J. PeriodotUal Res. 13: 215-223, 1978
Subcellular distribution and partial characterization of gingival mitochondrial and soluble malate dehydrogenases A. S. FINE, R. W . EGNOR, I. W. Scopi> AND S. S. STAHL
Dental Research Laboratory, Veterans Administration Hospital, New York, N.Y., and Department of Pcriodontics, New York University College of Dentistry, New York, N.Y., U.S.A. Partial purification and cliaracterization by polyacrylamide gel electrophoresis of human and rodent malate dehydrogenase revealed the presence of two distinct isoenzymes, one associated with the mitochondrial fraction (M-MDH) and one with the soluble fraction (S-MDH). Electrophoretic mobilities of the isoenzymes obtained from human and rodent gingiva were similar. Malate dehydrogenase distribtition patterns of the subcellular fractions .suggest that tlic S-MDH isoenzyme may be more active than the M-MDH isoenzyme in rodent gingiva. Malate dehydrogenase dislribution patterns in mild to moderately inflamed human gingiva were similar to those seen in dilantin hypeiplastic gingiva. S-MDH and M-MDH isoenzyme distribution patterns could not be fully evaluated because of the high activity of the M-MDH isoenzyme seen in the soluble fraction. However, the subcellular distribution patterns and electE-ophorctic mobility in human gingiva showed trends similar to those seen in rodent gingiva. Malate dehydrogenase specific activities in total homogenates of human inflamed and dilantin hyperplastic gingiva were similar. (Accepted for ptiblication Septcntbcr 27, 1977)
Introduction Malate dehydrogenase (E.C.I.1.1.37) has been previously studied within the various cell layers in skin (Hershey et al. 1960, Adachi et al. 1969) and within keratinized oral mucosa (Gerson et al. 1966, 1970, Bergquist 1972). However, this data was confined to total enzymatic activities of the dissected cell layers and did not differentiate the contributions of the mitochondrial and soluble enzymes (M-MDH and S-MDH respectively) within the tissues analyzed. Al-
though malate dehydrogenase is ktiown to 1^^ associated with oxidative phosphorylation (Fine et al. 1976) in isolated rat oral mucosal mitochondria, recent evidetice also suggests that M-MDH, and S-MDH isoenzymes act as important carriers of reducing equivalents from the mitochondria to the cytosol via the "malate shuttle" (Krebs et al. 1967, Rognstad & Katy 1973, Wheatley 1974). In this paper we wish to report upon the partial characterization of M-MDH and S-MDH isoenzymes and their subcellular distribution in gingiva obtained from two
216
FINE,
E G N O R ,
S C O P P
different (rat, human) species. Because of the inherent difficulty in obtaining human non-inflamed gingival samples we chose rat gingiva which is usually noninflamed for this portion of the study. Human samples were obtained from mild to moderately inflamed gingiva and dilantin hyperplastic gingival sites. The former specimens were chosen as a model in which oxidative metabolism declines with the increasing degree of inflammation (Fine et al. 1974b). The latter specimens were selected since oxidative metabolism is significantly greater in dilantin biopsy specimens than in either mildly or highly inflamed human gingival specimens (Fine et al. 1974a). Methods and Materials
Tissue Preparations Male Sherman strain rats (average weight 150 gms) were sacrificed as previously described (Fine et al. 1973). Tissues were quickly removed from 8 groups (5 animals per group) of rats and frozen on dry ice ( —70°C). Gingival (4-5 %) homogenates were prepared in cold 0.25 M suerose-lmM EDTA (disodium ethylenedinitrilo tetraacetate) pH 7.4. Subcellular fractions (cell debris, mitochondria, microsomes and soluble) were prepared as previously described (Fine et al. 1973) in rotor 40.2 (Spinco Model L2 65B preparative ultracentrifuge) using 2.0 ml microadapters. In addition, human gingival biopsy specimens were obtained from 11 male patients 23 to 72 years of age, undergoing periodontal treatment. Regional block anaesthesia was administered to avoid infiltration of the tissue. Tissues were washed in 0.075M phosphate buffer pH 7.0 to remove adhering blood, blotted on sterile gauze and quickly frozen at — 70°C. A representative tissue sample was taken for histological analysis and quickly fixed in buffered formalin. Six specimens were classified as
A N D
S T A H L
mild to moderately inflamed and five specimens were classified as dilantin hyperplastic Human gingival subcellular fractions (cell debris-nuclei, mitochondrial, microsomal and soluble) were prepared as previously described (Fine et al. 1974). Malate dehydrogenase subcellular distribution studies and the partial characterization of M-MDH and S-MDH by gel electrophoresis in human and rodent gingiva were analyzed in separate experiments. Malate Dehydrogetutse Enzyme activity was measured spectrophotometrieally using a Model 2400 Gilford spectrophotometer. The initial rate of oxidation of NADH was followed by measuring the decrease in absorbancy in Ej^n ,,,|| at 25°C, using 6.22 X lO^M-'cm-^ as the millimolar extinction coefficient for NADH. One unit of enzyme activity is that amount of enzyme required to convert 1 [xmole of NADH to NAD* per minute. Specific activity is expressed as (imoles of NADH oxidized per minute per mg protein. The enzymatic activity was determined by a modification of the method of Mehler et al. (]948). The reaction medium contained: 210 [il Na«HPO4-KH.,PO,, buffer pH 7.4, 20 nl oxalacetate (0.76 nM), 20 |il of tissue homogenate and 50 nl NADH (0.15 in a final volume of 300 i^d. Cytochrotne oxidase Cytochrome oxidase activity was determined spectrophotometrically by following the oxidation of dithionite reduced cytochrome c at Er,r,n,,|,, at 25°C as previously described (Fine et al. 1974). Polyacrylatnide Gel Preparatioti Polyacrylamide gels were prepared by a modification of the method of Davis (1969). Samples (50 nl) of the partially purified soluble and particulate fractions were layered on top of each gel and electro-
GINGIVAL
DEHYDROGENASES
phoresis was carried out in Tris-glycine buffer pH 8.3 for two hours at 3ma/tube in a Buchler-Searle disc gel apparatus at 10°C. Some gels were stained for protein (coomassie blue), and destained in methanol, acetic acid, HjO. Enzymatically active, and protein containing bands were both recorded by scanning the gels at E550i,]ii in a Gilford Spectrophotometer Model 2400 equipped witb a Model 2410 linear transport. Malate Dehydrogenase Visttalization in Polyacrylaniide Gels Enzymatic activity of the gel samples was determined by the method of De Jong and Olson (1972) using sodium malate as the substrate. The reaction medium contained: 100 mg NAD (Sigma type 1), 60 mg NBT (nitro blue tetrazolium), 4 mg PMS (phenazine methosulfate), 20 ml (lM) sodium malate, 140 ml H.O, 10 ml (O.IM)KCN, and 30 ml of (0.5M) Tris-Cl pH 8.1 giving a total volume of 200 mis. Gels were stained for two hours at room temperature. In the present experiments, the absorption peaks obtained after enzymatic staining were roughly proportional to the amount of enzyme applied to the gel, however, they should not be considered to be quantitative. Partial Purification of Soluble and Mitochondrial Malate Dehydrogenase Soluble Fractions The pooled soluble fractions obtained from rat and human gingival subcellular fractionation studies were diluted 1:1 with saturated (NH4)2SO4 solution, and centrifuged at 102,000g-30 min (Rotor 40.2 Spinco L265B). The sediment was resuspended in 0.25M sucrose-l mM EDTA pH 7.4 to the original volume. Supernates were subsequently fractionated at 60%, 70%, and 85 % (NH4)2SO4 by the addition of solid (NH4)2SO4. Supernates were stored overnight at 5°C after each addition of (NH4)2 SO4, and centrifuged at 102,000g-60 min to
217
remove the precipitates. Sediments were resuspended to their original volumes after 60 % fractionation and concentrated 7 X after the 70 % and 85 % fractionations. The supernate remaining after the 85 % fractionation was dialyzed 3 times against I liter of distilled water which was adjusted to pH 7.4 by the addition of IM Tris-base. Analysis of the fractions revealed that 81 % of MDH activity of the soluble fraction was found in the 85 % (NH4)2SO4 sediment fraction, and 3,2 and 14 % of the activities were found respectively in the 50%, 60%, and 70 % (NH4)2SO4 sediments in rat and human gingivae. Crude Mitochondrial MDH Preparations Crude mitochondrial pellets obtained from rat and human gingiva were concentrated to 1/8 of the original starting volume, by centrifugation at 25,500g-25 min (Rotor 40.2) and the pellets were resuspended in 0.25M sucrose-lmM EDTA pH 7.4 buffer containing 5 % deoxycholate. Note: An 87 % loss in enzymatic activity occurred after solubilization by 5 % deoxycholate of the crude mitochondrial pellets. The deoxycholate solubilized fraction was centrifuged 19,500 Xg-25 min. The supernate was removed and dialyzed overnight at 5°C in O.OIM Tris-Cl pH 8.0 and the sediment was discarded. The supernate contained 96 % of the enzymatic activity of the mitochondrial fraction. Protein Deterniinatiott Protein was determined by a micromodification of the Lowry et al. (1951) method, using crystalline albumin as a standard. Statistics: Enzyme recoveries are expressed as total units per fraction, concentration (units/ml) and % ± standard error of the total units/ml in each subcellular fraction. Differences in specific activity in the total homogenates of both human inflamed and
218
F I N E ,
E GN O R ,
S C O P P
S T A H L
HUMAN MDH's
dilantin hyperplastic gingiva were considered significant if they fell within p < 0.05 in the students "t" test. Histological Preparatiotts Light Microscopy Human biopsy specimens were fixed in buffered formalin, dehydrated and embedded in paraffin. Hematoxylin and eosin stained sections were made of human inflamed and dilantin hyperplastic gingival samples to determine the relative degree of inflammation and hyperplasia associated with each biopsy sample analyzed for MDH aetivity.
A N D
0.25
— Soluble — Crude Mitochondria
020-
ijj
o
Subcellular distribution and partial characterization of gingival mitochondrial and soluble malate dehydrogenases A. S. FINE, R. W . EGNOR, I. W. Scopi> AND S. S. STAHL
Dental Research Laboratory, Veterans Administration Hospital, New York, N.Y., and Department of Pcriodontics, New York University College of Dentistry, New York, N.Y., U.S.A. Partial purification and cliaracterization by polyacrylamide gel electrophoresis of human and rodent malate dehydrogenase revealed the presence of two distinct isoenzymes, one associated with the mitochondrial fraction (M-MDH) and one with the soluble fraction (S-MDH). Electrophoretic mobilities of the isoenzymes obtained from human and rodent gingiva were similar. Malate dehydrogenase distribtition patterns of the subcellular fractions .suggest that tlic S-MDH isoenzyme may be more active than the M-MDH isoenzyme in rodent gingiva. Malate dehydrogenase dislribution patterns in mild to moderately inflamed human gingiva were similar to those seen in dilantin hypeiplastic gingiva. S-MDH and M-MDH isoenzyme distribution patterns could not be fully evaluated because of the high activity of the M-MDH isoenzyme seen in the soluble fraction. However, the subcellular distribution patterns and electE-ophorctic mobility in human gingiva showed trends similar to those seen in rodent gingiva. Malate dehydrogenase specific activities in total homogenates of human inflamed and dilantin hyperplastic gingiva were similar. (Accepted for ptiblication Septcntbcr 27, 1977)
Introduction Malate dehydrogenase (E.C.I.1.1.37) has been previously studied within the various cell layers in skin (Hershey et al. 1960, Adachi et al. 1969) and within keratinized oral mucosa (Gerson et al. 1966, 1970, Bergquist 1972). However, this data was confined to total enzymatic activities of the dissected cell layers and did not differentiate the contributions of the mitochondrial and soluble enzymes (M-MDH and S-MDH respectively) within the tissues analyzed. Al-
though malate dehydrogenase is ktiown to 1^^ associated with oxidative phosphorylation (Fine et al. 1976) in isolated rat oral mucosal mitochondria, recent evidetice also suggests that M-MDH, and S-MDH isoenzymes act as important carriers of reducing equivalents from the mitochondria to the cytosol via the "malate shuttle" (Krebs et al. 1967, Rognstad & Katy 1973, Wheatley 1974). In this paper we wish to report upon the partial characterization of M-MDH and S-MDH isoenzymes and their subcellular distribution in gingiva obtained from two
216
FINE,
E G N O R ,
S C O P P
different (rat, human) species. Because of the inherent difficulty in obtaining human non-inflamed gingival samples we chose rat gingiva which is usually noninflamed for this portion of the study. Human samples were obtained from mild to moderately inflamed gingiva and dilantin hyperplastic gingival sites. The former specimens were chosen as a model in which oxidative metabolism declines with the increasing degree of inflammation (Fine et al. 1974b). The latter specimens were selected since oxidative metabolism is significantly greater in dilantin biopsy specimens than in either mildly or highly inflamed human gingival specimens (Fine et al. 1974a). Methods and Materials
Tissue Preparations Male Sherman strain rats (average weight 150 gms) were sacrificed as previously described (Fine et al. 1973). Tissues were quickly removed from 8 groups (5 animals per group) of rats and frozen on dry ice ( —70°C). Gingival (4-5 %) homogenates were prepared in cold 0.25 M suerose-lmM EDTA (disodium ethylenedinitrilo tetraacetate) pH 7.4. Subcellular fractions (cell debris, mitochondria, microsomes and soluble) were prepared as previously described (Fine et al. 1973) in rotor 40.2 (Spinco Model L2 65B preparative ultracentrifuge) using 2.0 ml microadapters. In addition, human gingival biopsy specimens were obtained from 11 male patients 23 to 72 years of age, undergoing periodontal treatment. Regional block anaesthesia was administered to avoid infiltration of the tissue. Tissues were washed in 0.075M phosphate buffer pH 7.0 to remove adhering blood, blotted on sterile gauze and quickly frozen at — 70°C. A representative tissue sample was taken for histological analysis and quickly fixed in buffered formalin. Six specimens were classified as
A N D
S T A H L
mild to moderately inflamed and five specimens were classified as dilantin hyperplastic Human gingival subcellular fractions (cell debris-nuclei, mitochondrial, microsomal and soluble) were prepared as previously described (Fine et al. 1974). Malate dehydrogenase subcellular distribution studies and the partial characterization of M-MDH and S-MDH by gel electrophoresis in human and rodent gingiva were analyzed in separate experiments. Malate Dehydrogetutse Enzyme activity was measured spectrophotometrieally using a Model 2400 Gilford spectrophotometer. The initial rate of oxidation of NADH was followed by measuring the decrease in absorbancy in Ej^n ,,,|| at 25°C, using 6.22 X lO^M-'cm-^ as the millimolar extinction coefficient for NADH. One unit of enzyme activity is that amount of enzyme required to convert 1 [xmole of NADH to NAD* per minute. Specific activity is expressed as (imoles of NADH oxidized per minute per mg protein. The enzymatic activity was determined by a modification of the method of Mehler et al. (]948). The reaction medium contained: 210 [il Na«HPO4-KH.,PO,, buffer pH 7.4, 20 nl oxalacetate (0.76 nM), 20 |il of tissue homogenate and 50 nl NADH (0.15 in a final volume of 300 i^d. Cytochrotne oxidase Cytochrome oxidase activity was determined spectrophotometrically by following the oxidation of dithionite reduced cytochrome c at Er,r,n,,|,, at 25°C as previously described (Fine et al. 1974). Polyacrylatnide Gel Preparatioti Polyacrylamide gels were prepared by a modification of the method of Davis (1969). Samples (50 nl) of the partially purified soluble and particulate fractions were layered on top of each gel and electro-
GINGIVAL
DEHYDROGENASES
phoresis was carried out in Tris-glycine buffer pH 8.3 for two hours at 3ma/tube in a Buchler-Searle disc gel apparatus at 10°C. Some gels were stained for protein (coomassie blue), and destained in methanol, acetic acid, HjO. Enzymatically active, and protein containing bands were both recorded by scanning the gels at E550i,]ii in a Gilford Spectrophotometer Model 2400 equipped witb a Model 2410 linear transport. Malate Dehydrogenase Visttalization in Polyacrylaniide Gels Enzymatic activity of the gel samples was determined by the method of De Jong and Olson (1972) using sodium malate as the substrate. The reaction medium contained: 100 mg NAD (Sigma type 1), 60 mg NBT (nitro blue tetrazolium), 4 mg PMS (phenazine methosulfate), 20 ml (lM) sodium malate, 140 ml H.O, 10 ml (O.IM)KCN, and 30 ml of (0.5M) Tris-Cl pH 8.1 giving a total volume of 200 mis. Gels were stained for two hours at room temperature. In the present experiments, the absorption peaks obtained after enzymatic staining were roughly proportional to the amount of enzyme applied to the gel, however, they should not be considered to be quantitative. Partial Purification of Soluble and Mitochondrial Malate Dehydrogenase Soluble Fractions The pooled soluble fractions obtained from rat and human gingival subcellular fractionation studies were diluted 1:1 with saturated (NH4)2SO4 solution, and centrifuged at 102,000g-30 min (Rotor 40.2 Spinco L265B). The sediment was resuspended in 0.25M sucrose-l mM EDTA pH 7.4 to the original volume. Supernates were subsequently fractionated at 60%, 70%, and 85 % (NH4)2SO4 by the addition of solid (NH4)2SO4. Supernates were stored overnight at 5°C after each addition of (NH4)2 SO4, and centrifuged at 102,000g-60 min to
217
remove the precipitates. Sediments were resuspended to their original volumes after 60 % fractionation and concentrated 7 X after the 70 % and 85 % fractionations. The supernate remaining after the 85 % fractionation was dialyzed 3 times against I liter of distilled water which was adjusted to pH 7.4 by the addition of IM Tris-base. Analysis of the fractions revealed that 81 % of MDH activity of the soluble fraction was found in the 85 % (NH4)2SO4 sediment fraction, and 3,2 and 14 % of the activities were found respectively in the 50%, 60%, and 70 % (NH4)2SO4 sediments in rat and human gingivae. Crude Mitochondrial MDH Preparations Crude mitochondrial pellets obtained from rat and human gingiva were concentrated to 1/8 of the original starting volume, by centrifugation at 25,500g-25 min (Rotor 40.2) and the pellets were resuspended in 0.25M sucrose-lmM EDTA pH 7.4 buffer containing 5 % deoxycholate. Note: An 87 % loss in enzymatic activity occurred after solubilization by 5 % deoxycholate of the crude mitochondrial pellets. The deoxycholate solubilized fraction was centrifuged 19,500 Xg-25 min. The supernate was removed and dialyzed overnight at 5°C in O.OIM Tris-Cl pH 8.0 and the sediment was discarded. The supernate contained 96 % of the enzymatic activity of the mitochondrial fraction. Protein Deterniinatiott Protein was determined by a micromodification of the Lowry et al. (1951) method, using crystalline albumin as a standard. Statistics: Enzyme recoveries are expressed as total units per fraction, concentration (units/ml) and % ± standard error of the total units/ml in each subcellular fraction. Differences in specific activity in the total homogenates of both human inflamed and
218
F I N E ,
E GN O R ,
S C O P P
S T A H L
HUMAN MDH's
dilantin hyperplastic gingiva were considered significant if they fell within p < 0.05 in the students "t" test. Histological Preparatiotts Light Microscopy Human biopsy specimens were fixed in buffered formalin, dehydrated and embedded in paraffin. Hematoxylin and eosin stained sections were made of human inflamed and dilantin hyperplastic gingival samples to determine the relative degree of inflammation and hyperplasia associated with each biopsy sample analyzed for MDH aetivity.
A N D
0.25
— Soluble — Crude Mitochondria
020-
ijj
o
