and beige (chediak-higashi) mice

Protein Degradation in Normal

and Beige (Chediak-Higashi) Mice

ROBERT T. LYONS and HENRY C. PITOT, Departments of Oncology and Pathology,McArdle Laboratory for Cancer Research, The Medical School,University of Wisconsin, Madison, Wisconsin 53706

A B S T R A C T The beige mouse, C57BL/6 (bg/bg),is ain aniimal model for the Chediak-Higashi syndromein main, a disease characterized morphologically bygiant lysosomes in most cell types. Half-lives for theturnover of [14C]bicarbonate-labeled total soluble liverprotein were determined in normal and beige mice.No significant differences were observed between theniormal and mutant strain for both rapidly and slowlyturniing-over classes of proteins. Glucagon treatmentdurinig the time-course of protein degradation hadsimiiilar effects on both normal and mutant strainsanid led to the conclusion that the rate of turnoverof endogenous intracellular protein in the beigemouse liver does not differ from normal.The rates of uptake and degradation of an exogenous

protein were determined in normal and beige mice byintravenously injecting 1251-bovine serum albumin andfollowinig, in peripheral blood, the loss with time ofphosphotungstic acid-insoluble bovine serum al-bumin and the parallel appearance of phospho-tungstic acid-soluble (degraded) material. No signif-icant differences were observed between beige andnormal mice in the uptake by liver lysosomes of 1251_bovine serum albumin (t,/2 = 3.9 and 2.8 h, respec-tively). However, it was found that lysosomes fromlivers of beige mice released phosphotungstic acid-soluble radioactivity at a rate significantly slowerthan normal (t,2 = 6.8 and 3.1 h, respectively). Thisdefect in beige mice could be corrected by chronicadmiiinistration of carbamyl choline (t,,2 = 3.5 h), acholinergic agonist which raises intracellular cyclicGMP levels. However, no significant differences be-

Parts of this work have been presented at the AnnualMeeting of the Federation of American Societies for Experi-mental Biology in San Francisco, Calif., June 1976, andhave been reported in abstract form. 1976. Fed. Proc. 35:1563. (1).

Dr. Lyons is a postdoctoral trainee of the National CancerInstitute (TO1 CA-5002).Received for publication 28 March 1977 and in revised

form 2 September 1977.

tween normal and beige mice were observed eitherin the ability of soluble extracts of liver and kidneyto bind [3H]cyclic GMP in vitro or in the basal levelsof cyclic AMP in both tissues. The relevance of theseobservations to the presurned biochemical defect un-derlying the Chediak-Higashi syndrome is discussed.

INTRODUCTIONThe beige mouse is an animal model for the humanChediak-Higashi (CH)' syndrome, a rare autosomalrecessively inherited disease. The disease is charac-terized by large granules (lysosomes) in most celltypes, partial albinism, increased susceptibility topyogenic infections, and several functional abnormal-ities of leukocytes including defective lysosomaldegranulation and impaired chemotaxis. Thus far, thedisease has been described in man, mink, cattle, andmice (2-7).Although the exact biochemical defect is not known,

recent reports have indicated that lysosomes mayaccumulate in the beige mouse because of defectiveexocytosis resulting either from decreased intracel-lular motility of lysosomes or from their improperfusion with the plasma membrane. Brandt et al. (8)have reported that beige mice treated with androgenhad significaritly higher levels of three hydrolyticlysosomal enzymes in kidney than the normal parentstrain (C57BL/6). Excretion into the urine is a majormechanism of removal of lysosomal enzymes fromnormal mouse kidney. Brandt et al. proposed thatthis process is impaired in beige mice because of anabnormal fusion of lysosomes with the plasma mem-brane of the kidney proximal tubule cells.

Oliver and co-workers (9, 10) studied the mobilityof concanavalin A receptor complexes on polymorpho-

I Abbreviations used in this paper: BSA, bovine serumalbumin; cAMP, cyclic AMP; cGMP, cyclic GMP; CH,Chediak-Higashi; PMN, polymorphonuclear leukocyte; PTA,phosphotungstic acid; S3, 105,000 g supernate of tissuehomogenate.

The Journal of Clinical Investigation Volume 61 February 1978 -260-268260

nuclear leukocyte (PMN) membranes as a test ofmicrotubule integrity. They reported that normalPMNs showed uniform distribution of membrane-bound concanavalin A. By contrast, concanavalin Awas aggregated into surface "caps" on both col-chicine-treated normal PMNs and untreated PMNsfrom either beige mice or human CH patients.The spontaneous capping response of PMNs of the

beige mouse in vitro was inhibited by cyclic (c)GMPand by cholinergic agonists such as carbamyl cholinethat raise intracellular cGMP levels. Oliver et al. alsoreported that these drugs and cGMP itself preventedthe formation of giant lysosomes in cultured embryonicfibroblasts froin beige mice (11).These responses can also be reproduced in vivo in

the beige mouse (9). Animals treated with low levelsof cholinergic agonists show normal granule mor-phology and a normal degree of concanavalin A-induced cap formation in peripheral blood PMN.These findings led to a hypothesis that defectivecGMP generation underlies all characteristic changesseen in the disease.Because ascorbic acid potentiates chemotaxis of

normal leukocytes, Boxer et al. (12) examined theeffects of ascorbate on PMNs from Chediak-Higashipatients. They reported that cAMP levels in PMNs ofsuch patients were an order of magnitude higherthan normal. Ascorbic acid treatment, both in vitroand in vivo, reduced cAMP levels and correctedfunctional defects in PMNs from patients with CHsyndrome. They hypothesized that cGMP, whichalso appeared to correct CH leukocyte function invitro, may act by antagonizing the effect of exces-sive cAMP. Thus cAMP may inhibit both micro-tubular assembly (13) and chemotaxis (14) whilecGMP potentiates these processes in normal leuko-cytes.The lysosome is widely believed to be involved in

the degradation of proteins of intracellular (15-18),serum (19, 20), and foreign (15, 21) origin. Becausethe beige mouse exhibits abnormally enlarged lyso-somes in most cell types, we have compared theturnover of endogenous and exogenous proteins innormal and beige mice. In addition, we have ex-amined both the levels of cAMP and the binding of[3H]cGMP in normal and beige mouse tissue ex-tracts. These assays were performed because the up-take of materials by lysosomes may involve cAMP andmicrotubule integrity (22, 23). Defective microtubuleformation in the beige mouse may in turn be theresult of abnormal cyclic nucleotide metabolism.

METHODS

Animals. Male inbred C57BL/6 mice were purchased fromSprague-Dawley (Madison, Wis.); male C57BL/6 (bg/bg) mice

were obtained from the Jackson Laboratory, Bar Harbor,Maine, and were bred at the McArdle Laboratory. Both strainswere utilized at 3-4 mo of age. The aniimals weremaintained on a 12-h light-12-h dark schedule, and werefed ad libitum on Purina chow (Ralston Purina Co.,St. Louis, Mo.).Turnover of endogenous liver proteins. Where indicated,

glucagon (200 ,ug/day per mouse) was administered sub-cutaneously as a 1:1 emulsion with trioctanioini (24). Allmice were injected with 100 ,uCi NaH'4CO3 (60( mCi/mmllol)and were killed by cervical dislocation at times indicated.Livers were homogenized in 4 ml of 0.25 M sucrose-0.05 NITris, pH 7.8-0.025 M KC1-0.005 M MgCl2 and werecentrifuged for 10 min at 10,000 rpm (Sorvall SS-34 rotor,DuPont Co., Instrument Products Div., Wilmington, Del.).The resulting supernates were then recentrifuged for 1 h at45,000 rpm (Beckman 50 Ti rotor, Beckman Instrumenits,Inc., Spinco Div., Palo Alto, Calif.). These final supernatanit(S3) fractions were stored frozen at -200C.

50-,lI aliquots of S3 were pipetted in duplicate on glassfiber discs (Whatman GF/C, 2.4 cm, Fisher Scientific Co.,Pittsburgh, Pa.), the proteins were precipitated in 10% tri-chloroacetic acid, and the discs were washed as describedlpreviously (24). Radioactivity was determined with toluene-2,5-diphenyloxazole on a Nuclear Chicago scinitillationcounter at about 25% efficiency.A second set of duplicate discs containing 50-,ul aliquots

of S3 was washed by the same procedure and was usedfor protein determinations. These washed discs were incu-bated overnight in scintillation vials containing 2 ml of 1 NNaOH to dissolve the protein. 100-,ul aliquots were re-moved for protein determination by the method of Lowryet al. (25).Uptake and degradation of 1251-BSA. All mice received

0.01% Nal (wt/vol) in their drinking water for at least 1 wkbefore use in these experiments to minimize uptake of iodideby the thyroid gland. Where indicated, some groups alsoreceived 0.02% (wt/vol) carbamyl choline for at least 1 wk intheir drinking water (9), which was changed daily. Animalswere lightly anesthetized with Nembutal and were in-jected via the tail vein with 50 ,ul of a solutioni con-taining 20 ,uCi 1251-bovine serum albumin (BSA) with-non-radioactive carrier BSA to a total of 100 ,ug protein. AHamilton microliter syringe was used when precise measure-ments of uptake were desired, as described by Davidsonet al. (26). At suitable intervals, duplicate 20O-/il bloodsamples were taken in heparinized disposable micropipetsby slicing 1 mm of tissue from the end of the tail. Bloodlsamples were immediately added to 1-ml aliquots of phos-photungstic acid (PTA) reagent (0.375% PTA in 0.5% HCI),vortexed and allowed to stand at least 15 min on ice. Thetubes were then centrifuged for 30 min at 2,800 rpm ina Sorvall GLC-1 centrifuge (DuPont Co., Instrument Prod-ucts Div.) located in a cold room. The precipitates werewashed twice in 1-ml aliquots of cold PTA reagent; tubescontaining these precipitates were placed in plastic tissueculture tubes (no. 3033, Falcon Plastics, Div. of BioQuest,Oxnard, Calif.). The three supernates (original plus twowashes) were combined in other culture tubes, and theradioactivity was determined with a gamma counter (Amer-sham/Searle Corp., Arlington Heights, Ill.) at 75% ef-ficiency for 1251.

In experiments designed to measure the organi distributioniof injected '251-BSA, animals were perfused through the heartwith cold physiological saline, as described by Davidsonet al. (26), until livers and kidneys had blanched free ofblood.

Cyclic GMP binding assay. The cGMP binding assay was

Protein Degradation in Normal and Beige (Chediak-Higashi) Mice 261

performed with a variation of the method of Lincoln et al.(27). Male mice were killed by cervical dislocation, andthe tissues were quickly excised and homogenized in 2 vol of25 mM Na phosphate, pH 7.4, containing 2 mM EDTAand 10 mM dithiothreitol. The homogenates were cen-trifuged at 10,000 rpm for 15 min (Sorvall SS-34 rotor), andthe resulting postmitochondrial supernate was centrifugedat 45,000 rpm for 90 min (Beckman 50 Ti rotor). Aliquotsof the resulting high-speed supernate (S3) were used forboth cGMP binding and protein determination by the biuretmethod (28).The binding assay was performed in a total volume of 0.12

ml and contained, in order of addition: 0.03 ml of 25 mM Naphosphate, pH 7.4; 2 mM EDTA; 0.02 ml of 2.5 mM 3-iso-butyl 1-methylxanthine; 0.02 ml of [3H]cGMP (0.1-5 pmol);and 0.05 ml S3. To estimate nonspecific binding of radio-activity, blank discs were assayed with aliquots of BSA(30 mg/ml) added in place of S3. Blank values were rou-tinely subtracted from the radioactivity values of the assaydiscs. 3-isobutyl 1-methylxanthine, an inhibitor of cGMPphosphodiesterase (29), was first dissolved in 20% ethanolwith phosphate-EDTA buffer (same as above) and then addedto volume. The assay mixture was incubated in 3-ml glasscentrifuge tubes for 60 min on ice. At the end of thistime period, 2 ml of cold phosphate-EDTA buffer was addedto each tube, and the contents were quickly filteredthrough Millipore filters (2.5-cm diameter, type HA, 0.45-,umpore size) with a Millipore sampling manifold (MilliporeCorp., Bedford, Mass., no. 2702550). The tubes and filterswere washed four times; twice with 2-ml aliquots ofphosphate-EDTA buffer and then twice with 2-ml aliquots of10 mM MgCl2. The filters were placed in scintillation vialsand then air dried at 90°C. After adding 5 ml of toluene-2,5-diphenyloxazole, bound radioactivity was determined in aNuclear-Chicago scintillation counter at about 25% efficiency.Glucagon was purchased from Eli Lilly and Co., Indianap-

olis, Ind.; trioctanoin from Eastman Chemical Products,Inc., Kingsport, Tenn.; phosphotungstic acid from Mal-linckrodt Chemical Works, St. Louis, Mo.; and 3-isobutyl-1-methylxanthine from Aldrich Chemical Co., Milwaukee,Wis. Carbamyl choline and Trizma base were products ofthe Sigma Chemical Co., St. Louis, Mo. 125I-BSA (6-60Ci/mmol) was obtained from New England Nuclear, Boston,Mass. Both NaH14CO3 (60 mCi/mmol) and [8-3H]cGMP(21 Ci/mmol) were purchased from Amersham/Searle Corp.

RESULTS

To determine whether or not the abnormally largelysosomes found in all tissues of the beige mouse areassociated with any aberrations in the overall rate ofintracellular protein degradation, half-lives for [14C]bi-carbonate-labeled soluble liver proteins were deter-mined. Mice were each injected with 100 ,Ci ofNaH14CO3 (30) and then killed at specified intervals.Radioactivity per milligram protein was determined inaliquots of liver cytosol as described under Methods.The results of a 137-h experiment designed to meas-

ure the mean half-life of a slowly turning-over classof liver proteins are shown in Fig. 1. A similar ex-periment that measures the mean half-life of morelabile liver proteins by following an 8-h time-courseis shown in Fig. 2. Table I summarizes the re-sults of a series of such protein degradation studies.

z

I-00.

1030D

0-

0

11/2 48.8 HOURS

0

20 40 60 80 100TIME (HOURS)

120 140

FIGURE 1 Turnover of total soluble liver protein in normalC57BL/6 mice. Mice were injected with 100l Ci NaH'4CO3and killed in groups of three at the times indicated. Pooledliver homogenates were centrifuged as described underMethods to obtain postmicrosomal supernatant fractions. Ali-quots of these supernatants were pipetted onto glass fiberdiscs, the proteins were precipitated with cold 10% tri-chloroacetic acid, and nonprotein-bound radioactivity wasremoved by sequential washings as described previously(24). Radioactivity per milligram protein on each disc wasdetermined as described under Methods.

No significant differences were observed betweennormal and beige mice in the turnover of eitherclass of soluble liver proteins. The effect of treat-ment of mice with glucagon was tested inasmuch asit has been reported that this hormone inducesautophagic vacuole formation and alters several physi-cal properties of lysosomes (31-33). A single intra-peritoneal injection of 100 ,g of glucagon per dayslowed the degradation of the more rapidly turning-over class of proteins somewhat, but had only aslight effect on the half-life of the more stable class(Table I). Again, no significant differences betweennormal and beige mice were observed.Because it was apparent that the degradation of

endogenous intracellular liver protein was not im-paired in beige mice, the ability of these animnalsto degrade exogenous protein was examined. 1251-BSA

262 R. T. Lyons and H. C. Pitot

z

H0a:CLL

<D0

0110.

103

t1/2 3.0 HOURS

2 4 6TIME (HOURS)

8

FIGURE 2 Turnover of total soluble liver protein in normalC57BL/6 mice. The protocol was exactly the same as in Fig. 1,except that groups of three mice were killed at 2-h intervalslip to only 8 h; thus more rapidly turniing-over proteinswere exatminie(l.

(100 ug proteini containinig 16 ,uCi) was injected intothe tail vein of' aniesthetized mice. Duplicate bloodsamples were takein fronm the tail at 15-nmim intervals,and the native proteins were precipitated inmmediatelywith cold PTA reagent and washed as describedunder Methods. The PTA-insoluble precipitate repre-sented inative 1251-BSA, while the PTA-soluble super-nate contaiined degraded fragments of this protein (26).A typical timle-course for the disappearance of native1251-BSA and the parallel appearance of degradedfragments in the peripheral blood is shown in Fig. 3.These processes were essentially linear for at least 4 hwhen plotted semilogarithmically.The removal of '25I-BSA from the blood is accom-

plished in vivo by pinocytosis and results in theformation of pinocytotic vacuoles in liver and kidneycells (34-36). These vacuoles fuse with primarylysosomes, resulting in the formation of secondarylysosomes. These bodies are the site of degradationof exogenous proteins (15, 21, 26, 37). The secondary

lysosomes may then migrate to and fuse with theplasmna memeibrane where the degraded fragments aredischarged into the extracellular compartment by exo-cvtosis. Tables II and III summarize the apparenthalf-lives for the processing of (1251)-BSA by thesemechanisms, as determined from the slopes of thelogarithmic plots.

It was observed (Table II) that untreated beigemice had a smiall but significant (P < 0.005) impair-ment in the rate of' uptake of 1251-BSA comparedwith normal. When mice were treated with 100 ,ug ofglucagon 1 h before 125I-BSA injection, there was asmall inlcrease in the half-life of circulating nativeBSA in both strains. This increase was significant(P < 0.0025) for niormal mice but not significant forthe mutant group. Wheni animals were treated withlow levels of the cholinergic agonist, carbamyl choline,in their drinkiing water, the rate of BSA uptake foreach strain was not significantly different from that ofuntreated controls.The half-life for the appearance of circulating PTA-

soluble fragments of BSA is shown in Table III. Thehalf-life for this process in beige mice was morethan twice that observed in normal mice. Glucagontreatment caused almost a fourfold increase in thishalf-life for normal mice with no change in thisparameter in beige mice. Carbamyl choline adminis-tration reversed this defect in the mutant mice inthat the release of PTA-soluble radioactivity underthese conditions occurred at a rate not significantlydifferent from normal.To determine which organ took up most of the in-

jected 1251-BSA, grouips of normal mice were killed at

TABLE ITurnover of Total Soluble Intracellular Proteini inl

Normal anld Beige Mlouse Liver

Half-life

Coniditioni Normal Beige

h

(A) UntreatedShort-term 3.0 3.7Long-terimi 48.8 46.0

(B) Glucagon-treatedShort-term 4.2 4.1Long-term 46.4 45.1

The protocol was the same as described under Figs. 1 and 2.Groups of mice receiving glucagon were injected once dailywith 200 Ag glucagon subcutaneously as a 1:1 emulsion intrioctanoin. The degradative rate constants (K) were deter-mined from the linear regression of ln (dpm/mg) against time,and half-lives were then calculated from the relationshipt1/2 = ln 2/K.


000Go-J

J.

a-0

UN DEGRADED1251 - BSA

PTA- SOLUBLE_ RADIOACTIVITY

*

I TIME (HOURS)FIGURE 3 Degradation of 1251-BSA in a normal C57BL/6mouse. 1251-BSA (100 ,ug protein containing 16 ,Ci) wasinjected into the tail vein. Blood samples were taken at inter-vals shown and were immediately precipitated in cold PTAreagent. Precipitates were isolated and washed by cen-trifugation as described under Methods. Radioactivity inPTA-inisoluble precipitates represents unldegraded nativeBSA; PTA-soluble radioactivity represents degraded frag-ments (26).

intervals after injection, and their livers and kidneyswere blanched in situ with physiological saline asdescribed in Methods. This perfusion removed mostof the radioactive blood from these organs and per-mitted an estimate of bound intracellular 125I-BSA.As shown in Fig. 4, 34% of the injected radio-activity remained in the extracellular (blood) com-partment after 5 min, with 18% and 6% in the liverand kidney, respectively. After 30 or 60 min, theliver still held over three times the radioactivityfound in the kidney. Thus the liver appears to be theprimary site of 125I-BSA degradation. When thisexperiment was repeated with beige mice, essentiallyidentical results were obtained (data not shown).

Oliver and Zurier (9) have reported that cGMPand two analogs of acetylcholine (carbamyl cholineand carbamyl-,8-methylcholine) which elevate intra-cellular cGMP levels (38) canl reverse the abnormal

TABLE IIUptake of 1251-Bovine Serum Albumin from Peripheral Blood

Half-life

Treatment Normal Beige

h

None (n = 7) 2.8±+0.3 3.9±+0.2Glucagon (n = 6) 4.4+±0.2 4.6+±0.4Carbamyl choline (n = 4) 2.6+0.2 3.5+±0.3

The protocol is described under Methods. Half-lives werecalculated as described under Table I. Values for each grouprepresent the mean±SEM of the number of animals shownin parentheses. The statistical significance of the differencesbetween group means was tested by using a one-tailedStudent's t test, and the results are reported in the text.

lysosomal morphology seen in PMNs from humanCH patients. In addition, we have just shown thatcarbamyl choline can reverse a functional defect inbeige mice. It therefore seemed possible that therecessive mutation which causes the CH syndromemay involve an alteration of cGMP binding proteins.To test this hypothesis, an assay for cGMP bindingproteins in mouse tissues was performed as outlinedunder Methods. The results shown in Table IVdemonstrate the specificity of the binding assay underthe conditions employed. Binding was assayed at pH7.4 because cGMP is reported to bind to type I cAMP-dependent protein kinase at pH 4 (39). We observedthat in the presence of a 20-fold molar excess ofcAMP over eGMP, 87% of [3H]cGMP bound to theproteins compared with the control without cAMP.Excess nonradioactive cGMP competed almost com-pletely with [3H]cGMP as expected. GMP caused someenhanced binding of [3H]cGMP, an observation similarto that reported by Lincoln et al. (27).

TABLE IIIRelease of PTA-Soluble 125I into Peripheral Blood

Half-life

Treatment Normal Beige

h

None (n = 5) 3.1±0.2 6.8±0.4Glucagon (n = 5) 11.8±1.1 6.7±0.5Carbamyl choline (n = 4) 3.4+±0.4 3.5±0.5

The protocol is described under Methods. Half-lives werecalculated as described under Table I. Values for each grouprepresent the mean±SEM of the number of animals shownin parentheses. The statistical significance of the differencesbetween group means was tested by using a one-tailedStudent's t test, and the results are reported in the text.

264 R. T. Lyonis autd H. C. Pitot

F . _BLOOD

> 30 r LIVERH ED KIDNEYS

o 26

2222

Hi 18-

0

(L

5 M NUTES 30MINUTES 60 M NUTES

TIME AFTER INJECTION

FIGURE 4 Distribuition of radioactivity after iintraveiiousiiijectioin of 125I-BSA into nornmal C57BL/6 imice. Mlice wereinjected with 8 ,uCi of 1251-BSA, killed at iDtervals showiland their organs were perfu-sed hi situ with cold physiologi-cal saline as described by Davidson et al. (26). Bloodsamples were taken with a 100-,ul capillary pipette from chestcavity bleeding immediately after cervical dislocation. Wholeorgans and blood samples were placed in plastic cultturettibes (Falcon Plastics), aind the radioactivity was countedin a gamma counter as described tinder Xlethods. Valuesfor each time point represent the mean-+SEM of threeanim--als.

A Scatchard plot for the binding of [3H]cGMP by7soluble proteiins from normal mouse liver cytosolis shown in Fig. 5. The apparent equilibrium dis-sociatioin coinstant (Kd) was determined from theslope, and the number of picomoles of [3H]cGMPbound per nmilligram protein was determined from the

14

° 12xI

0J 10

crL- 8 _

z 6

a-~~~~~~~Z

0

TmE AFEINETN

TABLE IVCompetition Assay for Binidinig of [3H]cGMP by

Mouse Tissue Extracts

Competitor added Molar excess [3H]cGMP bound

% of control

None 100

cAMP 20 87100 78500 471000 32

cGMP 20 11100 4500 210(0 2

GMP 20 128100 145500 158

1((0 163

Binding was measured as described underconcentration of [3H]cGMP used was 40 nM.

Methods. The

x intercept. Table V summarizes the results of a seriesof such Scatchard plots with soluble extracts fromliver, kidney, and lung of normal and beige mice. Boththe affinity of the binding sites and the number ofsites per milligram protein were essentially identicalfor normal as compared with beige mice in the threetissues surveyed. In agreement with Lincoln et al.(27), the lung extract contained the highest level ofcGMP binding protein(s).Boxer et al. (12) recently reported abnormally

2 3[3H]cGMP BOUND (pmo,x 102)/MG

FIGURE 5 Scatchard plot for [3H]cGMP binding to normal mouse liver cytosol proteins. Bindingwas determined as described under Methods.


TABLE VSummary of Scatchard Plots for [3H]cGMP Binding

by Mouse Tissue Extracts

[3H]cGMP boundTisstie Strain Kd per milligram protein

riM pmol

Liver Normal 2.28 0.045Beige 2.10 0.045

Kidney Normal 5.25 0.127Beige 5.38 0.134

Lung Normal 25.57 1.607Beige 24.33 1.555

[3H]cGMP binding data were determined as described underMethods, and Scatchard plots were drawn as shown in Fig. 5.The apparent equilibrium dissociation constants (Kd) weredetermined from the slopes, and the numbers of moles of[3H]cGMP bound per milligram protein were determinedfrom the x intercepts of these plots.

high levels of cAMP in PMNs from a human CHpatient. Both the high levels of cAMP and the func-tional defects in these cells were corrected by as-corbic acid therapy in vivo or in vitro. Inasmuch asmicrotubule assembly is reported to be inhibitedby cAMP and potentiated by cGMP (13), Boxer et al.hypothesized that the CH defect may be related to adefect in cAMP metabolism. To test whether or notthe beige mouse also had elevated levels of cAMP,this cyclic nucleotide was assayed in both liverand kidney of normal and beige mice. As shown inTable VI, cAMP levels in both organs were essen-tially identical for the two mouse strains.

DISCUSSION

The results of this investigation show that normal andbeige mice have essentially identical rates of degrada-

TABLE VICyclic AMP Levels of Normal and Beige

Mouse Liver and Kidney

Tissue Straini cAMP per gram tissue

pmol

Liver Normal 703±55Beige 707±+-52

Kidney Normal 743+43Beige 743+26

cAMP determinations were carried out with a cAMP radio-immunoassay kit (Schwartz/Mann Div., Becton, Dickinson &Co., Orangeburg, N. Y., no. 0750-13), following the recom-mended procedures. Each group represents the mean+SEMof three tissue samples, each assayed in duplicate.

tion of total soluble liver proteins (Table IA). Thissuggests that if lysosomes are involved in intracellularprotein degradation, they do not represent the rate-limiting step. Despite the presence of morphologicallyabnormal lysosomes in CH disease, there are contra-dictory reports concerning lysosonmal enzyme levels.Some researchers have found altered lysosomal en-zyme activity (40), but these reports have not beenconfirmed by other investigators (3, 41).Glucagon has been reported to induce autophagic

vacuole formation in rat liver (32, 42). The processinvolves transfer of enzyme from preexisting hydro-lase-containing components to newly formed auto-phagic vacuoles. Morphological changes and an in-crease in the osmotic sensitivity of lysosomes occurduring the first 50 min after glucagon injection (21, 32).Under the conditions in our studies (Table IB), gluca-gon treatment appeared to have no effect on theturnover of the more stable class of' soluble liverproteins and to prolong somewhat the mean half-lifeof the more rapidly turning over group. The lattereffect may be due to a hormonally-induced shift insynthesis to a slightly different class of proteins with adifferent mean half-life, or it may be an artefact dueto the enhanced fixation of [14C ]bicarbonate in thepresence of greater activity of pyruvate carboxylase.In either case, the coniclusion remainis that the turn-over of endogenous soluble liver proteins was unaf-fected by the beige mutation.

Padgett et al. (43) reported that 48 hr after normaland beige mice were injected intravenously withhorseradish peroxidase, the lysosomes of normal micehad digested almost all of the horseradish peroxi-dase, whereas large amounts were still present in CHmice at this time. Using ultrastructural cytochemistry,these authors did not find anly gross differencesbetween normal and beige animals in the pinocy-totic uptake of this foreign protein, but rather in thelysosomal processing. Consistent with these results,the present study concludes that the uptake of'anotherforeign protein, 1251-BSA, is slightly but significantlyreduced in beige mice (Table II), but that the releaseof PTA-soluble fragments is markedly impaired(Table III).The half-life for the degradation of '25I-BSA in

normal mice reported here is about 3 h. Gabathulerand Ryser (44) reported a half-life of 90 min for thedegradation of 131I-albumin by Sarcoma S180 cells inculture. These values are considerably greater than thehalf-lives reported by Davidson (45) for the degradationof 125I-ribonuclease by mouse kidney lysosomes: 14min in vivo and 11 min in vitro. These differencesmay possibly be related to the reported observationthat 55% of injected 1251-ribonuclease was absorbed bymouse kidney after 7 min (26), whereas in this presentstudy (Fig. 4) peak absorption of 125I-BSA did not

266 R. T. Lyons and H. C. Pitot

occur until about 30 min after injection. At this time,28% of the label was bound in liver and only 8% inkidney. The preferential uptake of BSA by liver andribonuclease by kidney may be related to the largemolecular weight difference between the two proteins(67,000 for BSA and 14,000 for ribonuclease).The nature of the presumed defect in cyclic nucleo-

tide metabolism in CH cells has not yet been eluci-dated. In addition, the precise role of cGMP inmicrotubule assembly is not understood. Inasmuch asmicrotubule assembly in vitro is promoted by reducedglutathione (46) and inhibited by calcium ions (47),cGMP may represent an intermediate in the reaction.Although we have found no significant differences intotal cGMP binding proteins of normal vs. beige mice(Table V), it is still possible that the cGMP-dependent protein kinase of beige mice has an alteredaffinity for cGMP. If this kinase were involved inmicrotubule assembly, this would explain the reversalof abnormalities in lysosomal morphology and functionobserved in vivo and in vitro when CH cells areexposed to agents which raise intracellular cGMPlevels. The cGMP-dependent protein kinases of nor-mal and beige mice are currently being compared inthis laboratory.In the studies described herein (Table VI), no

elevation of cAMP levels in beige mouse liver or kid-ney cells was seen. The report by Boxer et al. (12)of a 10-fold elevation of cAMP in human CH PMNscompared with normal may be a phenomenon char-acteristic of the human CH leukocytes only. Thehypothesis by Boxer et al. that excess cAMP inhibitsmicrotubule assembly in CH leukocytes is contrary tothe results of DiPasquale et al. (48), who report thatcAMP actually increases the ratio of polymerized tounpolymerized tubulin in cultivated Greene mela-noma cells. A complete description of the biochemi-cal defect underlying the CH syndrome may not beforthcoming until the mechanism of microtubule as-sembly is more fully understood.

ACKNOWLEDGMENT

This work was supported in part by grant CA 07175 fromthe National Cancer Institute.

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268 R. T. Lyon.s and H. C. Pitot

and beige (chediak-higashi) mice

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