Amino acid incorporation into ribonucleoprotein particles prepared from rabbit mammary glands during pregnancy and lactation

I52

BIOCHIMICAET BIOPHYSICAACTA

BBA 95941

AMINO ACID INCORPORATION INTO RIBONUCLEOPROTEIN PARTICLES PREPARED FROM RABBIT MAMMARY GLANDS DURING PREGNANCY AND LACTATION G. D. BAIRD ~.',u: I. D. ItERRIMAN Agvicultt~ral Re,ca,oh Counal, l~litute ft~r Reseavd~ on Animal Diseases, Compton, Newbuvy, Berkshire (Crvca~B~'ilain)

{ReceivedFebruary 19th, x968/

SUMMARY L Systems incorporating [14C]leucineinto acid-insoluble prote~iu in ~tre were prepared from the mammary glands of late-pregnant and lactating rabbits. In these systems ribonucleoproteiu particles were incubated with a supernatant fraction. 2. The optimui concentrations of the components of the system were determined. [lIC]Leucine incorporation was highty dependent on the presence of ATP and JTP. 3- [laC]Leucine incorporationwas directly proportional to the quantity of particulate fraction present, but was less sensitive to the quantity of supernatant fraction present. 4. [MC]Lcucine incorporation was found to be higher in systems prepared from lactating rabbits than in systems prepared from late-pregnant rabbits and to increase further as lactation progressed, The difference in file level of incorporation in latepregnant and lactating systems appeared to be due to changes occurring in both the particulate and supernataut fractions. Changes in the particulate fraction included an increase in the total quantity and in the specific activity of the ribonueleoprotein particles. The nature of the change in the supernatant fraction was not determined but the increased activity of this fraction was retained iv part after dialysis or Sephadex G-~5 treatment.

IN'rRODUCTION A numb~ of workers have previously studied amino acid incorporation i n ~itro in a variety of systems prepared from the mammary gland of the lactating guinea pig (e.g. FI~.SER AND GUTFREUNDI, TURBAAND HILPERTz, BP.ZWAND CAMPBELL3). The simplest system employed by these workers was one in which mierosomes were incubated with par t icie-free supernatant fluid. From thesestudies it can be concluded that the characteristics of protein biosynthesis in mammary gland tissue are similar to those in other mammalian tissues. This communication describes the properties of an i n vitro amino acid-incorpo., ration system prepared from rabbit manunary gland in which a fraction containing Abbreviation:PEP, phosphcenoipyruvate. Biochim. Riophys. Aaa 166 (~968) t62-x74

MAMMARY AMINO ACID II~GORPOPOkTION~t$ ~l~O

163

ribonucleoprotein particles is incubated with a supeam_atant fraction. ,rn v/Oo systems were prepared from the ~aammary glands of late-prcg/mnt and lactating rabbits, and it was found that the incorporating ability of the systems increased markedly from late-pregnancy to mid-lactation. The increase in incorporating ability was found to be due to contributions from both. the partietdate fraction and the supernat[nt fraction. Some of these findings have already been reported in abstract form°.

MATERIALSAND METHODS Chemicals ATP, GTP. GSH, the potassium salt of phosphoenalpyruvate (PEP), and pyruvate kinase were obtained from Bcehringer Corp. Ltd., Ealing, London. Solutions of ATP, GTP and PEP were adjusted to pFI 7.0 with o.x M N'aOI-L Uniformly labelled L-[lff3]leueine was obtained from The Radioebemieal Centre, Amersham, Bucks. (Great Britain). Sodium deoxycholate was obtained from British Drug Houses, Poole (Great Britain). Animals New Zealand White rabbits were used. Because rabbits have a constant ge:;tation period of 3x days the physiological status of a rabbit is known prepartum as well as postpartum, if the date of mating is recorded. l~eparation o/xoo ooo x 7, snpematant /ractio~ The rabbit was killed by a blow on the neck and the entire mammary gtand removed and immersed in ice-cold 0.44 M sucrose containing o.o05 M MgCI~,o.oz5 M KC1 and 0.05 M Tris-HCl buffer (pH 7.4) (sucrose buffer). The gland was cut up into a mince of semi-liquid consistency, which was repeatedly washed in sucrose buffer until relatively free from milk and cream. Finally, the mince was thorougbiy homogenised in 3 voL of sucrose buffer in a Potter-Elvehiem homogeniser. The homogenate was centrifuged for r5 rain at rsoo×g (av.), and the supernatant fluid obtained was centrifuged twice at io coo × g fay.) for 3° rain and r 5 vain, respectively. The supernatant fluid obtained after each of the above centrifugations was filtcred through glass wool to remove fat. A portion of the filtered IO oo0×g supernatant fluid, ustmliy amounting to x5-2o ml, was centrifuged at ion coo × g fay.) for 90 mill, yielding a final supernatant fraction which was used in the incorporation system. In the text. this fraction is usually termed the ioo Oddx g superaatant free, finn. Occasionally it is referred to as the untreated supernatant fraction, to distinguish it from xoooo0×gsupernatant fraction wkich has been diaiysed or treated with Sepbadex (see below).

Treatment o/the xoo ooo × g sup~natant /rtwtion In certain experiments use was made of supernatant fraction from which free amino acids and salts f~td been removed either by exhaustive dialysis or by gel Bioch~. Bio~hy~. A~i~, I66 (I058) t62-174

X64

G.D. BAIRD,L D. IffERRIMAN

filtration through Sephadex (Pharmacia, Uppsala, Sweden). In the ease of dialysis, 5 ml of the fraction were dialysed overnight in the cold and with stirring against 5oo ml of sucrosc buffer. The remainder of the fraction, together with the particulate fraction (see lmlow) was also kept in the cold. Assay of the fractions then took place on the day after preparation. In the ease of gel filtration, 5 ml of supernat ant fraction were passed at 4~ down a column of Sephadex G-25, of about ~o em × ~ cm, equilibrated with sucrose buffer. This took about ~ h and the fractions were then assayed on the day of preparation.

Preparation o] deoxyclwlate-treated xoo ooo × g particulate #action A particulate fraction, containing ribonucleoprotein particles, was prepared according to the method of ACS, NEIDLE AND W/~LSOH6. The remainder of the Io ooo × g supernatant fluid, prepared as described above and usually amounting to 6o-80 ml, was treated ~ t h that amount of xo % dooxycholate required to give a final concentration of o.5 % (ref. 6). This concentration of dcoxycbolate corresponded to a proportion of about o.35 mg deoxycholate to I mg protein in the xoooo×g supernatant fluid. After centrifuging for 9o rain at Ioo ouo × g (av.) the supernatant solution was discarded, and the pellets, which had a white opaque appearance, were rinsed 3 times in sucrose buffer before being finally resuspended in 2 ml of the same buffer for every IO g of tissue from which the pellets were derived. Preparation o/deoxycholate-treated zo 000 × g pacticulate #action The pellets obtained from tile two Io ooo × g (av.) centrifugations above wer,; pooled and resuspended to 50 ml in sucrose buffer. Having been made 0.5 % witl~, respect to dooxycholate, half the suspension (which was taken as a convenient volumel was then centrifuged at ioo ooo ×g (av.) for 90 rain, The resulting pellets were rinsed and then resuspended in x ml of sucrose buffer for every xo g of tissue. Conditions/or the incorporation o/[~(C]leucln¢ The total volume of the in vitro incorporation system was L55 ml, which consisted of z.z5 ml of incubation medium to which weEe added o.z ml of particniatc fraction, containing approx. Lx mg protein, and 0.3 ml of supernatant fraction, containing approx. 3.7 nag protein. The incubation medium contained ATP, ~/zmoles; PEP, xo pmoles; GSH, zo pmoles; GTP, LO pmole; KC1,25 pmoles; MgClz,7.5 pmoles; Tris-HCl buffer (pH 7.4) 23o pmoles; pyrnvate kinase, o.I rag, and uniformly labelled L-[x'C]leucine(spec. activ, x65 mC/mmole), 5o0 ropE. Taking into account the K + associated with the PEP, the quantity of K + in the incubation medium was 35/~moles. The incubations were carried out in air with shaking at 37° for various time iuterv~ds up to 60 rain and the reaction was terminated by the addition of an equal volume of xo % trichloroacctic acid. Assay o] eadioacti~ity The samples were prepared for assay by a method similar to that described by HSRmMAN A~D HuN'rERT: The trichloroaceti¢ aeid-preeipitable material obtained BiocMm. Biophys.Aaa, 166 (~9681 ~62-i74

MA.~4MAR~AMINOACID INCORPORATION~ ~/~0

t65

from each incubation sample was sedimented :rid t h e n washed a n d resuspended in 5 % trichloroacetic acid. After heating at 95 ° for x5 mln, the suspension was centriinged and t h e resulting pellet dissolved in o.5 m l o.x M N a O H . o.2 m l of this solution was then transferred to a counting p o t containing 5.o ml of se]ntillatinn fluids a n d counted. Results were expressed as counts/rain incorporated over a given period of time, usually 6o rain. This value was frequently related t o unit protein or R N A content of the particulate fraction.

Assay o/protein and R N A Particulate a u d supernatant fractions were a n a l y - ~ l for protein content b y the m e t h o d of L o w a v e~ aI. s using erystallised bovine plasma albumin as a standard, R N A was extracted from the fractions b y the acid extrartion m e t h o d of CEmoTrt a°, based on t h a t of OGUR AND ROSEN*z. R N A in the extracts was estimated b y the orcinol m e t h o d of CRRIO~-rI~° or b y measuring optical density a t 26o m/~. Similar values were obtained for R N A content b y either method. Yeast R N A was used as a standard.

Analysis o/~he [ractlons T h e yields of protein and R N A in zoo coo × g particulate fractions a n d xco ooo × g s u p e r n a tan t fractions prepared from rabbit m a m m a r y glands are given i n Ta bl e I. I n the table, the values obtained for fractions prepared from pregnant rabbits have been averaged, as lmve the values obtained for fractions preparod from lactating rabbits. As can be seen. there was little difference in the yield of protein in the particulate fractious of late-pregnant a n d of lactating rabbits. On the other hand, the yield of RN'A was ~ m e 2.z times greater in lactation t h a n in pregnancy. As a conse-

TABLE I AVERAGEYIELDS OF PROTB|H AND ~ A 1N Ioo o00 Xg PARTICULATEAND SUI~I~STATANTF R A C T I O N S P R I ~ P A R ~ D FROM R A B B I T I~'AMMARYGLAND Details of the preparation of the fractions are given in ~EXHOr~S.Yields of protein and ~ ' A in the fractions are ¢xpress~l as mg derived from z g vet weight of n~amm~vy gland. In the case of the particulate fraction, the values are Oven :t:S.D., together with the statistical significance between the means for the pregrmnt rabbits and the lactating rabbits. The number of observations in each case is recorded in the brackets.

Particulate [faction Protein yield RNA yield

supernedant /faction " RNA/ Protein RNA RNA[ protein yield yield prot,~n

Pregnant rabbits (½ to 4 days prepartura} 3.984-L3O(XS) o.37:Eo.13(8) o.093 Lactating rabbits (½ to z5 day~ postpartum) 3.494-o.94(~) o,8~-So.24(14) 0.235 S i ~ i c a u c o of difference P = 0,2 p = < o.oox Bio~him.

4x.2(7)

2-I (4}

43.3{x2) 2.x(7)

o,o51 0.04.9

Biophys. Acta, 166 (1968) x6~-t74

X6~

G.D. BAIRD, I, D, HERRIMAN

y'"'"

t

... 14

Fig. i. Effect of the stage of pregnancy or lactation on the R1qA/proteinratio of ioo ooox~" particulate fractionsprepared from rabbit mammary gland. Each point represents one prepaxa, rich. quenee, the average value for the RNA/psotein ratio was higher for the fractions from lactating rabbits. Very similar protein and RIgA yields were obtained for supernatant fractions prepared from either pregnant or lactEting rabbits. In Fig. x, values for the ratio of the RNA and protein contents of ioo ooo×g paxtienlate fractions are plotted against the physiological status of the rabbit from which each fraction was prepared. It will be noted from Table I and Fig. x that the values of the RNA/protein ratio for the particulate fractions are uniformly low as compared with the value (between 0.65 and 0.9) obtained for particulate fractions preFared in a similar way from the liver12. A possible reason for this was that the particulate material sedimenting from the Io ooo ×g supernatant fluid treated with 0.5 % dcoxycholate stUI contained microsomal membranes. Alternatively, this material might contain non-microsomal protein sedimenting with free ribosomes. To test these possibilities, the effect of varying the concentration of deexycholate in the IO ooo×g supernatant fluid was examined. From Table If it can be seen that increasing the concentration of dcoxycholate from 0.5 to x.5 % does cause some shedding of protein, with a consequent increase in the RNA/pv)tein ratio. However, the loss of protein in absolute terms was only half that occurring when the dcoxychelate concentration was increased from o to 0.5 %. Besides this, the RN'A/protein ratio for the particulate fraction was still well below that normally expected for a ribosomal preparation, even when i. 5 % deoxycholate was employed. When incorporating activity was expressed as counts per rain per mg partienlate-fractian RNA or g wet weight mammary gland, the differences in the activities of the particulate fractions prepared in the presence of the various dcoxycholate concentrations became less marked, Particulate fractions prepared in the presence of 0,5 % deoxyeholate were also subjected to sucrose density gradient fractionation according to the method of BLOn~L ~ V POTTER~. Following centrifugation, typical absorbauca profiles for an h~erogeneeas population of ribosomes were obtained and at least 60-70 ~o of the RNA content of the particulate fraction w ~ present within the gradient. The material within the gradient also had an overall RNA/protein ratio similar to that of the original particulate fraction, After mild treatment of particulate fraction with rlboBiochi,~. Biophys. Ada, i6fi (I96~) 16z-z74

M A M M A R Y AMINO ACID INCORPORATION It~ ~ Y O

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nuclease, the peaks i a the absorbance profile attributed t o poly~omal components failed t o appear, and the monomeric peak was enlarged.

Characteriaics oJ ~ ¢ in vilro amino acid-lncor~ora~ing system Incorporation of ~ ] l e u ¢ i n e into the complete ~ ~'~'o system was found to be linea.r for about 2o rain. Subsequently, the rate of incorporation declined and the radioaet;.vity of the isolated protein had reached a maximum level after 6o rain. Components of the in ~ifro incorporation system were varied in order to determine optimal conditions for incorporation. A plot .is given in Fig. ~ of the effect of

/

w~ct~3

3.7.

f

8.1 oo0n~il~~¢forti¢~lefroct]~Mo~;teory oMtgl

Fig. 2. Effect of variation of the concentration of KCI, MgCI~and GSH in the in vitro system on the level o~ t:6C]leucineincorporation in the system. The concentration of each of the 3 compounds was varied in turn while those of the other ~ were kept at the tontine value, The arrow indieatas the routine eoneentr~.tion employed. The plots were obtained with a.~ in vitro system prepared from a f-day-postpartum rabbit, Incubations were for 60 rain at 37°. II-II. KCI; &-&, MgCI~; 0 - 0 , GSH. Fig. 3. Proportionality between the quantity of tooooo×g particttl~.ta fraction present in the Sn vitro system and the level of [uC]icuctae incorporation. The plots are for systems prepared from a t-day-prepexthm rabbit iO-O) an6 a x2-day-postl~artum rabbit (O-O), respectively. Increasing concentrations of particulate fraction were u~ed while other components of the system were kept constant. The arrow indurates the point corresponding to the quantity of particulate fraction used routinely, o-o.96 mg par ticulate-fracticn protein were taken in the pregnant eyetem and o-z,t9 mg in the lactating system, Incubations were for 60 rain at 37% TABLE l i t Dg~B~NCg OF ~I*C]LgUCINBlUCORFORATIO~ON T~g PRBS~NCBOF ATP, ATP-~m;~U~RATmO SYSTEMA~IDGTP Components were omitted from in vitro systems prepared from the mammary glands of s 4-dxyprepartum rabbit and a 5-d~y-postpartum rabbit, respectively, in the same experiment. Incubations were for 60 nlin at 37%

Omissions

None A~IP, PEP and pyruvate klnase PEP and pyruvata kina~e GTP

Counlslmin

P~rcenl el incorporation oJ complela *ystem

Pregnani

Laclating

Pregnan#

Lactcaing

396 ~3 4t o

2845 36 t7 o t04

Xoo 5,8 Jo, 3 o

xoo t-3 6.0 5,8

Biochim. Biophys. Acta ,1(~6 (I96~~ t6~-z74

MAMMARYAMINOACID INCORPORATIONi~ I~/~0

~

varying the concentration of KC1, MgCl~ and GSH m the medium on incorporation of [t'Clleuc~ne in a system from a lactating rabbit, I t can be seen that the concentrations employed routinely approximate to the optimal concentrations of these 3 components. Essentially identical results, tho~.~h at a lower level of incerpor~tian, were obtained with a system prepared from a e-~sy-preparturn rabbit. The concent:~tians of "Iris buffer, ATP, G T P and P E P used in the i n ~itro system all corr~penclecl approximately to the optimalconcentrations for incorpvration determined by separate variation of the concentrations of these components in th~ system. The effects on in. corporation of omitting GTP, ATP-J-ATP-regenerating system (i.e. P E P and py~lvate kinase) and ATP.regenerating system alone are recorded in Table III. As can be seen from Table I I I LUC~leucine incorporation in systems prepared from either pregnant or lactating rabbits was hlghiy dependent on the presence of all these components. I t was observed that over a wide range of ~0o ooo × g particulate fraction concentration the quantity of [UC]leucine incorporated over 6o rain was directly propo~ion~d to quantity of particulate fraction present. This is demonstrated for particulate fractions prepared from pregnant and lactating rabbits in Fig. 3The effects of varying the quantity of untreated and dia!ysed zoo oco × g supernatant fractions i n / n vitro systems prepared from a pregnant rabbit and a lactating rabbit are plotted in Fig. 4, from which it can be seen that in the absence of supernatant fraction no incorporation occurred. In general, variation of the quantity of the fraction in the system from o.x to 0.6 m]. caused only moderate changes in the degree of incorporation. The greatest change was observed where dialysed supernatant fraction was used in the system prepared from the lactating rabbit. In this case, incor-

2s

/: /.. •

VOlUme of ~0~¢Ootontf~ctI~ Imli

f

boys ~ys ~stpart~ premature

Fig. 4, Effect of v~'i~tion of the quantity of d lalysed and undialy~d ioo coo × g supernstant fraction in the/Ja mfro system on the level of [~C]leucine incorporation. The plots are for systems prepared from a 3-day-pregartum ral~bit (closed symbols) and ~ 4-day-postpartum rabbit (open symbols), o-7. 3 mg ~upernatant -fraction protein were taken in both the pregnant and the lactating systems. Incub~tione were for 6o rain at 37°. For details of dialysis, see 14~THODS.0 - - 0 and 0 - 0 , dialysed supen~tant fraction; A - A and A-A, undialysed supcrnatant fraction. Fig. 5. Effect of the stage of pregnancy or lactation on the level of [~C]lsucine incorporation in the in vitro system. Incorporation was related ~o unit quantity of protein in the ~ooooo×g p~rti¢olate tro~tion in each c~.se, EaCh point represente a single experiment. Incubations were for 6o rain at 37°. Biochim. Biophys. Acta, x66 (1968) 16z-x74

I~0

G . D . BAIRD, I. D, HERR|MAN

puration appeared to be proportional to the quantity of the fraction present, between o.i and 0.5 ml. De,pendence el ~14C]lsueine incorporation on the slagz o/~regnangy or lactation In a series of e:rperimcuts the synthetic abilities of in vitro systemsprepared from the mammary glands of late-pregnant and lactating rabbits were compared by relating ~C]leucine incorporation to unit protein content of the zoo coo × g particulate fraction in each case. The values which were obtained are plotted in Fig. 5. It was found that incorporation proceeded at a rather low level in systems from latepregnant rabbits. In contrast, incorporation had already begun to rise in systems prepared from immediately postparturient rabbits. Incorporation then increased as lactation progressed and in systems prepared from rabbits at io to zg days postpartum, incorporation was proceeding at a rate which was more than Io times higher than that prepartum. In view of the prewensly observed increase in the value of the RNA/protcin ratio for ioo coo × g particulate fractions with the onset of lactation (Fig. z), it could be concluded that the increase in incorporation related to protein content (Fig, 5) was probably due in part to an increase in the, ribosomal content of the particulate fractions after parturition. Further contributions to this increase in incorporation could, however, also be made by an increase in the specific incorporation activity of the ribosomes present in the particulate fraction and by changes in the properties of the supernatant fraction. In order to assess the relative importance of these various contributions to the observed increase in incorporation related to protein content, iooooo×g particulate and supernatant fractions from late-pregnant and lactating rabbits were incubated together in mixed-match experiments. Prior to incubation, portions of the supernatant fractions in some experiments were either dialysed exhaastively or subjected to gel filtration on Sephadex G-z5 (see M~TnOVS). Subsequently, particulate fractions were incubated with homologous and beterologous supernatant fractions in the in vitro system, Several conclusions could be drawn from these experiments. Thus, the Io0 ooo × g partie~date fraction from the lactating rabbit was always found to he more active in promoting incorporation than the corresponding fraction frem the pregnant rabbit, when incubated in the presence of the same type of supernatant fraction. This was the case wbetber incorporation was expressed as counts/rain per mg protein or per rng RI~A in the particulate fractions, Furthermore, the untreated supernatant fraction from the lactating rabbit was always more active in promoting incorporation than the corresponding fraction from the pregnant rabbit, when incubated with similax particulate fraction. Although dialysis or passage through Sephadex increased the activity of supernatant fractions from both pregnant and lactating rabbits, the activity of fractions from pregnant rabbits was increased to a proportionately greater extent. For thh reason, the difference in the ability of fractions from the z sources to stimulate incorporation was reduced somewhat under these conditions. Differences in the activity of supematant fractions from pregnant and lactating rabbits could not be accounted for by differences in protein or RNA content, These results are summarized in Table IV. The values for each experiment in Table IV are in each case Biorl~ira. BiopI~ys, Acts. 166 (z968) ~6z-174

MAMMARYAMINO ACID INCORPORATIONi~ ~trO

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averages of all t h e appropriate values obtained when the particulate fractions were incubated with the various types of supornatant fraction,

TABLE IV IHCICEAOE I N |NCORPOIC.C.TION 1N "I'~Z p l f . ~ S E ~ C ] ~ O F IO0 ooo X ~ PARTICTJLATE A N D S U p E I ~ I A T A ~ I T F R A C T I O N S Y R O M L A C T A T I N G R A B B I T S O V E R T H A T O B S E R V E D IN T H E P R E S E N C E O F T H E C O R R E S P O N n ING YRACTgONS FROM PREGNANT RABBITS

Fractions were prepared from the mammary glands of a pregnant and a Lactstin 8 rabbit in each experiment. For description of procedure see text. The ~cesults are expressed as the percent inc~a~e in incotiporatlon occurring when a given fraction from the Lactating rabbit was employed in the i~ vitro system in p]sce of the corresponding fraction from the pregnant rabbit. Incorporation was memsured either as counte/min per mg partisuLate-fra~tion protein or per mg partictdate-fraction RNA. The physiological status ot the rabbits w a s a s follows: Expt. 1 : 4 days prepartum and 5 days postpartum; Expt z: 2 days prepertum and 8 days postpartum; Expt. 3: z days prepattum and z3 days postpartum. Incubations were for 6o rain at 37% l~xpt, nunY~er

1 z 3

Parliaulate lrc~tion (percent increase in i~corporatlon)

Stepcrnata,t fraaion (percen~ inaregse in i,corporatio,)

Incorporation measured as counts#hi. per mg particulate[faction RNA

Incorpoyation Untreated nwasw.ed a s ~aunts/min per mg particulatelva~tion protein

Dialysed

x87 Iz 3 7z

3o4 I77 38t

-239

66 964 26z

--

5epkadextreated

-33

Com/parison o! lhe incorlSorating abilities o/ t he I s ooo × g and t o o 00o × g particulate tracaons The work of FRASER AND GUTFREUND I a n d BREW AND CAMPBELL~ showc~ t h a t material ~edimenting from homogenates of lactating guinea-pig m a m m a r y g h a d in w h a t is conventionally tak en to be the mitochondrial fraction, i.e. between I5oo × g a n d xo ooo × g in this present study, was in fact as active in promoting incorporation in the in ~i~ro system as was t h a t material sodimenting in the conventional mierosomal fraction. B ~ w ANO CAMVBELLa concluded t h a t the activity of the mitochondrial fraction was due largely t o extensive contamination with m i c r o m m a l l~tateriai. I t was necessary, therefore, to determine whether the fraction sedimcuting at xo ooo × g in the present s t u d y possessed a n incorporating activity of a similar order to t h a t of the xoo c o o x g particulate fraction. It was also necessary to determine whether t h ein co r p o r atin g activ lty of the xo ooo × g fraction increased after parturition in the same w a y as did t h a t of the zoo o o o × g particulate fraction. T h e results obtained with ro s o s x g and zoo ooo × g particulate fractions F e pared from the m a m m a r y glands of 3 rabbits a t different physiological stages ~re recorded in Table V. F r o m Table V it is evident t h a t the incorpoi:ating activity of the x o o o o x g fraction increased after parturition in a similar m a n n e r t o t h a t of the Biockim. Biophys. Aaa, t66 (~968) I6z-x74

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nAIRD, I. D. HERRIMAN

TABLE V ANALYSIS OF THE IOO O00 X g PARTICULATE FRACTION AND "fh~lg IO O*XI.X g PAR'rt CU'LAX'~ PRACT|ON AND COMPARISON OF "fll~lR INCORpORA'I'1N'O ACTIVITIES a n d s u b e e q u e n t [ y i n c u b a t e d i n in vit~o s y s t e m s a s de, scribed i n Mg¢uor~s. Ptoteitt and llNA yields in the fractions axe expressed as mg derived from t g wet we/ght of mammary gland. Inoubatione were for 6o mln at 37°. The iraotlans were prepared

3 days preparlum too ooo × g fraction ioooo×gfraction z days postpar~ur~ too ooo x g fraction xo ooo x g traction go days postpartum ~rooooo x g fraction xo ooo × g fraction

Protein y~eld

RNA yield

RNA[ protein

Counts/rain per rag fraclion protein

Count$/mln per mg ~racllon RNA

Counts/rain per g ~ t weight tissue

3.7° 4-73

o.38 0.34

o.lo 3 0.072

800 488

7 850 6450

3 ooo 214o

4,77 4.06

o,98 o.54

o.eo5 O.t33

8 900 3 ~20

43 too ~4 43°

42 250 13 leo

3,~4 9,5o

0.77 o,95

o.~45 o,loo

t3 0o0 5 580

53 5oo 55 5°o

39 zso 54 5°o

zoo coo × g fraction, whether incorporation was related to protein or RNA, Incorporation related to protein content was about half as great in t h e xo ooo x g fraction as in the sou ooo × g fraction, wtdle related to RIgA content it was of the same order in the 2 fractions. Incorporation related back t o wet weight of m a m m a r y gl and tissue also gave similar values foc the e fractions. I t can be inferred t h a t the x o o o o × g fraction contains ab o u t half the total q u a n t i t y of ribonucleoprotein material and t h a t the incorporating ability of this fraction changes with physiological status in a m a n n e r similar to t h a t in which the incorporating ability of the zoo ooo × g fraction changes,

DISCUSSIOn*

Anabysis o[ t ~ /maions T h e RNA/protein ratio for the zoo ooo × g particulate fractions was uniformly low, a n d even the highest value obtained (about o.37 for a x3-day-postpartum rabbit) was below the value normally obtained for ribosomal preparations. I t is concluded tentatively t h a t the RNA/protein ratio is low because of contamination of the particulate fraction with non-microsomai protein. This conclusion is based on the following observations, (x) E v e n when high concentrations of deoxychelate are employed, t h e value of the ratio is still low, (z) Using routine particulate fractions, distributions are obtained on sucrose density gradients wl~ich are typical fur heterogeneous ribosomal populations. Furthermore, the m a j o r portion of the R N A content of tile particulate fraction remains within t h e gradient. (3) There is a m a r k e d difference in the q u a n t i t y of protein present in the zoo o o o × g particulate fraction prepared with 0.5 % deoxycholate a n d present in this fraction prepared w i t h o u t dcoxycholate. This difference is sufficient t o be consistent with mierosomes (RNA/ protein = o.2), in the absence of deoxyclwlate, being transformed in t h e presence Bio~him. llD~hys. Aaa, ~66

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MAMMARY AMINO ACiD INCORPORATION ~@vitro

:t73

of O.5 % dcoxycholate into ribosomes (RNAt'protein = x,o), if it is assumed that the quantity of non-microsomal protein present is similar in both e~aes. .... The presence of a variable quantity of contaminating protein does not appear to have any marked effect on the activity of the paxticalate fraction. Thus; although protein is shed from the particulate fraction with increasing concentrations of deoxyeholate, little change occurs in the incorporating activity related to particulatefraction RNA or to wet weight of mammary gland (Table II) . . . . . . From Table I and Fig. x it is evident that the quantity of RNA in the zoo ooo x g particulate fraction rises between late pregnancy and mid lactation. There is, therefore, on average a greater proportion of ribonucleoprotein paxtieles in fractions prepared from lactating rabbits than in fi'aetions prepared from pregnant rabbits. This finding suggests that increase in size of the mammary gland with the onset of lactation is accompanied by an increase in the concentration of ribosomes within the gland,

Ckaracteristies o! the io~corporation system In general, the properties of the/~ vitro incorporation system, as described in this communication, axe closely similar to those of systems of the same type prepared from other mammalian tissues, However, there seems to be a greater need for added GTP and ATP-regenerating system than is the case with liver and brain (e.g. KORNERTM, STENZEL, ARONSONAND RUBINll). A feature of the present work is the demonstration that the rate of amino acid incorporation in a given/~ vitro system is directly proportional to the quantity of too ooo × g particulate fraction present over a wide concentration range, This finding is the justification for expressing incorporating ability as counts/rain incorporated per unit quantity of particulate-fraction protein or RNA. In contrast, there was no direct proportionality between ~*C]leucine incorporation and the quantity of supernatant fraction present. Dependence o/ [uC]leucine incorporation on ~he stage of pyegnancy or lactation The results plotted in Fig. 5 clearly show that the activity of the in ~iOo incorporating system increases rapidly from parturition to mid-lactation. In contrast, the activity of systems prepared from the mammary glands of tare-pregnant rabbits is relatively low over the last 4 days of pregnancy, and does not show any clear rLse as parturition app~'oaches. The mammary gland of the pregnant rabbit is by no means inert. The gland itself is rapidly developing. Enzymes associated with milk production increase in ~ctivity (e.g. HECrZ~aANiS) and in late pregnancy the gland contains milk-like fluid. In spite of this, it seems that the potential rate of protein synthesis associated with lactation is of a markedly higher order than that associated with the developing gland in late-pregnancy. This conclusion is supported by the work of I)ENAMURla, who found that the value of the RNA/DNA ratkJ increased in rabbit mammary gland from a value of about x,o to a value of about 4.o over the first 15 days of lactation. I'~YA[ DNA ratios considerably greater than unity are taken to indicate a rapid rate of protein synthesis (el. GREI~NBAUMAND SLATER~7). 13ioch~m. Biophys. Aaa, ~66 (1968) xdz-~74

x74

o. D, RAIRD~ L D. HERRIMAN

The increase in the rate of protein synthesis is d e a r l y due in p a r t to the increase in the q u a n t i t y o~ ribanaeleoprotein particles present in unit weight of t h e m a m m a r y gland. However, the mixed-match experiments showed that, under identical incubation conditions, the ioo o o o X g particulate fraction from the lactating rabbit is more act~v,e t h a n t h a t from t h e pregnant rabbit when a c t i 'dt y is expressed as incorporation related to RlgA content. F r o m this i t m a y be concluded t h a t the specifit; activity of the cibonuclcoprotein particles is greater in laets, tion t h a n i n pregnancy, One exp/anation of this finding mi ght be t h a t ribonudcoprotein particle preparatio~s from tLe lactating rabbit contain a higher ratio of polysnmes to monomerie ribosomes t h a n do similar preparations from the pregnant rabbit. T h e resnits suggest t h a t changes in the m a m m a r y gland at the level of the ribosomes during the transition from pregnan',':y to lactation m¢~y be similar t o those observed in the liver on administration of growth hormone or thyroid hormone to hypophysectomised or thyrnidectomised t~,~ts, respeetively~e,aL

ACKNOWLEDGEMENTS The authors thank Miss JANgCI~ LIZI~for excellent technical assistance, and Dr. G. D. HUNTER for encouragement and advice. ILEF~RENCES I 2 3 't

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Biochim. Biophys. Ac4a, t66 (1968) z6z-J74

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