SUBMITOCHONDRIAL DISTRIBUTION OF MAGNESIUM AND CALCIUM: CHANGES DURING MAGNESIUM DEFICIENCY Department

FRANK W. HEATON and GHAZI A. GEORGE of Biological Sciences. University of Lancaster. Lancaster

LAI 4YQ. U.K.

Abstract-l. Magnesium was present mainly in the intermembranal compartment normal mitochondria, and most of the calcium was in the inner membrane. 2. During magnesium depletion, magnesium was lost primarily from ihe inner ditional calcium was deposited in the inner membrane and the matrix.

lNTRODUCTION Evidence is accumulating to indicate that a fundamental metabolic disturbance occurs within the mitochondria of magnesium-deficient animals. Histological studies have revealed early mitochondrial lesions in the kidney (Hess et al., 1959) and heart (Heggtieit et al., 1964: S,usin & Herdson, 1967) of magnesium-

deficient rats, and cellular fractionation shows that the magnesium loss and calcium deposition in liver occur predominantly in the mitochondrial fraction of the cells (George & Heaton, 1975). Mjtochondria from magnesium~eficient rats are swollen and have a lowered Pi0 ratio, whereas microsomai preparations function normally when provided with a source of energy, and these observations led to the hypothesis that the inhibited protein synthesis and changes in cellular potassium and sodium concentrations that occur during magnesium deficiency are all secondary to a primary disturbance in energy metabolism (George & Heaton, 1978). Magnesium activates so many enzymes in t&o (Mahler, 1961) that it is difficult to predict which are most likely to be impeded during magnesium deficiency in vice. An investigation was therefore undertaken with liver m~to~hondria to determine whether any of the four main regions, the outer membrane, inner membrane, intermembranal space or the matrix are selectively affected by the deficiency. MATERIALS AND METHODS

Twelve male Wistar albino rats weighing about 100 g were randomly divided into Mg-deficient and control groups. and were pair-fed with the appropriate diets for 18 days using automatic feeding apparatus (Loveless ef nl.. 1972). Distilled water was provided ad libitum. The synthetic diets were prepared by mixing (g/kg): casein, 200; sucrose, 660; arachis oil, 80; cod-liver oil, 20: salt mixture, 40 and purified vitamins as described previously (Heaton & Anderson, 1965). They were identical in composition, apart from the addition of MgClz to the control diet, and the Mg content determined by analysis was 1.0 mg/kg (deficient) and 790 mg/kg (control).

Rats were killed by cervical dislocation guinated

from the heart: heparinized

plasma

and exsanwas separated

and

the matrix

membrane

and

of ad-

for mineral analysis. The liver was perfused through the aorta with 40 ml of ice-cold 0.25 M sucrose before removal from the body.

The liver of each rat was immed~ateiy homogenized with ice-cold 0.25 M sucrose in a Potter-Elvehjem homogenizer fitted with a Teflon pestle to give a 25”,, (w:v) homogenate. The homogenates were centrifuged at 190g for 10min to remove nuclei, then at 9OOOg for 1Omin to sediment mitochondria: the mitochondrial pellets were washed once with sucrose equal lo one-half the volume of the discarded supernatant. ~itochondria from each rat were disrupted in two ways. by osmotic Iysis alone and by osmotic lysis plus sonic&on. Mitochondria containing about 70mg protein were suspended in 1Oml of 1 mM Tris-HCI buffer. pH 7.6, and allowed to stand at 0 C for 15 min. Five ml of each suspension was sonicated at 20 kcyclesjsec for 30 set at 0 C using an MSE ultrasonic disintegrator and the two samples from each rat were centrifuged at 150,OOOy for I hr to sediment all particulate matter. Each pellet was resuspended in 5 ml of 0.25 M sucrose and the suspensions centrifuged. first at 10,000 y for IOmin to give the heavy fractions. then at 150.000y for 1hr to give the light fractions; the supernatants were combined with the corresponding Tris supernatants from the original centrifugation. The fractionation procedure is summarized djagramalically in Fig. I. The composition of the various fractions was determined by measuring the activity of marker enzymes. Rotenoneinsensitive NADH-cytochrome c reductase and succinatecytochrome c reductase activities were used as markers for the outer and inner membranes respectively (Sottocasa et ul.. 1967a). adenylate kinase for the intermembranal space and malate dehydrogenase for the matrix (Sottocasa et at., i967b).

Protein was measured in the various mitochondrial fractions by the Lowry method (Lowry et ul., 1951) using bovine serum albumin as a standard. Other portions of the same fractions were dry-ashed by heati,. in a muMe furnace at 500 C for 24 hr and the ash was dissolved in 2 M-HCI. Mg and Ca were determined in the ash solutions, and in plasma deproteinized with 10”~ (w/v) trichloroacetic acid, with a Unicam SP 90 atomic absorption spectrophotometer; test and standard solutions contained 0.1 M-HCI, and for determination of Ca 2500mg/l of Sr was added (as SrCI,), to prevent interference by other constituents of the samples. The statistical significance of differences was assessed by Student’s t-test.

FRANK W. HEATON and GHAZI A. GEORGE

276

MI-A

(inO.OJlMTris/HCl)

SUPEINATANI LIQiIFIwrIav

SOLIJBLE’FRXTION s2

Fig. I. Scheme

RESULTS

AND

for fractionation

DISCUSSION

The distribution of marker enzymes between the various fractions indicated that the two treatments disrupted mitochondria in different ways. With OSmotic lysis alone (Fig. l), fraction H, contained nearly all the inner membrane and matrix. L, contained most of the fragmented outer membrane and St contained most of the intermembranal material. When osmotic lysis was combined with sonication, L, contained fragmented outer and inner membranes, S, contained intermembranal and matrix materials, and H, contained very little of anything. By estimating magnesium, calcium and protein in all fractions and combining the data from the two disruption procedures, it was therefore possible to determine their distribution between the four main regions of the mitochondria: L, gave the outer membrane, LB-L, the inner membrane, S, the intermembranal compartment and S,-S, gave the matrix material. The recoveries of magnesium. calcium and protein after the two fractionation procedures varied from 81 to 92% of the amounts in the original mitochondria. The distribution of the three substances between the different regions of the mitochondria varied. Most of the protein was found in the inner membrane and matrix, whereas most of the magnesium was present in the intermembranal compartment and the matrix, and the majority of the calcium was in the inner Table 1. Distribution

of magnesium,

calcium

of mitochondria

membrane (Table I). These observations are in general agreement with the results of previous investigations on protein (Sottocasa et al., 1967a) and magnesium distribution (Bogucka & Wojtczak, 1971) which used different procedures for mitochondrial fractionation. The actual concentrations of magnesium and calcium, relative to protein, were highest in the intermembranal compartment and the inner membrane respectively (Table 2). A severe state of magnesium deficiency was produced in the experimental animals, as indicated by the mean plasma magnesium concentrations of 5.8 If: 0.7 and 21.0 f 0.8 mg/l in the deficient and control rats respectively, and the decrease in magnesium concentration and tendency towards an increase in calcium concentration in the whole mitochondria (Table 2) were similar to those observed previously .(George & Heaton, 1975). The only statistically significant decrease in magnesium concentration found within the mitochondria was in the inner membrane, but the proportion of total mitochondrial magnesium fell in both the inner membrane and the intermembranal compartment (Table 1). Conversely the additional calcium was deposited approximately equally between the inner membrane and the matrix. The rise in both magnesium and calcium concentrations within the outer membrane of the deficient rats appeared to be mainly due to the reduced protein content of this subfraction.

and protein in subfractions and control rats Percentage

Constituent

Group

Mg

Control Mg-deficient Control Mg-deficient Control Mg-deficient

Ca Protein

of rats

Outer 4.29 4.60 3.10 2.70 4.44 2.89

membrane + + f + * +

0.32 0.36 0.31 0.20 0.30 0.26’

Inner

of liver mitochondria

of amount

membrane

14.70 11.90 63.90 70.30 36..77 33.12

f & + k f +

from magnesium-deficient

in whole mitochondria Intermembranal compartment

0.41 0.33*** 1.50 2.30* 0.80 0.91

Mean values f S.E.M., n = 6 for each group. Values significantly different from control: *P < 0.05; **P < 0.01; ***P < 0.001

36.47 31.00 2.50 1.60 11.18 9.43

+ f + + + f

1.10 0.97** 0.45 0.63 0.97 0.76

Matrix 28.53 37.80 11.50 17.60 39.27 42.86

* k + 4 f f

2.10 2.90*** 1.40 l.SO** 1.80 2.00

Mg and Ca within Table 2. Concentrations

of magnesium

277

mitochondria

and calcium

dria from magnesium-deficient

Concentration Group

Fraction Whole

mitochondria

Outer

membrane

Inner

membrane

Intermembranal compartment Matrix

Mean control:

of rats

460 443 450 690 183 160 1440 1410 354 399

values + S.E.M.. n = 6 for each group. *P < 0.05: **P < 0.01 : ***P < 0.001.

SUMMARY

1. Rat liver mitochondria were fractionated by a combination of osmotic lysis, osmotic lysis plus sonication, and differential centrifugation to give the four main mitochondrial regions. 2. Most of the magnesium was found in the intermembranal compartment and the matrix, and the majority of the calcium was in the inner membrane. 3. Dietary magnesium depletion caused magnesium loss from the inner membrane, and possibly from the intermembranal compartment, and additional calcium was deposited in the inner membrane and the matrix. 4. These observations indicate that metabolic processes associated with the inner mitochondrial membrane are most likely to be inhibited during magnesium deficiency.

(pg/pg

Mg

Control ME-deficient Control Mg-deficient Control Mg-deficient Control Mg-deficient Control Mg-deficient

Changes in the concentration, rather than the distribution, of a substance are likely to be indicative of a selective effect on its metabolism, and it is striking that the fall in magnesium concentration within the inner membrane was accompanied by a corresponding rise in calcium concentration. This indicates that magnesium depletion occurs preferentially in the inner membrane and suggests that metabolic processes which occur in this region of the mitochondrion, such as electron transport and oxidative phosphorylation, will be most susceptible to disruption during magnesium deficiency. Several authors have reported a partial uncoupling of oxidative phosphorylation in mitochondrial preparations from magnesium-deficient animals (Vitale et a/., 1957; DiGiorgio et al., 1962; George & Heaton, 1978), but the normal oxygen uptake of similar preparations (George & Heaton, 1978) indicates that the electron transport chain functions normally. Further studies are necessary to show whether magnesium is required to maintain the permeability of the inner mitochondrial membrane, as suggested from work with the ionophore A 23187 (Binet & Volfin, 1975; Wehrle et al., 1976; Duszynski & Wojtczak, 1977), or whether it is involved enzymically in the process of oxidative phosphorylation.

of liver mitochon-

in subfractions

and control rats

143 159 112 173 288 325 36 32 5 35

+_ 4 * 4* * 10 i 20*** f 6 + 7* + 90 * 70 * 15 &- 20

Values

protein) Ca

significantly

i * & + i i & f f *

3 10 9 10** 10 9* 2 3 0.3 4***

different

from

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278

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mitoBio-