK p-Phenyl pyruva:e p-CMB Iodoacetic acid EDTA Dithiothreitol
1
2
3
4
5
100 38 23 25
100 26 19 15
100 38 11 25
10
7 40 8 15 0 0
37 7 12 0 0
I00 23 18 28 10 42 10 14 0 0
100 34 28 20 8 58 10 15
56 10 18 0 0
10
0 0
The microbial inhibitors of pepstatin, leupeptin, and antipain were 10fig/mi; the other inhibitor
Hypothalamic Cathepsin D: Assay and lsoenzyme Cornposition T. N. Akopyan, N. A. Barchudaryan, L. V. Karabashyan, A. A. Arutunyan, A. Lajtha, and A. A. Galoyan Institute of Biochemistry, Yerevan, USSR; and Center for Neurochemistry, WardS Island, New York 12
sensitive and convenient method of endopeptidase assay using as substrate
glohin modified with pyridoxal-5-phosphate was used for determination of acid proteinases in bovine hypothalamus separated by isoelectric focusing. The soluble protein fraction of hypothalamus upon elution from Sephadex gave five peaks of proteinase activity at p H 3.2. 'The properties indicate that these peaks of endopeptidase activity are isoenzyme forms of cathepsin D. Key words: cathepsin D, endopeptidasc assay, acid proteinases
I NTROD UCTlON
Cathepsin D, one of the main intr-acellular proteinases. plays an important role in the breakdown of proteins in normal cells [ l , 21 and also in thc pathologic processes of tissue breakdown [ 3,4]. Although the successful purification of this enzyme has been reported from different sources [5-9] , data concerning the isoenzyme composition of this enzyme are lacking. Previously, we reported thc purification and some properties of acid proteinase from bovine hypothalamus, including its action o n somatostatin and substance P [ l o ] . This enzyme was identified b y us a s cathepsin D (EC 3.4.2.3). During purification of calhepsin D from hypothalamus we observed multiple peaks of enzyme activity. This observation was extended t o study the isoenzyme composition of cathepsin D in hypothalamus. For resolution of enzymes having similar activities, isoelectric focusing is a suitable and powerful method. A difficulty of this method is that fractions from focusing columns contain ampholines and sucrose, which interfere with those methods of measuring proteinase activity that utilize amino group and tyrosinc-detecting reagents. In this communication, we report the development of a simple and sensitive method for endopeptidase assay and the use of this method for determination of acid proteinases of bovine hypothalamus separated by an isoelectric focusing column.
Address reprint requests to T.N. Akopyan, Institute of Biochemistry, Baruyr Sevag sir 5/1, Yerevan 375044, Armenia, LJSSR. 0360-4012/79/040S/6-036~$01.40 0 1979 Alan R . Liss, Inc.
366
Akopyan et a1
M A T E R I A L S AND METHODS
Sephadex G- 100 (fine grade) and anipholine were obtained from Pharmacia; PMSF (phenylmethyl sulfonylfluoride), TPCK (L-1 -tosylamide-2-phenylethyl chlormethyl ketone), and 0-phenylpyruvate were secured from Sigma. Pepstatin, leupeptin. and antipain were kindly supplied by Dr. H. Uinezawa (Institute of Microbial Chemistry, Tokyo). Pepsin and chymotrypsin were from Worthington; pyridoxal-5'-phosphate, from Reanal (Budapest). Preparation of Pyridoxal-Globin
Globin powder (4 gm) was dissolved in water (300 ml) containing 3 M guanidine C1, with the pH adjusted to 9.8 with 2 N NaOH. The pII was maintained at 9.8 while 0.8 gm pyridoxal-5-phospbate was added. After two hours Na BH4 was added t o the solution until the brilliant yellow color disappeared. After prolonged dialysis. the pyridoxal-globin obtained was stored at -10°C. Enzyme Assay
Endopeptidase assay was performed in 0.6 nil of reaction mixture containing 0.05 M citrate buffer, pH 3.5 (for pepsin and hypothalamus acid proteinases). or 0.1 M phosphate buffer, pH 7.6 (for chyrnotrypsin), and 100 pg pyridoxal-globin. After incubation at 37°C for one hour the reaction was stopped by addition of 0.15 ml of 30% (w/v) trichloroacetic acid. After centrifugation the pH of the supernatant was adjusted to 5.5 with 1 M sodium citrate, and the fluorescence was measured at 41 0 nm (excitation at 330 nm) on a Farrand Optical Co., Inc. fluorescence spectrophotometer. Controls were the substrate and enzymes incubated separately as above. Electrofocusing Procedure
Bovine hypothalamus was homogenized for 90 seconds in 2 volumes of cold 0.025 M phosphate buffer (pH 7.6) containing 0.3 M sucrose and 0.276 Triton X-100. The crude extract was centrifuged for one hour at 100,OOOg. The obtained supernatant was desalted by passage through a column of Sephadex G-25 (3 X 2.5 cm). Isoelectric focusing was performed in a 110-in1 column with cooling jacket (LKB) using ampholines mixtures (1%) producing pH gradients of 3-10 or 4-8 and sucrose-density gradient (40-0%). The protein sample (40-60 mg) was applied on the column as the component of light solution. The electrophoresis was carried out for 20 -24 hours at 4°C and a potential of 500 V. The experiments were stopped when the current through the column reached a constant value. Molecular Weight Determination
The method used was based on that described by Andrew [ I 11. The hypothalamicsoluble proteins (100,OOOg) were precipitated by (NI14)2SO4 (0-80%) and added to a column of Sephadex G-100 ( 3 X 90 cm) equilibrated previously with 0.025 M phosphate buffer (pH 7.6). The column was calibrated with Blue Dextran, bovine serum albumin, ovalbumin, chymotrppsinogen A, and myoglobin (proteins from Serva). RESULTS AND DISCUSSION
Pyridoxal-5'-phosphate reacts at basic pH with the €-amino group of lysine residues of proteins, resulting in the formation of Schiff bases. We used globin because this protein is a substrate for a large number of endopeptidases and contains a relatively high amount of
Hypothalamic Cathepsin D
367
1.5 W
v
$
1-0
m p:
~.0 - 5
m Q
k*-”nm 300
8
_1-I-
w
10
LL
U
5
15
25
35
02
45
04
PYRIDOXAL-GLOB1N
U
L
ENZYME
U
0.8
06
1.0
(m)
Fig. 1. a: Absorbance spectra of pyridoxal-globin in 0.1 M citmte-phosphate buffer pH (5.5). b: pII-dependence of absorbance of ppridoxal-globin at 330 nin; 0.1 M citrate-phosphate buffers (pH 3-9) were used. c: The concentration dependence of fluorescence of pyridoxal-globin. d: Dependence of the cleavage of pyridoxal-glohin on pepsin (0-3) and chymotrypsin ( 0 - * ) concentrations.
I1,7 = i a
15
I
30
40
50
60
J 70
F R A C T I O N NUMBER Fig. 2. Resolution o f acid protease activity of the hypothalamus by isoelectric focusing.
lysine residues. The modified globin obtained had maximum absorbance at 330 nm in the pH region 5 6 (Fig. la, b). Under our conditions about 10 mol pyridoxal-5’-phosphate coupled with 1 in01 globin. It was possible to detect the presence of 1 pmol of protein (Fig. Ic). Pyridoxal-globin was tested as substrate with pepsin and chymotrypsin. A linear relationship was found between the fluorescence of acid-soluble pyridoxal peptide products and enzyme concentration in the regions tested (Fig. Id). Hypothalamic acid protease was investigated by isoelectric focusing. In preliminary experiments pH 3--10 was used, but proteinase activity was detected only in the region of
368
Akopyan e t a1
* -> c
I-
U
*z N
Z 04 h
0
m N
4
50
100
150
200
250
ml
w n 20
40
60
80
FRACTION NUMBER Fig. 3. Molecular weight estimation of hypothalamus acid protease. Soluble proteins (1 00,000g) of hypothalamus were precipitated by (NH4)2S04 and t h e dissolved proteins (100 mg) were applied to a column of Sephadex G-100; the volume of each fraction was 5 ml.
pH 4.5-7. Therefore the resolution of the enzyme was carried out using carrier ampholine, pH 4-8 (Fig. 2). Five distinct peaks of endopeptidase activity were found with isoelectric points of 4.8, 5.2, 5.7, 6.2, and 6.7. Although five peaks were obtained in all the experiments, the isoelectric points varied by f 0.1 pH units. We performed several experiments to test for possible experimental artifacts. Five peaks of activity were obtained if electrofocusing was run so that enzymes were focused in different regions of the sucrose gradient. The medium in which hypothalamus was homogenized did not affect the elution pattcrn of enzymes. There was a similar picture of resolution whether distilled water, 0.025 M phosphate, or tris-HCI buffer (pH 7.6) was used. Hypothalamic acid protease activity upon gel filtration on a column with Sephadex G-100 before isoelectric focusing was eluted in one sharp peak corresponding to a molecular weight of about 50,000 (Fig. 3). After gel filtration the resolution by isoelectric focusing into five peaks was not altered. To detect any possible differences in the properties of the five enzyme peaks, we tested the effect of some inhibitors. The pattern of inhibition was similar in each of t h e five peaks analyzed. Little or no effect was observed with ethylene diaminetetracetic acid (EDTA), iodoacetate, parachlormercuribenzoate, or TPCK. Only small inhibition was found with antipain and PMSF, but more significant inhibition with leupeptin and phenylpyruvate (Table I). Pepstatin completely inhibited activity at very low concentrations (ID5,, equal to 6-30 ngiml). The high affinity of pepstatin to the hypothalamic acid protease is parallel to its inhibition of pepsin; cathepsin D from liver, uterus. and whole brain; gastricin; and renin [ 12-1 51 . Three endopeptidases are believed to initiate proteolysis at acid pH: cathepsin B l ? cathepsin D, and cathepsin E [16]. All five peaks of activity obtained by isoelectric focusing in our work were identified as endopeptidases having properties similar to those of
Hypothalamic Cathepsin D
3 69
TABLE I. Inhibition of the Five Peaks of Hypothalamus Acid Proteinwe Activity Per cent ag c inhibition
Pepstatin Leupeptin .4ntipain
PMSF TPCK p-Phenyl pyruva:e p-CMB Iodoacetic acid EDTA Dithiothreitol
1
2
3
4
5
100 38 23 25
100 26 19 15
100 38 11 25
10
7 40 8 15 0 0
37 7 12 0 0
I00 23 18 28 10 42 10 14 0 0
100 34 28 20 8 58 10 15
56 10 18 0 0
10
0 0
The microbial inhibitors of pepstatin, leupeptin, and antipain were 10fig/mi; the other inhibitor
