Studies on the flavins in rat liver mitochondrial outer membranes

437

Biochimica et Biophysica Acta, 497 (1977) 4 3 7 - - 4 4 6 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 28217

STUDIES ON THE FLAVINS IN RAT L I V E R MITOCHONDRIAL O U T E R MEMBRANES

P A T R I C I A M A R T I N E Z and R O Y M c C A U L E Y *

Department of Pharmacology, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, N.Y. 11203 (U.S.A.) (Received O c t o b e r 4 t h , 1976)

Summary The incorporation of radioactivity derived from [2-14C] riboflavin into the flavins o f rat liver mitochondrial outer membranes was studied. These membranes were found to contain a b o u t 0.6 nmol of non-covalently b o u n d flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain a b o u t 1.5 nmol per mg of covalently b o u n d flavins. After labeling, radioactive flavins appeared in the non-covalently b o u n d flavins for a b o u t 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labeling was affected b y cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently b o u n d flavins, radioactivity was incorporated into the coenzymes for at least 18 h, b u t the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently b o u n d flavins for a b o u t 2 h, b u t then stopped. Labeling of both types of flavins after [14C]riboflavin was considerably slower than the incorporation of [3H]leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenzymes is complete.

Introduction

Flavin coenzymes can interact with their apoenzymes either covalently or through non-covalent bonds. In mammalian mitochondria, succinate dehydrogenase [ 1 ] , monoamine oxidases [2,3] and sarcosine dehydrogenase [4] all contain covalently b o u n d flavins which can be extracted as flavo* Present address: Department of Pharmacology, Wayne State University School of Medicine, Detroit, Mich. 48201, U.S.A.

438 peptides after proteolysis. The majority of the mitochondrial flavins are apparently linked in their respective proteins by non-covalent forces [5] and can usually be extracted from their apoenzyme after denaturation in strong acids. While the in vivo relationships between the flavins and apoproteins have been well studied in several instances, (for example, succinate dehydrogenase [6] and cytochrome bs reductase [7]), almost nothing is known about the processes involved in the assembly of flavoproteins in vivo. Theoretically, the cofactors in mitochondrial flavoproteins might become associated with the apoenzyme during its synthesis while the protein moiety is still attached to polysomes. Alternatively, the cofactors may be incorporated at some time after synthesis of the apoenzyme, either in transit to or after incorporation into the mitochondrion. In the experiments reported here, the incorporation of radioactive flavins into the mitochondrial outer membranes was studied. The outer membranes were selected for study because they can be readily purified and because they contain examples of enzymes with both covalently bound flavins, monoamine oxidases [8], and non-covalently bound flavins, kynurenine-3-monooxygenase [9] and others. The flavin c o n t e n t of the outer membranes was also estimated. Preliminary accounts of this work have been presented at the F A S E B meetings in Anaheim, Calif. [11]. Methods

Treatment o f animals. Male Sprague-Dawley rats weighing about 300 g were routinely fasted 18 h before killing. All injections were made intraperitoneally in water solutions. Preparation o f mitochondrial outer membranes. Mitochondrial outer membranes were prepared by a modification of the m e t h o d described by Sottocasa et al. [10] using from 2 to 12 rat livers. The rats were stunned by a blow on the head and decapitated; each liver was then perfused via the portal vein with about 25 ml of cold saline. All subsequent operations were at 4 ° C. The pooled livers were passed through a Harvard tissue press prior to homogenization with a Teflon-glass tissue grinder in 50 ml of 0.25 M sucrose for each liver. Nuclei and cell debris were removed by centrifugation at 500 X gay for 10 min. The resulting supernatant was then centrifuged at 7500 X gay for 10 min. The 7500 X gay supernatant was usually discarded but in experiments requiring microsomes, the supernatant was centrifuged at 12 000 X gay for 10 min to remove small mitochondria and then an additional 60 min at 105 000 X ga~ to sediment the microsomes. In the preparation of outer membranes, the 7500 X gay mitochondrial pellet was resuspended by gentle homogenization in 25 ml 0.25 M sucrose per liver and the mitochondria were again collected at 7500 X ga~ for 10 min. The procedure was repeated with 12.5 ml 0.25 M sucrose per liver. At each step, the " f l u f f y layer" was decanted as completely as possible. The mitochondria were resuspended in 7.5 ml 10 mM Tris/phosphate (7.5) per liver and allowed to sit on ice for 10 min; then 2.5 ml of a solution containing 1.8 M sucrose, 4 mM ATP and 4 mM MgSO4 was added per liver. The suspension was mixed well and allowed to sit for 10 min on ice. 5-ml aliquots

439 were sonicated for 15 s with the microprobe of a Branson W185D sonifier at the No. 3 setting. 10 ml of the sonicated mitochondria were layered over 15 ml of 1.2 M sucrose in polycarbonate centrifuge tubes and the discontinous gradient was centrifuged for 60 min at 78 000 X gay in a Beckman Instruments Inc., No. 30 rotor. The hazy yellow layer at the interface between the sucrose solutions was carefully collected with a Pasteur pipet, mixed with two volumes of cold distilled water, and centrifuged again at 78 000 X gay in a No. 30 rotor. The resulting yellowish pellets were resuspended in 0.5--1.0 ml 0.25 M sucrose per liver. These membranes usually contained about 30--40% of the monoamine oxidase and 3--5% of the protein originally in the sonicated mitochondria. Estimation o f radioactive flavins. Animals were administered 20 pCi per kg of D-[2-'4C] riboflavin (Amersham/Searle Corp.) with a specific radioactivity of 26--29 Ci/pmol. At various times afterwards mitochondrial outer membranes were prepared from the pooled livers of three similarly treated animals and the outer membranes were frozen (--20°C) for analysis the next day. 2.1 ml cold methanol were mixed with 0.9 ml of a suspension of radioactive outer membrane (usually containing 3--4 mg protein per ml). The mixture was incubated on ice for about 10 min before centrifugation at 27 000 × g~v for 10 min. The clear supernatant was saved and the pellet was washed three times, once with 3 ml of 70% cold methanol and twice with 3 ml of 5% HC104. After each wash the pellet was precipitated at 27 000 × g ~ for 10 min and the washes were combined with the original methanol supernatant. The methanolperchloric acid extracts were neutralized with KOH, and aliquots were mixed with Aquasol (New England Nuclear Inc.) prior to estimation of radioactivity. These aiiquots are considered to represent the non-covalently bound or " e x t r a c t a b l e " flavins of the outer membranes. It should be mentioned that 70% methanol followed by 5% HC104 extracted more radioactivity than either methanol or HC104 alone. The residue after the methanol and HC104 extractions contains the covalently bound or "non-extractable" flavins and was dissolved in a small volume of 0.5 M NaOH at 35°C prior to being transferred to a scintillation vial. Aquasol and sufficient HC1 to neutralize the mixture were added, and radioactivity was estimated by liquid scintillation spectroscopy at an efficiency of between 80 and 85%. Data for both extractable and non-extractable flavins are expressed as counts per min per mg outer membrane protein. In some experiments the distribution of radioactivity in riboflavin, FMN and FAD was measured. In these instances about 5 mg of radioactive outer membranes were extracted twice with 4 ml of 5% HC104. The extracts were carefully neutralized with KOH and the KC104 pellet was removed. The supernatant was lyophilized and frozen for chromatography on DEAE-Sephadex as described by Fazekas and Sandor [12]. Fractions containing riboflavin, FMN and FAD were collected and radioactivity was estimated in Aquasol. Estimation o f radioactive protein. Animals were given 3.3 mCi per kg L-[4,5-3H2] leucine (New England Nuclear Inc.) with a specific radioactivity of 50 Ci/mol. At times of up to 4 h afterwards the animals were killed and mitochondrial outer membranes prepared from the livers of three similarly treated rats. The membranes were frozen for analysis the following day. 1 ml of outer membranes containing from 3 to 4 mg protein per ml was

440 mixed with 2 ml 7.5% trichloroacetic acid, and the mixture was centrifuged (approx. 2000 X gay for 10 min). The pellet was washed twice more with 3 ml 5% trichloroacetic acid, heated for 20 min at 90°C in 3 ml 5% trichloroacetic acid, chilled and centrifuged. The resulting pellet was washed once with ethanol/ ether (1 : 1, v/v) and finally dissolved in 0.5 M NaOH at 35°C. The mixture was transferred to a scintillation vial containing Aquasol and sufficient HC1 to bring the mix to neutrality. The radioactivity in the samples was estimated by scintillation counting at 30--35% efficiency for 3H. Data are expressed as counts per min per mg protein. Estimation o f flavin content. The relative amounts of non-extractable and extractable flavins were estimated by a modification of the m e t h o d of Appaji Rao et al. [ 1 3 ] . 5 mg of bovine pancreatic trypsin (Sigma Chemical Co., T y p e III) was dissolved in one o f two 1-ml samples each containing 3--5 mg outer m e m b r a n e protein. Both samples were incubated for 90 min at 35°C, and then 1 ml of 20% HC104 was added, and the m i xture were chilled on ice. The residue, after centrifugation at 5000 X gav for 10 min, was washed twice with 2 ml 5% HClO4, and the pooled supernatants were neutralized with KOH. After the KC104 was removed by centrifugation, the extracts were lyophilized, and dissolved in 1 ml 0.1 M Tris • HC1 (pH 7.5). The absorbance at 450 nm in the absence and presence of 1 mg per ml Na2S204 was measured, and E m , o x i d . . r e d . = 9.8 • 103 M -1 • cm -1 was e m p l o y e d to calculate the flavin cont ent . The extracts o f membranes which had n o t been incubated with trypsin were used as estimates o f extractable flavins while the difference in flavin c o n t e n t between the two extracts represented non-extractable flavins released as flavopeptides by trypsin. In an ef f o r t to determine the total a m o u n t of non-extractable flavin, including th at which might n o t be released by t rypt i c digestion, samples containing 3--5 mg out er m em br ane protein were precipitated in 2 ml of cold 5% trichloroacetic acid. The residue was collected by centrifugation, washed twice with 2 ml 5% trichloroacetic acid, and the precipitate was dissolved in 1 ml 1% sodium d o d e c y l sulfate/4% NaHCO3. The flavin c o n t e n t was estimated by absorbance at 450 nm as described above. The amo u n ts of FAD, FMN, and riboflavin in the extractable flavins was estimated in 5% trichloroacetic acid extracts o f out er membranes by the procedure o f Burch [ 1 4 ] . Flavin c o n t e n t in all instances is expressed as nmol per mg o u ter m e m b r a n e protein. Other analytical methods. Monoamine oxidase was estimated polarographically by the m e t h o d o f T i p t o n and Spires [ 1 5 ] . Glucose-6-phosphatase [16] and succinate c y t o c h r o m e c reductase [10] were measured spectrophotometrically. Hexobarbital hydr oxyl as e was measured radiometrically [ 17 ]. RNA was estimated by absorbance at 260 nm in alkaline digests of HCIO¢ precipitates [ 1 8 ] , and protein was estimated by the m e t h o d of L o w r y et al. [19] using bovine serum albumin as a standard. Results

Characterization o f the mitochondrial outer membranes The m e t h o d used to prepare the out er membranes is sufficiently different

441

from those previously reported t h a t an evaluation of the purity of the isolated membranes seemed appropriate. The activities of several enzymes characteristic of microsomal membranes and of the inner and outer mitochondrial membranes were measured. The data, in Table I, indicate a substantial purification of the outer membranes. Monoamine oxidase, a marker for the outer membranes, was purified 8--9 times while the specific activity of the inner membrane marker, succinate cytochrome c reductase, was considerably diminished. Based on estimates of glucose-6-phosphatase, the outer membrane preparation would appear to be extensively contaminated with microsomes. On the other hand, judged by RNA c o n t e n t and hexobarbital hydroxylase activity the microsomal contamination is considerably less. The apparent paradox can be explained if the glucose 6-phosphate was hydrolyzed by non-specific phosphatases. Alternatively, glucose-6-phosphatase may genuinely be a constituent of the outer membrane. This would be consistant with reports of small amounts of glucose-6-phosphatase associated with rat liver mitochondria [10].

Estimates o f flavin content Estimates of the concentrations of both "extractable" and "non-extractable" flavins in the outer membranes are in Table II. Extractable flavins are defined here as those which are released by treatment with perchloric or trichloroacetic acid under conditions described in Methods while non-extractable flavins are either n o t released at all or require digestion by trypsin in order to be released by the acid extraction. As judged by two separate analytical techniques, the outer membranes contain about 0.6 nmol per mg protein of extractable flavin. About 70% of these flavins are in the form of FAD while FMN comprises about 25% of the total. After proteolysis with trypsin, an additional 0.3 nmol flavin per mg protein

TABLE

I

MARKER ENZYMES MICROSOMES

IN

MITOCHONDRIAL

OUTER

MEMBRANES,

MITOCHONDRIA

AND

E n z y m a t i c a c t i v i t i e s w e r e m e a s u r e d in m i t o c h o n d r i a l outer membranes, mitoehondria and microsomes p r e p a r e d f r o m t h e p o o l e d livers o f six rats. E a c h v a l u e is t h e average o f t w o d e t e r m i n a t i o n s . Enzymatic

activities

Mitochondrial outer membranes

Mitochondria

83 54

10 6

3 1

Succinate cytochrome c reductase (nmol cytochrome c reduced per min per mg)

6

45

1

Glucose-6-phosphatase ( n m o l Pi p e r 2 0 r a i n p e r m g )

Monoamine oxidase (natoms min per mg) Benzylamine Serotonin

Microsomes

0 per

2.6

--

5.9

Hex obarbital hydrox ylase (nmol oxyhexobarbital p e r 2 0 rain per rag)

1.5

--

18.3

RNA (rag per rag)

0.03

0.02

0.25

442 TABLE n FLAVIN CONTENT

OF MITOCHONDRIAL

OUTER MEMBRANES

Estimates were made in mitochondrial outer membranes prepared from the pooled livers of three rats. D a t a are e x p r e s s e d as n m o l p e r m g p r o t e i n . E a c h v a l u e is t h e a v e r a g e (+- S . E . ) o f f i v e d e t e r m i n a t i o n s . Outer membrane

flavins

n m o l p e r m g (± S . E . )

(1) E x t r a c t a b l e f l a v i n s Total FAD and FMN and riboflavin FAD FMN Riboflavin

0.62 0.59 0.43 0.14 0.03

(2) N o n - e x t r a c t a b l e f l a v i n s Released by trypsin Total

0 . 2 9 -+ 0 . 0 5 a 1.52 ± 0.17 ac

+ 0.10 -+ 0 . 0 4 ÷ 0.04 + 0.01 +- 0 . 0 1

a b b b b

a D e t e r m i n e d b y s p e c t r o p h o t o m e t r i c assay d e s c r i b e d in M e t h o d s . b Determined by fluorimetric assay [14]. c Estimated in triehloroacetic acid-extracted, sodium dodecyl

sulfate/NaHCO3-solubilized

mem-

branes.

could be ex tr act ed from the out er membranes. Finally, if out er membranes were extracted with trichloroacetic acid and then the residue was dissolved in sodium d o d e c y l sulfate/NaHCO3 and assayed, the membranes apparently contained 1.5 nmol flavin per mg protein. From these data it seemed t hat a b o u t 70% o f the o u te r m e m b r a n e flavins were non-extractable, evidently covalently linked to their a p o e n z y m e , and t ha t only a b o u t 20% o f these non-extractable flavins could be readily released by trypsin. In an e f f o r t to test the ability of trypsin to release non-extractable flavins fr o m o u ter membranes, six pairs of rats were administered 20 pCi of radioactive riboflavin per kg and were killed 4 h later. O ut er membranes were prepared f r o m each pair and the non-extractable radioactivity was estimated as described in Methods. The average specific activity for the six o u t e r m e m b r a n e preparations was 62 + 12 counts per min per mg protein. The o u t e r membranes were incubated with trypsin as described in Methods for bot h 90 min and 20 h, and the non-extractable radioactivity was again determined. After the shorter incubation the non-extractable specific radioactivity was n o t statistically different from unincubated membranes (39 + 10 cpm per mg), but after 20 h only 23% o f ~he non-extractable radioactivity remained (14 -+ 4 cpm per mg). apparently the bulk of the non-extractable flavin is b o u n d so t hat it is only p o o r l y released by tryptic digestion.

Incorporation o f radioactive riboflavin into extractable flavins At various intervals after the administration of radioactive riboflavin, hepatic o u ter membranes were prepared and ext r a ct ed with m et hanol and HC104 as described in Methods. In Fig. I the specific radioactivity o f the extracts, expressed as counts per min per mg of protein, is shown. The extractable radioactivity presumably represents flavins which com bi ne with their apoproteins in a non-covalent or dissociable manner. These extractable flavins increased in specific activity most rapidly over the first 2 h of labeling, and account ed for

443 A

1500

1500

E

E

E

i

~ 1000

I000

o. ¢.)

v

._~ > o

500

50C O. -r

LI_ 0 I

I

I

2

i

4 Hours

I

I

6

I

i

I

2 Hours

4

Fig. 1. I n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o e x t r a c t a b l e flavin c o f a c t o r s . R a t s w e r e given 2 0 /~Ci p e r k g [ 1 4 C ] r i b o f l a v i n i n t r a p e r i t o n e a l l y a n d killed a t v a r i o u s t i m e s t h e r e a f t e r . T h e m i t o c h o n d r i a l o u t e r m e m b r a n e s w e r e p r e p a r e d f r o m livers p o o l e d f r o m t h r e e similarly t r e a t e d a n i m a l s . T h e s e m e m b r a n e s w e r e e x t r a c t e d w i t h m e t h a n o l a n d H C 1 0 4 as i n d i c a t e d in M e t h o d s . T h e a p p e a r a n c e o f r a d i o a c t i v i t y in t h e e x t r a c t s is s h o w n (o o). In s o m e e x p e r i m e n t s c y c l o h e x i m i d e (1 m g p e r kg) w a s a d m i n i s t e r e d 3 0 rain p r i o r to [ 1 4 C ] r i b o f l a v i n (~ ~). E a c h v a l u e is t h e a v e r a g e o f t w o d e t e r m i n a t i o n s , a n d d a t a are e x p r e s s e d as c o u n t s p e r rain p e r m g p r o t e i n . Fig. 2. I n c o r p o r a t i o n o f r a d i o a c t i v e p r o t e i n s i n t o m i t o c h o n d r i a l o u t e r m e m b r a n e s . R a t s w e r e given 3 3 0 #Ci p e r kg [ 3 H I l e u c i n e i n t r a p e r i t o n e a l l y a n d killed a t v a r i o u s t i m e s t h e r e a f t e r . T h e o u t e r m e m b r a n e s w e r e p r e p a r e d f r o m t h r e e p o o l e d livers, a n d t h e r a d i o a c t i v i t y p r e s e n t in p r o t e i n w a s e s t i m a t e d as d e s c r i b e d in M e t h o d s . E a c h v a l u e is t h e a v e r a g e o f t w o d e t e r m i n a t i o n s , a n d d a t a are e x p r e s s e d as c o u n t s p e r rain p e r mg protein.

the bulk of the radioactivity in the outer membranes. By a b o u t 4 h after labeling the specific radioactivity had begun to plateau, and remained essentially constant for the next 18 h (1580 cpm per mg after 18 h; mean of three determinations). The distribution of radioactive flavins among the most c o m m o n flavins in the outer membranes was measured at intervals during the first 4 h after labeling. These data, in Table III, show that radioactivity was distributed according to the concentration of the various nucleotides; that is, at all three intervals, a b o u t 80% of the counts were in FAD while slightly less than 20% were in FMN. This distribution of radioactivity is more consistant with the labeling of flavoproteins than with a non-specific adsorbtion of flavins to the outer membranes. TABLE III D I S T R I B U T I O N OF R A D I O A C T I V I T Y A M O N G T H E E X T R A C T A B L E F L A V I N S E s t i m a t e s o f the specific r a d i o a c t i v i t y of t h e flavins i n d i c a t e d b e l o w w e r e as d e s c r i b e d in M e t h o d s . M i t o c h o n d r i a l o u t e r m e m b r a n e s w e r e p r e p a r e d f r o m t h e p o o l e d livers o f t w o r a t s given 20 /~Ci p e r kg [ 1 4 C ] r i b o f l a v i n i n t r a p e r i t o n e a l l y a t v a r i o u s t i m e s p r i o r t o killing. D a t a are e x p r e s s e d as c o u n t s p e r rain p e r m g p r o t e i n . E a c h v a l u e is t h e a v e r a g e o f t w o d e t e r m i n a t i o n s w i t h d i f f e r e n t s a m p l e s . Hours after injection

1 2 4

cpm per mg Total flavin

FAD

FMN

Riboflavin

630 725 1175

485 570 900

105 125 240

40 40 40

444 An effort was made to determine whether the appearance of flavins in the outer membranes was closely linked to the synthesis of apoproteins and their subsequent incorporation into the outer membranes. The incorporation of radioactive extractable flavin was measured in animals given a dose of cycloheximide sufficient to inhibit protein synthesis by a b o u t 90% (1 mg/kg). It can be seen in Fig. 1 that this treatment did not affect the extent to which radioactivity appeared in the outer membranes after labeling with [14C] riboflavin. In another series of experiments, the rate of appearance of proteins labeled with [3 H] leucine in the outer membranes was measured, and as shown in Fig. 2, the incorporation of radioactive proteins reached a plateau by 2 h after administration of the radioactive amino acid. It seems, as judged by these experiments, that the appearance of extractable flavins in the outer membranes is not closely coupled to the synthesis of apoproteins, and, in fact, the appearance of the coenzymes in the outer membranes is measureably slower than the appearance of new synthesized proteins.

Incorporation of radioactivity into non-extractable flavins In animals labeled with [14C] riboflavin, a relatively small percentage of the radioactivity remained in the outer membranes after methanol and HC104 extraction (Fig. 3). This radioactivity is probably in the form of covalently linked flavins since, as was previously mentioned, it could only be released by prolonged incubation with trypsin. Furthermore, the non-extracted flavins continued to be labeled over the 6 h interval shown, although the rate decreased somewhat in later periods. Consequently, the percentage of non-extractable radioactivity increased from a b o u t 3% of the total counts at 1 h to 8% at 6 h and a b o u t 15% (230 cpm per mg protein; mean of three determinations) at 18 h. Unlike the extractable flavins, prior administration of cycloheximide reduced the incorporation of radioactivity into the non-extractable cofactors. After 2 h of an apparently normal rate of labeling, the incorporation of radioactive flavin was abruptly halted. Cycloheximide evidently did n o t affect the

/

I00

~

50 c

,,

I

2

Hours Fig. 3. I n c o r P o r a t i o n o f r a d i o a c t i v i t y i n t o non-extractable f l a v i n eofaetors. Rats were given 20/~Ci per kg [14C] riboflavin intraperitoneaUy and killed at various times thereafter. The mitochondrial outer membranes were prepared from livers Pooled from three similarly treated animals. These membranes were extracted with methanol and HClO 4 as indicated in Methods. The appearance of radioactivity in the r e s i d u e is s h o w n ( o o ) . T h e e f f e c t o f a d m i n i s t r a t i o n o f c y c l o h e x i m i d e (1 m g p e r k g ) 3 0 m i n p r i o r t o [ 14C] r i b o f l a v i n is a l s o s h o w n (A ~). E a c h v a l u e is t h e a v e r a g e o f t w o d e t e r m i n a t i o n s , a n d d a t a a r e e x p r e s s e d as c o u n t s p e r m i n p e r m g p r o t e i n .

445 availability of [14C] flavin nucleotides since there was sufficient cofactor to label the extractable flavins (Fig. 1). It is more likely that cycloheximide inhibited the synthesis of appropriate apoproteins, and when apoprotein pools were exhausted, the assembly of flavoproteins stopped. Discussion The chemical and enzymatic properties of the mitochondrial outer membranes have been described in some detail [20] ; however, the flavin c o n t e n t of these membranes had not been previously measured. Estimates presented here indicate that rat liver mitochondrial outer membranes contain about 2.1 nmol of flavin per mg protein. Only about 30% of these coenzymes can be extracted by acid; these flavins (principally in the form of FAD) are apparently bound to their apoproteins by non-covalent bonds. The bulk of the outer membrane flavins are more firmly associated with the membranes, and, in fact, these nonextractable flavins cannot be released by a 90 min incubation with trypsin prior to acid extraction. However, prolonged incubation with trypsin (20 h) did release the majority of the non,extractable flavins. These data suggest that these flavins are firmly, probably covalently, bound in flavoproteins which are poorly hydrolyzed by trypsin. It is interesting that monoamine oxidases which contain covalently linked flavins have been shown to be resistant to tryptic digestion [22,23]. Since these enzymes may comprise a substantial percentage of the outer membrane protein [21], it is possible that they account for the trypsin-resistant membrane flavins. On the other hand, neither the chemical form nor the nature of the bonding of the non-extractable flavins of rat liver mitochondrial outer membranes have been established, and it is n o t even possible to decide whether the apparent resistance to trypsin is due to the nature of the flavoprotein or its orientation in the membrane. An a t t e m p t was made to study the in vivo assembly of outer membrane flavoproteins using ['4C]riboflavin as a label for the prosthetic groups. The non-extractable flavin cofactors, or covalently bound flavins, continued to be labeled for at least 18 h after the administration of [ ~4C] riboflavin. Evidently, radioactive flavins are available for incorporation into flavoproteins for sometime after labeling of the flavin pool. It is also interesting the flavins continue to be incorporated into flavoproteins with non-extractable cofactors for several hours after interruption of protein synthesis. These data suggest that in the case of flavoproteins with covalently bound cofactors, the assembly of the holoprotein does not occur immediately after synthesis of the apoprotein. Instead, the proteins probably enter a pool of sufficient size to maintain flavoprotein assembly for several hours after apoprotein synthesis has stopped. The incorporation of radioactivity into extractable flavins, or non-covalently bound flavins, is not affected by cycloheximide, and it is likely that the rate of appearance of radioactive flavins in these outer membrane proteins is determined by the rate of dissociation of the apoenzymes and their cofactors. At least in mitochondrial outer membranes, it is probable that the various flavoproteins are assembled from apoprotein and cofactor pools instead of being closely linked to de novo apoprotein synthesis.

446

Acknowledgements The authors would like to thank Dr. J. Pinto for his helpful advice in this work. This study was supported by U.S.P.H.S.N.I.H. Grant AM 17468. References 1 2 3 4 5 6

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