Incorporation of Amino Acids by Isolated Submitochondrial Fractions of Lactating Goat Mammary Gland in Vitro1

Incorporation of Amino Acids by Isolated Submitochondrial Fractions of Lactating Goat Mammary Gland in Vitro 1 H. F. H A G G A G = and N. C. G A N G U L I National Dairy Research Institute Karnal-132001, India

products of submitochondrial protein synthesis in liver system of rats also were investigated (9). In our detailed studies on incorporation of amino acids by goat mammary gland during lactation (12), the ability of mitochondria and the submitochondrial fractions isolated from the tissue in incorporating amino acids also was examined. This report indicates the prominence of the inner membrane of mammary mitochondria in incorporation of amino acids during lactation.

ABSTRACT

Incorporation of amino acids by mitochondria and its subfractions from mammary gland of the goat during lactation was studied in vitro. Assessment of distribution of radioactivity incorporated by intact mitochondria into its subfractions revealed maximum specific activity in the inner membrane. During lactation the inner membrane of mitochondria exhibited further stimulation in such incorporation. Matrix was the next active fraction whereas outer membrane and peripheral space had negligible radioactivity. In isolated submitochondrial fractions from mammary gland incubated with radioactive amino acids under similar conditions of assay, the inner membrane was the most potent subfraction in incorporation. In mammary tissue during lactation this fraction was the most active site. The relative sequence of the subfractions appeared to remain unaltered in lactation and was in the order of inner membrane > matrix > outer membrane > peripheral space. The ratio of specific activity in inner to outer membrane appears to increase significantly during lactation.

MATERIALS AND METHODS

INTRODUCTION

Several convincing reports (2, 25) have established the ability of mitochondria to incorporate amino acids into proteins. Studies of incorporation with isolated liver mitochondria of rats have demonstrated that incorporation of amino acids is associated exclusively with the inner membrane (4, 10, 19). The

Received February 7, 1978. l NDRI Pub. No. 77-104. 2 Food Science Department, Faculty of Agriculture (Ein-Shams University) Shubra-Khaima, Cairo, Egypt. 1978 J Dairy Sci 61:1384--1391

The mammary gland from nonlactating and lactating goat was procured from the local slaughter house ice cold. The tissue immediately was fractionated to isolate mitochondria according to Mockel (18). The submitochondrial fractions were prepared by the method of Melnick et al. (17). The outer membrane and motoplast were isolated by osmotic lysis described by Parsons et al. (21). The peripheral space was collected from supernatant by sedimentation and concentrated in dialysis tubing with Polywax-6000 in cold (4 C) overnight. The matrix was separated from the inner membrane by sonicating the mitoplasts followed by differential ultracentrifugation. These fractions were isolated from both nonlactating and lactating tissues. The fractions also were tested for typical activities of marker enzymes for characterization. Monoamine oxidase was used as the marker enzyme for the fraction of outer membrane. The method adopted was that of Tabor et al. (31). The method for assessing the activity of cytochrome oxidase as the marker of the inner membrane was that of Schnaitman et al. (26). The procedure of Sottocasa et al. (27) was used for measuring the activity of glutamate dehydrogenase for the matrix. Adenylate kinase of the intermembrane space was assayed by the method of Sottocasa et al. (27), and NADPH-cytochrome C reductase was measured to assess microsomal contamination by the method of Omura et al. (20). To measure the

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SUBMITOCHONDRIAL PROTEIN SYNTHESIS activity of these enzymes, the membranes were treated first with Triton X-100 (final concentration .between .01 and .05%). All enzymes were assayed in the freshly isolated fractions according to the procedure cited for the fraction of the cell. Chlorella protein hydrolysateC 14 (.5 mc/ml) was purchased from Bhabha Atomic Research Centre, Bombay. The ATP, ADP, hexokinase type llI, glucose-6-phosphate dehydrogenase type V from bakers yeast, benzylamine, cytochrome-C type IV, Lglutamic acid monosodium salt, NAD +, NADP+, NADH, and cycloheximide were from Sigma Chemical Co., U. S. Other chemicals were of analytical grade and procured locally. The assay system for amino acid incorporation was that of WaUace et al. (32). It consisted of 4.0 mg either of mitochondria or its fraction, 50 mM KCI, 20 mM K2HPO4, 5 mM MgC12, 100 mM sucrose, 10 mM succinate, 500 pM (nonlactating) and 700 pM ATP (lactating), 1.2 mg cycloheximide, and 1.0 pc of [C 14] chlorella hydrolysate, in a total volume of 2.0 ml adjusted to pH 7.2. Our assay system contained cycloheximide, which inhibits protein synthesis in the cytoribosome cell sap system (30) but has no effect on protein synthesis by isolated mitochondria (1, 5, 15) even at high concentration in eukaroytic ceils (1). The reaction was terminated by adding 2.0 ml of 10% trichloracetic acid after incubation 1 h at 37 C. The precipitated protein was processed according to Beattie et al. (3) and finally dissolved in .4 N NaOH. The radioactivity was measured in liquid scintillation counter in an aqueous system according to Bray (6). The protein content of these samples was determined by the method of

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Lowry et al. (16) after they were dissolved in 3% NaOH. RESULTS Enzyme Characterization of Microsomal Centaminatien

The determination of NADPH-cytochrome C reductase gives a measure of microsomes in the mitochondrial fraction. The preparation of mitochondria was used for the assay, and the specific activity for NADPH-cytochrome C reductase was recorded. Results in Table 1 show that recovery of the enzyme was 96.6% for the lactating animal while it was 97.5% for the nonlactating animal, but the mitochondrial fraction had low activity, thus showing the high purity of these fractions. Enzyme Characterization of Mitochendrial Subfractions

The purity of the different submitochondrial fractions was assessed by their respective marker enzymes (Table 2). The marker enzymes for the outer membrane, matrix, peripheral space, and inner membrane isolated from the lactating and nonlactating mammary gland had high purity as apparent from Table 2. Ability of Isolated Mitechendria frem Mammary Gland to Incorporate Amino Acids

The relative ability of the subcellular fractions of the mammary gland from goat to incorporate amino acids in the trichloroacetic acid insoluble fraction was studied first. Data in Table 3 show that all the fractions, namely

TABLE 1. Activity of NADPH-Cytochrome C reductase in microsomes and mitochondria isolated from goat mammary gland. Physiological status

Total protein (mg)

Specific activity a

Total SU

Activity (%)b

Microsome

Lactating Nonlactating

32.5 28.0

24.8 13.9

803.4 382.5

96.6 97.5

Mitochondria

Lactating Nonlactating

19.0 14.5

1.4 .9

26.8 14.2

3.4 2.5

Fraction

aone spectrophotometric unit (SU) of enzyme is the amount which gives a change in OD of .001 per min. bpercent of the total units. Journal of Dairy Science Vol. 61, No. 10

P-

o

.o

TABLE 2. Specific activity a of marker enzymes in submitochondrial fractions o f lactating and nonlactating goat mammary gland.

on Z P ~

Adenylate kinase

Cytochrome C nxidase

146.9 94.5

1,339.0 1,270.0

1,204.5 367.0

3.2 2.8

110.5 86.5

32.5 25.0

2,500.0 698.5

Lactating Nonlactating

89.5 56.2

20.0 18.0

166.0 79.5

Negligible Negligible

Peripheral space

Lactating Nonlactating

30.7 22.0

45.0 32.5

6,876.0 2,730.0

Negligible Negligible

Matrix

Lkctating Nonlactating

Negligible Negligible

1,050.0 721.5

10.9 8.5

Physiological status

Monoamine oxidase

Mitochondria

Lactating Nonlactating

33.3 27.3

Inner membrane

Lactating Nonlactating

Outer membrane

Fraction

Glutamate dehydrogenase

o

643.0 147.0

aone spectrophotometric unit of each enzyme is the amount which gives a change in OD of .001 per min in case of glutamate dehydrogenase, adenylate kinase, monoamine oxidase, and cytochrome C oxidase at 340 nm, 340 rim, 250 nm, and 550 nm, respectively.

rr > ~3

Z Z

r-

SUBMITOCHONDRIAL PROTEIN SYNTHESIS

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TABLE 3. Relative abilities of isolated mammary gland subcellular fractions from goat to incorporate labeled amino acids. Specific radioactivity (cpm/mg protein) Cell fractions

Fold increase

Nonlactating

Lactating

Nuclear

4,000--20,273 12,000 (3) a

20,273-112,000 87,570 (7)

7

Mitochondria

16,000--91,000 58,200 (5)

43,000-318,000 185,400 (5)

3

Ribosomesb

7,534--56,865 27,628 (4)

3,567--98,297 31,939 (9)

2.10 4.85

5.80 2.11

Ratio values Mitocondria Ribosomes Nuclear

1.15

aFigures in parentheses refer to the number of samples analyzed. bCycloheximide inhibits protein synthesis by ribosomal system and hence has been omitted in the assay mixture for ribosomal protein synthesis.

nuclear, mitochondria, and ribosomes, are capable of incorporating amino acids. In the gland from nonlactating animals, the mitochondria was the most active fraction and the nuclear fraction was least active. Lactating m a m m a r y tissue exhibited a dramatic increase in incorporation activity in nuclear (7-fold) and in mitochondrial fractions (3-fold) compared to the nonlactating tissue. However, incorporation by ribosomes did not alter. In both nonlactating and lactating animal, mitochondria was the most active fraction in incorporating amino acids. Distribution of Incorporated Amino Acids in Submitochondrial Fractions

Whole mitochondria isolated from mammary gland of both nonlactating and lactating animals were tested for amino acid incorporation. The assay mixture was processed for assessing the distribution of the incorporated amino acids in both whole mitochondria and their subfractions. Data in Table 4 immediately suggest that the inner membrane was the most active site in such incorporation phenomena irrespective of the physiological status of the animal. The next potent fraction was the matrix.

Incorporation of Amino Acids by the Submitochondrial Fractions Isolated from the Mammary Gland

In the next experiments, the incorporation of amino acids was studies with the submitochondrial fractions, namely peripheral space, matrix, outer membrane, and inner membrane. Data in Table 5 indicate that in nonlactating animals, the matrix and the inner membrane synthesized proteins with almost similar specific activity (cpm/mg protein). The peripheral space was least active whereas the outer membrane was also fairly active. In lactation, the inner membrane was most active in synthesizing proteins with highest specific activity compared to the other submitochondrial fractions. There was a correlation between subfractions in their ability to incorporate amino acids. During lactation there was a rise in the incorporation activity in the inner membrane. The ratio values of specific activities in these subfractions in Table 6 elicit highly convincing data on the relative performance of protein synthesis. During lactation the ratio of inner to outer membrane increased. Ratio values for inner to matrix increased in lactation whereas there was no change in the relative activity between matrix and peripheral space. The ability to incorporate amino acids Journal of Dairy Science Vol. 61, No. 10

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HAGGAG A N D GANGULI

TABLE 4. Distribution of radioactivity of the incorporated amino acids in the submitochondrial fractions. Specific radioactivity (cpm/mg protein)

Experiment Fractions

no.

Nonlactating

Lactating

Mitochondria

1 II

12,984 36,539

40,748 47,840

Inner membrane

1 I1

39,509 49,145

79,025 75,886

Outer membrane

I 11

1,885 5,416

16, 716 5,591

Peripheral space

1 II

2,911 3,075

2,294 4,250

Matrix

I 11

20,965 26,150

44,440 28,409

by the inner membrane is stimulated during lactation in the mammary gland. DISCUSSION

Sottocasa et al. (28) and Parsons et al. (21) have shown that the outer mitochondrial membrane was similar to microsomes in that both contained NADH-cytochrome C reductase activity. Only the microsomes contained NADPH-cytochrome C reductase. Therefore, it appears reasonable to assume that the determination of NADPH-cytochrome C reductase gives a measure of the presence of microsomes in the mitochondrial fraction. Results in Table 1 show that the mitochondrial fractions had low activity (3.4%) in lactating tissue and also in the nonlactating mitochondrial preparation (2.5%), thus showing high purity of the

fractions. Similarly (Table 2), the purity of the different submitochondrial fractions was assessed by their respective marker enzymes. All fractions were of high purity. From the results in Tables 1 and 2, all enzymes in the fractions isolated from lactating mammary gland had higher activity than the respective enzymes of the nonlactating tissue. The labeled amino acids are incorporated by the isolated subcellular fractions of goat mammary gland and predominantly by the mitochondria (Table 3). During lactation both the nuclear and mitochondrial fractions exhibit increased ability to incorporate amino acids, such stimulation being more striking for nuclear fraction (7-fold). From the ratio values, the mitochondria from the lactating animal is the most active fraction as compared with ribo-

TABLE 5. Incorporation of amino acids by submitochondrial fractions isolated from goat mammary gland. Submitochondrial fractions used

Peripheral space Matrix Outer membrane Inner membrane

Nonlactating 1

2,057 45,130 12,056 49,664

Journal of Dairy Science Vol. 61, No. 10

Lactating II

I

(cpm/mg protein) 1,898 2,175 51,812 39,444 26,248 6,354 55,184 65,616

II

13,532 262,302 33,779 480,241

SUBMITOCHONDRIALPROTEIN SYNTHESIS

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TABLE 6. Ratios of specific activities for the subfractions of mitochondria (data from Table 4). Ratio values Inner

Physiological status

Experiment no.

Nonlactaring

I

II l II

Lactating

membrane/ outer membrane

Inner membrane/ matrix

Matrix/ peripheral

4.1

1. I 0

2.1

1.06

21.93 27.32

10.3 14.2

1.66 1.83

18.13 19.38

somes, although comparison with the nuclear fraction gave a different picture. Fraser and Gutfreund (11) demonstrated that the mitochondrial fraction had the highest radioactivity when homogenate of guinea pig mammary gland was incubated with radioactive amino acids. Net synthesis of ~-lactalbumin by both the mitochondrial and microsomal fractions prepared from lactating mammary gland of guinea pig was reported by Bew and Campbell (7). Hence, the past reports on in vitro synthesis of protein by the mitochondrial system and its augmented performance in lactating animals (14) are supported by our findings. Huang and Keenan (13) pointed out that preparation of actual mitochondria from mammary tissue involves altered techniques. However, since our system contained added cycloheximide, both mitochondria and the nuclear fractions appear to be involved in protein synthesis during lactation. In a recent paper, Singh and Ganguli (29) also reported that the mitochondrial fraction from the goat mammary gland is the most potent unit at subcellular level for amino acid incorporation. It is convincing from the data on the distribution of radioactivity in the submitochondrial fraction (Table 4) that the inner membrane is the most potent fraction having a much higher specific activity than other fractions. The matrix appears to be the next active fraction. In lactating animals, there was further increase in incorporation in the inner membrane which is two to three times more than the intact mitochondria. The data on the submitochondrial fractions (Table 5) provide convincing evidence that the inner membrane is the most active site for

space

amino acid incorporation in mammary gland tissue. This report is the first one on a tissue as specific as mammary gland to demonstrate once again the unique dialogue of the inner membrane in protein synthesis, as for liver mitochondria of rat (4, 19). Our report also delineated results on the relative ability of the submitochondrial fractions in its de n o v o synthesis of proteins from amino acids. During lactation there is a phenomenal increase in incorporation activity in the inner membrane compared to the outer membrane as is evident from the ratio values of the specific activities of synthesized proteins (Table 6). Although matrix also exhibits stimulated synthesis in lactation (Table 5), it cannot, however, surpass the inner membrane, as is apparent from ratio values (Table 6). The peripheral space did not seem to be participating actively in such synthetic operations even during lactation. These findings have provided evidence that the inner and outer membranes of isolated mitochondria in mammary gland of goat also are synthesized by different systems like the liver tissue (10, 22). Rosano and Jones (23) recently reported a significant increase in organaUe density during mitochondrial maturation phase during the transition from late pregnancy to 8-day lactation in mammary gland of mouse. During the transition into lactation, a large expansion of the inner mitochondrial membrane and perhaps the matrix material occurs in the differentiating parenchymal cells. The same authors (24) further showed that the mitochondrial proteins increase much faster than monoamino oxidase from pregnancy to lactation. Both enzymic and morphological studies support the concept of Journal of Dairy Science Vol. 61, No. 10

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an e x p a n s i o n o f m i t o c h o n d r i a l i n n e r m e m b r a n e in t h e m a m m a r y gland o f m o u s e . T h e t u r n o v e r of mitochondrial protein of inner membrane f o r rat liver is also higher (8). Our o b s e r v a t i o n s w i t h t h e m a m m a r y gland f r o m g o a t are consist e n t w i t h p r o t e i n s y n t h e s i s at m i t o c h o n d r i a l level o f m a m m a r y gland b y o t h e r species. ACKNOWLEDGMENTS

The a u t h o r s are grateful t o D. S u n d a r e s a n , Director, for his k i n d e n c o u r a g e m e n t in this w o r k . H. F. Haggag is also grateful to t h e g o v e r n m e n t s o f E g y p t a n d India f o r financial s u p p o r t u n d e r the I N D O - A R E Cultural Exchange P r o g r a m m e . REFERENCES

1 Aswell, M. A., and T. S. Work. 1968. Contrasting effects of cycloheximide on mitochondrial protein synthesis in vivo and in vitro. Biochem. Biophys. Res. Commun. 32:1006. 2 Aswell, M. A., and T. S. Work. 1970. The biogenesis of mitochondria. Annu. Rev. Biochem. 39:251. 3 Beattie, D. S., R. E. Basford, and S. B. Kortiz. 1966. Studies on the biosynthesis of mitochondrial protein components. Biochemistry 5:926. 4 Beattie, D. S., R. E. Basford, and S. B. Kortiz. 1967. The inner membrane as the site of the in vitro incorporation of L-(C 14) leucine into mitochondrial protein. Biochemistry 6: 3099. 5 Borst, P., A. M. Kroon, and G. J. C. M. Ruttenberg. 1967. Page 81 in Genetic elements, properties and functions. D. Shugar, ed. Academic and P. W. N., London and Warsaw. 6 Bray, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:279. 7 Brew, K., and P. N. Campbell. 1967. Studies of the biosynthesis of protein by lactating guinea-pig mammary gland. Characteristics of the synthesis of aqactalbumin and total protein by slices and cell-free systems. Biochem. J. 102:265. 8 Brunner, G., and W. Neupert. 1968. FEBS Letters 1:153. Taken from: Schatz, G., and T. L. Mason. 1974. The biosynthesis of mitochondrial proteins. Annu. Rev. Biochem. 43:51. 9 Burke, J. P., and D. S. Beattie. 1974. Products of rat liver mitochondriai protein synthesis: Electrophoretic analysis of the number and size of these proteins on their solubility in chloroform:methanol. Arch. Biochem. Biophys. 164:1. 10 Clark-Walker, G. D., and A. W. Linnane. 1967. The biogenesis of mitochondria in Saccharomyces cerevisiae. A comparison between cytoplasmic respiratory-deficient mutant yeast and chloramphenicolinhibited wild type cells. J. Cell Biol. 34:1. 11 Fraser, M. J., and H. Gutfreund. 1958. Proc. Roy Soc. Ser. 8149:392. Taken from: B. L. Larson and G. N. Jorgensen. 1974. Biosynthesis of milk protein. Page 115 in Lactation. Vol. 2. B. L. Journal of Dairy Science Vol. 61, No. 10

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