Rates of degradation of glycoproteins from normal and regenerating rat livers: A study using double isotopes

ARCHIVES OF BIOCHEMISTRY Vol. 199, No. 2, February,

AND BIOPHYSICS pp. 384-392, 1980

Ra ‘es of Degradation of Glycoproteins from Normal and Regenerating Rat Livers: A Study Using Double Isotopes NORMAND Laboratory

MARCEAU,

JULIEN

of Medical The Department

DESCHfiNES,

Biophysics, Centre Hospitalier of Medicine, Lava1 University, Received

July

AND JACQUES de l'liniversitb Quebec,

Laval, Canada

LANDRY and

19, 1979

The average decay rates (half-lives) of mixed glycoproteins were measured using double isotopes of fucose and glucosamine and compared to those of mixed overall proteins measured with leucine and NaH’“CQ, in whole homogenates and plasma membranes from normal and regenerating rat livers. A large reutilization of leucine was observed under both normal and regenerating conditions. Fucose seems to be recycling most predominantly in regenerating liver, whereas glucosamine was found to be very little if not at all reutilized under both conditions. Comparison of the results obtained with NaH1”CO, and glucosamine demonstrated that glycoproteins from normal liver homogenate are degraded at a faster rate than mixed proteins. Contrary to that of mixed proteins, the half-life of glycoproteins remains unchanged during liver regeneration, and the use of glucosamine revealed that the degradation of plasma membrane glycoproteins is identical to that found in whole homogenate under both normal and regenerating conditions. Finally, the relative degradation rates of fractionated plasma membrane proteins and glycoproteins were evaluated under the same conditions. During liver regeneration some readjustments are observed in the that the synthesis and relative degradation rates of individual specie s which suggest degradation of the various surface membrane glycoproteins proceed at rates that are ._ controlled independently.

Studies by Swick and Ip (1) and Scornik and Butbol (2) have shown that proteins from liver undergoing regeneration are degraded at a rate estimated to be 30650% slower than in normal liver. The obtention of such measurement was possible with the use of NaH14C0,, (into arginine), an amino acid precursor found to be recycled at a very small extent during liver compensatory grovvth. Previous measurements, using prelabeled [gua&o-‘“Clarginine, had led to an apparent zero protein degradation of mixed proteins under the same condition (3), and Swick and Ip explained these results in terms of a difference in the initial body distribution of the two respective precursors (1). These workers have demonstrated that during regeneration, the prelabeled arginine is mobilized from the extrahepatic tissues, whereas NaH’4C0, is a precursor primarily taken up by the liver, where it is incorporated equally into arginine, glutamic 0003-9861/80/020384-09$02.00/O Copyright All rights

9 1980 by Academic Press, of reproduction in any form

Inc. reserved.

acid, and aspartic acid, which in turn have very low probability of recycling in the same organ (4). Scornik and Butbol stressed the point however that their studies using NaH14C0,, (into arginine) concerned only measurements of average decay rates of mixed proteins and that a harmonious increase of liver components during regrowth requires some readjustment in the relative rates at which different classes of proteins are renewed. Part of the present work concerns measurements on the average decay rates of a particular class of proteins, namely glycoproteins, in normal and regenerating livers using double-isotope method with fucose and glucosamine. Previous measurements on relative degradation rates of normal rodent liver subcellular organelles have indicated that membrane proteins, especially plasma membrane proteins, are degraded more rapidly than protein from the whole homogenate 384

GLYCOPROTEIN

DEGRADATION

IN NORMAI,

and the soluble fraction (5, 6). No similar measurements have been made for mixed proteins or glycoproteins during liver regeneration, Therefore, the average decay rates of mixed proteins and glycoproteins in plasma membrane were measured and compared with those obtained in whole homogenate under both normal and regenerating conditions. Moreover, studies on plasma membrane proteins have previously shown that individual species experience differential turnover with the higher molecular weight species undergoing more rapid degradation (7, 8). Similar studies have further demonstrated that this mode of degradation still holds for glycoproteins, i.e., fucose (9) and glucosamine-containing species (6, 9). Considering the above proposal on a possible readjustment in the decay rates of different classes of proteins during liver regeneration, it was of interest to evaluate the influence of this compensatory growth on the relative degradation rates of individual protein and glycoprotein species from the plasma membrane. MATERIALS

AND

METHODS

Male Wistar rats weighing 15 to 200 g were used in all experiments. They had access to food and u-ater nd libitum. L-[4,2H]Leucine (31 Ci/mmol). D-[6-“Hlglucosamine (12.6 Ciimmoi), L-[l-llC]leucinc (60 mCi/mmol). D-jl-‘JC]glucosamine (58 mCi/mmol), and L-[1-14C]fucose (57 mCi/mmol) were obtained from Amersham/Searle. L-[1,5,6-“H]Fucose (5 Ciimmol) was from New En$md Nuclear Corporation. NaH”CO,, (25-S mCi/pmol) was obtained from ICN Pharmaceuticals Inc.

Decay Rate Measuremenf Glycoproteks

oj’ Mixed

Normnl liver. The modified version (7) of the double isotope technique of ilrias et nl. (8) was used here to measure metabolic decays of mixed proteins (glyco) in liver Lvhole homogenates and plasma membranes from normal rats. A first labeled precursor (‘%) was administered to a rat anti allowed to decay for 3 days. Then, the second isotopic form (“H) of the same precursor was injected to the same animal. The animal was sacrificed 3.5 h later and its liver was homogenized and processed to obtain

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385

isotopic ratios, after correction for the :‘H and “(1 injected doses and counting efficiencies. Although this approach yields only a two-point metabolic deca> curve (Appendix A) the estimated half-lives obtained in a :3-day period are comparable to those measured following injection of a single precursor (Table I; Refs. (7. IO)). Rege,~ernti,cg liver. Metabolic decay rates of liver whole homogenate and plasma membrane mixed proteins (glyco) from partially hepatectomizetl rats were measured in two ways. In initial experiments, decay measurements over a :&lay period were done using a single precursor in a single rat, according to procedures described by others (1, 3). Although this approach is acceptable for decay detr*rmination in \vhole homogenate. its use in plasma membrane> is hazardous since two membrane isolations are recluiwl at a X-day interval. Therefort> in later t~sperimentx, the tlouble isotope technique of Glass and Doyle (71 \ras atlaptetl. The first animal was irljectetl \vith either [“Hllcucine, [~‘H]glucosamine, or [:‘H]fucose and subjected to hepatrctomy after a 36-h period (0 h). Thirty-six hours later (:36 h), another normal :mimal receiretl the I’(: isotopic form of the same precursor. Both animals were killed :I6 h later (7% h from 0 h). Done this v,xy, th(x ‘I C isotope IYJ~IYWII~~ the status of labeled proteins at :I(i h postinjection (time of hepatectomy) and th(a :‘H isotope, the level ot labeling after a supplementary 72 h of liver wgencration. The isotope ratio is therefore reprcsentativt~ of the 72 h tlegrxlation of thtl labeled species present at the time of hepatectomy. This experimental procrtlurc~ insures that animals lvere used untlrr identical physiological conditions at the time of both injections. which is necessary for the use of thr doubleisotope techniques (7, 8). Half-lives for total hepatic proteins (ylyco) ~vere calculated from the ,‘H:‘-‘(’ xtivitj ratios measured in aliquots of the pooled homogenate (4.5 g from each liver). after correction for irljectecl doses, wuntin~ efficiencies. and isotopic tlilution during gro\vth regcbneration (1, 2). The other advantage of this procrtlure is that single manipulation is required to obtain metabolic decays for proteins (glyco) in the pooled homogenate and the plasma membrane fraction. Also. thcl use of this method is most appropriate for the determination of the relative rates of tlegrxlation of liver plasma membrane glycoprotein species under regenerating condition.

Relnfive Rates oj’ Deglndutiow Jdembrane Glycoproteirls

oj’ Plnsmn

In experiments on regenerating liver, a plasma fraction \vas obtained from the pooled livers and the different proteins (glyco) were separated by polyacrylamide gel electrophoresis in the presence membrane

386

MARCEAU,

DESCHGNES.

of sodium dodecyl sulfate (SDS-PAGE),’ as (lone before on normal liver (9). The :JH:*4C activities along the gels yielded a measure of the relative rates of degradation of the separated subunits during the first 3 days following hepatectomy (Appendix A).

Partial

Hepatectomy

Partial hepatectomy (20) was performed under ether anesthesia according to a standard procedure (11). A small abdominal incision was practiced and the median and left lateral lobes of the liver were removed. Control rats were sham operated.

Sample

Preparation

Whole homogenate. Liver tissue was first homogenized in 10 vol of 1 mM NaHCO, (pH 7.5) as described by Ray (12). Mixed liver proteins were prepared from an aliquot of this solution by precipitation with trichloroacetic acid (58, final concentration). The precipitation followed by successive extractions with ethanol and ether yielded essentially the same results. The precipitate was solubilized in Protosol (New England Nuclear) and counted in 10 ml Aquasol. Plasma membrane. A plasma membrane fraction \vas isolated from homogenate of regenerating liver using a modified version (12) of Neville’s method (13). Yields of 1.1 and 1.3 mg protein membrane were obtained per gram of normal and regenerating liver wet weight. The specific activity in 5’.nucleotidase was 21.5 higher in membrane than in total homogenate for normal liver, in comparison to 17.2 in plasma membrane preparation from regenerating liver. The relative specific activities in glucose 6-phosphatase were 0.6 and 0.7 for membrane preparations from normal and regenerating livers, respectively. Under electron microscopy the two preparations were morphologically indistinguishable and revealed some extended smooth membranes sheets frequently linked by desmosomes and the presence of membrane vesicules. The membrane fraction was solubilized with SDS and dialyzed according to the procedure of Glossman and Neville (15), except that urea was omitted in the dialysis buffer (9). Radioactivity in plasma membrane was measured by counting 100.~1 aliquots of the solubilized membrane fraction. Individual mernbTane protein (glyco). Aliquots, 100 ~1, of the membrane solution were subjected to 7% SDS-PAGE (9). Gels were sectioned in one hundred l-mm-thick slices, digested in H,O,, and then counted as described previously (9). ’ Abbreviations gel electrophoresis sulfate.

used: SDS-PAGE, in the presence

polyacrylamide of sodium tlodecyl

AND

LANDRY RESULTS

AND

DISCUSSION

Average Decay Rate of Mixed Liver Proteins and Glycoproteins Whole Homogenate Rats were injected intravenously with amino acid and carbohydrate isotopic precursors, according to time schedules described under Materials and Methods. Some of the animals were partially hepatectomized while others were sham operated and used as a control group. Livers were processed to measure decay rates of mixed proteins and glycoproteins during a 3-day period. The left part of Table I shows the values obtained using leucine, NaH’“CO,, fucose, and glucosamine, respectively. For means of comparison with data published by others, the estimated apparent half-lives corresponding to exponential decays (Appendix A), are presented on the right portion of the table. Sham-operated animals. The use of leucine yields an apparent half-life of 6.7 days, which is similar to that obtained before with the same precursor (7). NaH’“CO,, leads to a value twice as low as that obtained with leucine. Other workers have found intermediate values for [guanido“Clarginine (1,2, 7,8). Therefore, as found previously (1,2), NaH14C0,, seemsto be the least recycled amino acid precursor. The use of fucose and glucosamine yields the same half-lives for glycoproteins, and the 2-day value is even lower than that found for the overall proteins using NaH14C0,. This already suggests that the carbohydratecontaining protein class in liver is degraded at a faster rate than the overall liver proteins. It does not take into account however, a possible reutilization of the carbohydrate precursors and a good way to assessthis possibility is to measure decay rate after a two-third hepatectomy. Partially heputectomized animals. Under condition of partial hepatectomy, the observed half-life with leucine, a precursor known to be greatly reutilized (16), compares well with that obtained by others for arginine (l-3), the value being the infinite. NaH14C0, yields a value twice (5.6 days)

GI,Y(‘()PR()TEIN

I)EGRADATION

IN NORMAL TABLE

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RAT

387

LIVERS

I

DECAY RATES(HALF-LIVES)~F MIXEDPROTEINS AND GLYCOPROTEWS OF LIVER HOMOGENATES AKD PLASMA MEMBRANES FROM NORMAL@HAM-OPERATED)ANDTWO-THIRDS HEPATECTOMIZED RATS, L:SI7iG VAIZIOCS ISOTOPES Percentage

injectetl”

Lcwcine

Homogenate Plasma membraw

7:3 i 612

NaH’Y‘O.,

Homogenatca

FllCOS~~

Homogenate Plasma membrane

33 -t 19i

(;lucos;amint~

Homogenate Plasma membrane

xii-+ ‘i 38 2 12

precursor

at 3 days

Tu-o-thirds hcpatrctomizrd rats

Shamopwatrd rats

Isotopic

activity

j 7

49 3 4

Estimatrtl Shamoperated rats

half-life

(days)

Two-thirtls hrpatectomized rats

112 -t I6 112 * 6

6.7 4.2

1 1

(i!)

i?. 9

T,.fi

(ilk 5 76 I 10

2.0 1.3

4.2 ‘i..i

37-t iI6 z

2.0 2.1

2.1 2.0

4 4

” \Vith lrucine. fucosr. or gluwsamine. sham-operated animals were given 50 FCi of the “H form :I clays after the alministration of 15 PCi of the “C form; the partially hepatectomized rats received 23 PCi of “C form :i days aftrr thr atlministrdtion of $5 &i of the “H form accortling to the time schedule under Materials ant1 Methods. The mean half-life xvas calculated from the change in the radioactivity during thr S-day pvricld. as tIescrib under Appendix A. In the case of NaH”C’O,,, rats wew given injections of 260 PcIi per rat. Four to six xCmals Lvere used per group, except for NaH”C‘Q,, whew only t\vo animals \verc used. Valurs for thp Igltnr~ido-“(‘larginine precursor were takrbn from the Ref. (8). The pc~rcentagc~ :I-clay activitirs are tht, mean f SF:.

as high as that seen in sham-operated animals (2.9 clays). This value meets those half-lives reported by Swick and Ip (1) and Scornik and Butbol(2) for the overall mixed proteins using the same precursor. Under the samecondition, fucoseyields an estimated half-life of 4.2 days for mixed glycoproteins compared to the Z-day value obtained in the control group. With glucosamine, the measured half-life is essentially the same as that obtained in controls (‘2.0-2.1 days). In spite of the fact that NaH’“C0, can be recycled to an extent of about 7%’ (l), it still remains that the average rate for mixed proteins is reduced following partial hepatectomy (1, 2). The 4.2-&y half-life obtained with fucose may also reflect some recycling, due to a mobilization of nonhepatic fucose during liver regeneration. Indeed, it has been shown before (17) that in the first hours following an oral 01 intravenous administration of [‘“C]fucose in normal rat, more than 80% of the label is first detected in estrahepatic tissues. Thus,

it is most conceivable that during liver regeneration some excess fucose returned from the labeled estrahepatic tissue, \vhich leadsto an apparent increase in the estimated half-life for liver glycoproteins. On the contrary, \vith glucosamine, the fact that the same half-life is obtained for mixed glycoproteins in both normal ant1 regenerating livers strongly suggest that the precursor is not reutlllzed at all. Again, this conclusion is supported by previous \vork on the fate of glucosamine after its entry into the blood circulation. Glucosamine is mainly taken up by the liver where part of it is readily incorporated into glycoproteins after acetylation (18). Another fraction is converted into either N-acet~lgalactosamine 01 sialic acid before protein incorporation (19). All these conversions greatly reduce the possible recycling of labeled glucosamine since the glycoprotein breakdown products, N-acetylgalactosamine and N-acetylglucosamine are taken up very little by the liver and sialic acid is not at all ((20), IV. Marceau

388

IMARCEAU.DESCHI?NES,ANDLANDRY

and R. Blais, unpublished results). All these observations make glucosamine a precursor of choice in studies on hepatic glycoprotein breakdown. Plasma

Membrane

The average decay rates of mixed proteins and glycoproteins were measured in plasma membranes from normal and regenerating livers. The results, presented in Table I, show that as found by others in normal rat (5, 6), the estimated half-life for mixed proteins is lower in plasma membrane than in whole homogenate (Table I). Moreover, the fact that the same half-life is found, using [ guanido-‘4C]arginine (1.8 days) or glucosamine (2.1 days), support the findings that most glycoproteins are contained in plasma membranes (6, 15) and that the protein and carbohydrate portions of glycoproteins are degraded in a concerted fashion (10). Following partial hepatectomy, the half-life obtained with leucine is infinite, just as found for mixed proteins from whole homogenate (Table I). Under the same condition, the use of fucose to obtain mean degradation rates of plasma membrane glycoproteins confirms the above observation that this precursor is reutilized to a good extent (7.5 vs 1.3 days). With glucosamine the estimated half-life in partially hepatectomized rats (2.0 days) is the same as that determined in the control group (2.1 days) and the value is also identical to those measured for glycoproteins from whole homogenate of both normal and regenerating livers (Table I). The fact that the decay rate of plasma membrane glycoproteins is not modified during liver regeneration suggests readjustments in the decay rate of proteins in other subcellular fractions for example, soluble and microsomal fractions, and/or as observed for mixed homogenate proteins and some plasma proteins (21, 22), an increase in the biosynthetic rates of membrane glycoproteins. More studies are required to further clarify this point. Nevertheless, it remains that the rate measured for plasma membrane glycoproteins represents an average value and perhaps there are

some readjustments in the relative decay rates of individual species within the glycoprotein class in plasma membrane. A good way to test this possibility is to evaluate the relative degradation rates of plasma membrane glycoprotein species under both normal and regenerating conditions. Relative

Degradation Rates of Plasma Membrane Glycoproteins

We have reported before data on the relative degradation rates of plasma membrane glycoproteins from normal rat liver using the two isotopic forms (:‘H and I%) of leucine, fucose, and glucosamine, respectively (9). Such measurements \vere made by taking the actual ratios of isotope activities detected along polyacrylamide gels (9). However, further mathematical considerations on the data demonstrated that we cannot use the actual ratios but instead the In radioisotope ratios, corrected for doses and counting efficiencies (Appendix B). Others have also been mistaken in the representation of similar data (5, 6, 8) and the error introduced has some importance in trying to evaluate the relationship between degradation rates vs molecular weights of the various species fractionated by SDS-polyacrylamide gel electrophoresis. Therefore, our previous data on the relative degradation rates of plasma membrane glycoprotein from normal rat liver were reevaluatetl by using the In isotope ratios and compared to those obtained after partial hepatectomy. Figures lA-C show the relative degradation rates obtained in normal liver with leucine, fucose, and glucosamine, respectively. By using the In ‘“C13H disintegrations per minute ratio representation corrected for closes, a In ‘“C/“H ratio equals to zero corresponds to no apparent degradation, and a high negative logarithm is equivalent to a high rate of degradation. As indicated in Fig. lA, relatively small deviations in In ratios are obtained along the gel with leucine and on the average all the values are not that far from zero. This is not the case with fucose and glucosamine, where large cleviations from the zero line are

GLYCOPROTEIN

DEGRADATION

IN NORMAL

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REGENERATING

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389

FIG. 1. (A-C) Relative degradation of proteinand carbohydrate-containing species of plasma membrane isolated from normal rat liver, a:: expressed in terms of ‘-‘C-:‘H profiles (9). (A) A rat was given 250 PCi of [“Hlleucine 3 days after being injected with 60 PCi of 1’ ‘C]leucine to yield l’C::‘H ratios. (K) The animal \vas given 250 &i of [:‘H]fucose 3 days after administration of 50 PCi [“C]fucose. (C) The animal ~vas injected with 200 &i of [C’H]glucosamine :1 days after the administration of 10 pCi of \‘%]glucosamine. (D-F) Relative degrxlation of proteinand carbohydratccontaining species of plasma membrane isolated from regenerating rat liver. (D) A first animal was injected with 700 PCi of [,‘H]leucine 36 h before hepatectomy. Thirty-six hours later, a second animal wx:, injected Mith 100 PCi of [ ‘lC]lwcine. The two animals were killed 86 h later, the liver homogenates ~vere pooled and processed to obtain the plasma membrane fraction. (E) The time schedule deswihrtl in (A) \vah fo11oLvetl except that 720 FCi of [:‘H]fucose and 100 PCi of [ ‘~C]fuwse xvere il?jectetl. (F) The same time schedule was follo\\etl except that 320 /*Ci of [“H]glucosamine and 100 ,uCi of [“C]glucosamine \vere injectetl. The In :IH:“C ratios are calculatetl from the tlistrihution of each isotopic precursor activity detected in SDS-pol!~acr~lanli(l~~ gel after el(drophoresis (Appendix II). The direction of migration is from left to right. ~lolecular lveights are given on top in a nonlinear scale.

observed along the gel. (Figs. 1R and C). From these results there is no doubt that glycoprotein species of higher molecular weights are degraded at faster rates. Moreover, by considering the data presented in Table I, the lower deviation obtained for leucine when compared with those for fucose and glucosamine can be explained in terms of a difference in precursor reutiliza-

tion. Indeed, proteins of higher molecular weights turn over at a faster rate and thus the large reutilization of a precursor minimizes the observed difference in turnover rates between small and large protein species. The same type of analysis \\-a$ done in the case of regenerating liver. Rats \vere injected with two isotopes (“H and lY’) of

390

MARCEAU,DESCH~NES,ANDLANDRY

the same precursor, according to the time schedule presented under Materials and Methods. The liver plasma membranes were isolated and fractionated by SDSpolyacrylamide gel electrophoresis. The “H and 14C radioactivities were counted in l-mm gel slices. In terms of electrophoretic mobility, the fractionated membrane species labeled with leucine, fucose, and glucosamine yielded radioactivity profiles (data not shown) much similar to those observed before in normal liver (9). Figures lD-F shovv the relative In ratios obtained along the gels with leucine, fucose, and glucosamine, respectively. After partial hepatectomy, the heterogeneity in degradation rates observed among the different species of plasma membrane from normal liver has vanished, as confirmed by the very low deviations in In ratios along the gels. Moreover, vve note that with leucine all the In :‘H/‘“C ratios are positive, meaning that the uniform degradation obtained with this precursor could well be explained in terms of an extensive recycling during partial hepatectomy. This conclusion is supported by the apparent infinite half-life obtained in vvhole homogenate and nonfractionated plasma membranes (Table I). The same explanation could hold for the data obtained with fucose (Fig. 1D and Table I). However, a comparison of the results obtained with glucosamine between normal and regenerating conditions leads to a more appropriate picture of the phenomenum. While the relative In ratios of the different labeled subunits become homogeneous along the gel in the case of regenerating liver (Fig. lF), when compared to those observed in normal liver (Fig. lC), the average half-life for the mixed plasma membrane glycoproteins remain the same under both situations (Table I). Moreover, a detailed comparison between the In ratio distributions obtained along the gel revealed that the degradation rates for subunits of 225K and above are slower in regenerating liver than in normal liver. For species of 1OOK to 150K, the decay rates are on the contrary slightly faster, and finally those for species smaller than 60K remain essentially the same. All this means that some

readjustments are taking place in the relative degradation rates of individual surface membrane glycoproteins implying that, as found by others for mixed proteins in homogenate (22) and plasma proteins (23), the synthesis and degradation of various surface membrane glycoproteins proceed at rates that are controlled independently. All these data on measurements of degradation rates of glycoproteins in normal and regenerating rat livers were obtained by the double-isotope method of Glass and Doyle (7). The present adaptation for measurements of degradation rates of mixed glycoproteins in regenerating liver is most convenient. As discussed in Appendices A and B, the half-life measurements depend on tmo data points, assuming a single exponential (first order) decay for each glycoprotein species (7). However, when one considers a group of mixed species, each having their own half-life, such approximation does not hold as well for a long degradation period (1, 2). In the present studies, the time intervals were 3 h-3 days and 1.5-4.5 days for measurements in normal and regenerating livers, respectively. Therefore, although a 3-day degradation was considered, the calculated half-lives in the respective cases are not exactly equivalent. At any rate, the values obtained with the various precursors still yield a very good evaluation of their degree of reutilization (Table I). The recent results reported by Tauber and Reutter (24) on the measurements of decay rates (half-lives) of mixed proteins and glycoproteins using single isotopes of leucine, methionine, fucose, and N-acetylmannosamine are in close agreement with those reported here (Table I). However, the use of a double-isotope approach is most convenient for measurements of relative degradation rates of individual proteins and glycoproteins after fractionation on SDS-polyacrylamide gel electrophoresis. Considering that the degradation of a protein followed a first-order kinetics with few exceptions (25, 26), measurements of the relative degradation rate obtained at any time interval should be representative of the whole degradation

GLYCOPROTEIN

DEGRADATION

IN NORMAL

process of one species. However, the obtention of the true values is masked when one uses a precursor which is recycled to a large extent. The results in the case of with leucine, especially regenerating liver, is a good example since after correction for doses, counting efficiency, and liver regrowth-mass, the degradation rates of all the fractionated species are negative (Fig. 1D). Glucosamine does not seem to be recycled and its use here indicates a readjustment in degradation rates of the various species. These observations are supported by the recent ones of Tauber and Reutter obtained with a single isotope (27). In the light of all these data obtained i?z vivo, one may wonder if such readjustment in the relative degraclation rate of liver plasma membrane proteins (glyco) can also take place in an in vitro situation. It is worth noticing the work of Tweto and Doyle (28) on HTC hepatoma cells in culture, indicating that all surface membrane proteins are degraded at similar rates. APPENDIX

A

Assuming a first-order kinetics for the degradation of proteins and glycoproteins, rate constants can be calculated from the following equation: N(f) = ,V(O)e - K,,.t

[ll

AND

per &i

APPENDIX

R

As discussed under Materials and Methods, the use of a double-isotope method allows one to measure the relative degradation rates of the various proteins and glycoproteins fractionated by SDS-PAGE. Using the expression stated in Appendix A -K,, = [In z],,,/t

LIVERS

391

K,, = [lng],/I where N(0) is the number of labeled molecules at time zero, N(t) the number remaining after a period t, and Kc1is the rate constant of degradation. In the present experiments, K,! were evaluated from the fraction of the radioactivity (N(t)/N(O)) remaining in the whole homogenate ancl plasma membrane after a 72-h period. The K,! constants were then converted to half-lives (tlj2) by the expression tlr2 = (ln 2)/K,,. In the case of the normal liver, the fraction (N(f)/N(O)) remaining after a 3-day degradation period is obtained by taking the ratio of “C and :{H radioactivities normalized for the injected doses and counting efficiencies N(t) -N(0)

total activity (dpm) in ‘-‘C at Day 3 per &i injected total activity (dpm) in :‘H ’ at Day 0 per &i injected

In the case of regenerating liver, one has also to take into account the increase in liver weight. However, the tlvo-thirds of the organ has been removed and the live1 homogenates were made from the same amount of tissues from each liver processed as discussed under Materials and Methods. Therefore, the remaining fraction is given by:

injected)

Note that this protocol is in fact eyuivalent to that used before with a single isotope (2, 3, 24).

RAT

01”

3 x (total dpm in :‘H activity per FCi injected) (total dpm in 14Cactivity

REGENERATING

x total weight of regenerating livel total weight of normal liver

for the degradation rate constant of a protein, the expression for the species 1 becomes: -K,,,

= [In $$$/f,

for the species 2 -K,,:

= /III %1/t,

and finally for the species ?I -K,,,, = [ln %1,/t.

392

MARCEAU.

DESCHGNES.

The K, expressions for all the fractionated species from plasma membranes of normal and regenerating livers are presented in Fig. 1. As discussed in Appendix A, the Kc1 values are obtained from the In ratios of 14C and “H radioactivities normalized for the injected doses, counting efficiency, and liver regrowth (regenerating liver). Previous reports (5, 8) have presented data on the relative degradation rates as the actual ‘“C::‘H or :3H:14C ratios without normalization, which means that the K,, expressions for the n fractionated species were equivalent to

K, = N(t)1 No,’ N(t), . K, = . . . K,, = No,, N(O), N(O),,

In those reported experiments, a relation was established between degradation rate and molecular weight of a fractionated by SDS-PAGE (5, 8, 9). In other words, N(t),

L N(O),

was compared to N(t),

N(O), 01” N(t),, No,,’

Such comparison is misleading and it becomes apparent that the exact relation is obtained by comparing instead the In radioactivity ratios. ACKNOWLEDGMENTS We thank Mr. D. Mailhot for his technical assistance. This work was supported by the Medical Research Council of Canada, MA-4967. REFERENCES 1. SWICK, R. W., AND IP, M. M. (1974)J. 249, 6836-6841.

Biol.

Chem.

AND

LANDRY

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