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Urea cycle enzymes in retina, ciliary body-iris, lens and senile cataracts
Eq.
Ey
RP~. (1984)
39, 483&K%
Urea Cycle Enzymes in Retina, Ciliary and Senile Cataracts GADIPARTHI
F:yr
Biochemistry
IU.KAO
Laboratorirs,
Medical
AND
Departmud
C’ollrge. Sew
(Received 2 January
EDWART)
Body-Iris, C'OTLIER
of Ophthdmoloyy.
Cornell
York, ,VY 10081.
1984 and accepted 4 April
Lens
1 TnioPrsity
(‘.S.d. 1984. Xew
York)
(‘arbamylation of lens proteins induces conformational changes and may play a role in the development of cataracts in uremic patients. Thus, the activities of the urea cycle enzymes: carbamyl phosphate synthetase I, ornithine transcarbamylase. argininosuccinate synthetsse, argininosuccinase and arginase. were determined in lens, retina and riliary body-iris of calf and rabbit. No ornithine transcarbamylaae activity was found in ciliary body-iris, lens and retina of calf and rabbit whereas carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase activities in calf lens were 502rtO.21. S%50+0.29. 9.17 +@lS and 632+0.19 &mol (g protein)-’ hr-‘1. respectively. Except arginase. the activities of carbamyl phosphate synthetase I, argininosuccinate synthetase and argininosuccinase in lens were 3&50 “/, of the values in retina or ciliary body-iris. The K,,, for each of the substrates was obtained for argininosuccinate synthetase. argininosuccinase and arginase of calf lens. Activities of carbamyl phosphate synthetase 1, argininosuccinate synthetase. argininosuccinase and arginase in clear human lenses, aged 67-87 years, were 0.11 + WOJ (0.67 + @Ol, @20 f 001 and 03Xf@03 (pmol lens-’ hr-’ ) , res spectively. Two-fold increase in the activity of arginase was found in senile cataracts, but all other enzymes had 38-87 “” decreases in a&vi&s. It is likely that the rise in arginase activity in cataracts could facilitate polyamine synthesis through ornithine and ornithine decarboxylase and additional formation of cyanate. a carbamylating compound, both of which have been implicated in cataract formation. Further. derreased activities of argininosuccinate synthetasr and argininosuwinase together with increased arginase activity could lead to the depletion of arginine in senile cataracts. Key words; arginase. argininosuwinate synthetase. argininosuc~cinase. urea cycle enzymes. retina. lens, ciliary hodyGris. calf. rahhit. human.
1. Introduction Although the Krebs-Hensleit urea cycle plays an import,ant role in the excretion of excessive ammonia from body tissues (Schimke, 1962; Nuzum and Snodgrass, 1971: Pierson and Brien, 1980; Stewart and Walser, 1980), little is known on the presence and functions of this pathway in lens despite its high protein content (Zigler and Goosey, 1981) and the high levels of such amino acids as ornithine and arginine (Barber, 1968). Previously, we have reported increased activities of arginase in senile cataractous lenses (Rao, Swamy, Siva Reddy and Sadasivudu. 1981). Furthermore, high polyamine levels have been found in senile cataracts (Kremzner, Roy and Spector, 1983) and Harding and Rixon (1980) f ound carbamylated lens proteins in cat’aracts and postulated that carbamylation may be a possible factor in cataractogenesis in the uremic condition. Act’ually potassium cyanate at 1 mM concentration induced opacity formation in rabbit lenses in culture (Kinoshita and Merola, 1973). Sodium cyanate intake induced cataracts in humans and dogs (Nicholson, Harkness. Benson and Peterson, 1976; Kern, Bellhorn and Peterson, 1977). In gyrate atrophy. an inborn error of metabolism due to the deficiency of ornithine S-aminotransferase
Address University
correspondence Medical College,
0014-4835/84/040483
to: Gadiparthi K. Rae, PhD.. Sew York, KY 10021. CT.S.A.
+ 13 $03.00/O
Department
@ 1984 Academic
of Ophthalmology,
Press Inc. (London)
Cornell
Limited
-Ml
activity. Ornithine further of the senile
t:. s subcapsular is derived understand urea cycle cataractous
cataract from
I<.\0
fi)rmation
arginasr.
.\SI)
II (‘o’I’I,II~:I:
has been the
last,
ol~rrvrd
enzymatic.
the role of the urea c*yc*le in the eye in the ocaular tissucss of calf ant1 rabbit
(Takki reaction
and
Sirnett.
in t’hts urea
we dett~rminrtl and in human
l!+ifi). (,y(ale.
‘1’0
t#hr enzymes normal ntltl
lenses.
2. Materials
and Methods
r,-Arginine hydrochloride. r,-aspartic acid. 1,.citrulline. r.-ornithine hydrochloride. ATP (disodium salt). N-acetylglutamate, oligomycin, carbamyl phosphate (dilithium salt). argininosuccinate (harium salt), arpinase. argininosuccinate lyase. omithine carhamyltransferase, glycinr, glycylglycine, imidazolr. Tris. a-isonitrosoprof)iophenone, diacetylmonoxime and dimethylglyoxime were purchased frotn Sigma (Ihemical Company. All other reagents used were of analytical grade. Rabbits with 2-3 kg body weight (New Zealand) were used throughout the study. Calf eyes were obtained from slaughterhouse. Normal human lenses were supplied by the Connecticut, Eye Bank & Visual Research Foundation, Inc., New Britain, Connecticut. The lens, ciliary hodg-iris and retina were dissected out from the eyeballs of rabbit and calf carefully. The capsule-epithelium, cortex and nucleus regions of the lens were separated and pooled. The dissected tissues were used either fresh or frozen. C’ataractous lenses were supplied by Yale-New Haven Hospital and categorized according to (licchrtti, Sharma and Cotlier (1982). The tissues were homogenized in cold. double distilled water using a Potter-Elvehjem homogenizer unless otherwise mentioned. Homogenization in cold. double distilled water did not cause any losses in urea cycle enzymf~ activities. l3nzym.e
assays
Carbamyl phosphate synthetasr I (il:.(‘. h’ .3. I. I/?). Carbamyl phosphate synthetase activity was determined as per the method of Stewart and Walser (1980). The assay mixture (1.0 ml. pH 7.2) consisting of 150~mol K(I. lO,~mol NH&I. 20,~mol KHCO,, 20,umol ornithine. 15 pmol MgCI,. 100 pmol Tris, 10 pmol N-acetylglutamate, 5 ,ug ml-’ of oligomycin (added as ethanolic solution), 5 ,amol ATP and excess of ornithine transcarbamylase was incubated with 0.2 ml of 3O%, (w/v) homogenate for 30 min at, 37°C’ in a waterbath. The reaction was terminated by the addition of 1.2 ml of 10”; (w/v) trichloroacetic acid. A zero time trichloroaceticacid added control was maintained always. In the absence of A-acetylglutamatr in the assay mixture. no carbamyl ph0sphat.e qnthetase I act,ivity was observed. After centrifugation, the clear supernatants were used for citrulline estimation. The citrulline was determined according to the procedure of Kulhanek and Vojtiskova (1964). To 0.25 ml of the supernatant. 0.25 ml of 1 ‘:$ (w/v) dimethylglyoxime in 96 9, (v/v) ethanol and 2.5 ml of acid mixture (155 ml of 96”& cont. H,SO,+ 25 ml of 80?, H,PO, + 320 ml of double distilled H,O + 8 g of antipyrine) were added and heated in a boiling waterbath at 100°C for 15 min. The tubes were cooled to room temperature and optical densities were measured at 447 nm on Gilford model 260 spectrophotomrter. Llsing citrulline standards, the enzyme act)ivity was calculated and expressed as micromoles of uitrulline formed per gram of protein per hour. Ornithine carbamyltransjerasr (E.f’. ;?, I, 3.3). Ornithinr carbamyltransferase activity was assayed as per the method described by Levin (1971). The assay mixture consisted of 02 ml of 60 mM ornithine in 60 mM glycylglycine huffer (pH 8.0) and 0.2 ml of lo’:,& (w/v) homogenate. The enzyme reaction was started with the addition of 61 ml of 190 mM freshly prepared carbamyl phosphate solution. After 30 min incubation at 37°C’ the enzyme activity was terminated by the addition of 10 o/C, (w/v) trichloroacetic acid. At the end of incubation in the control tube trichloroacetic acid was added, followed hy homogenate. The cent,rifuged supernatants were taken for citrulline analysis. The c:itrulline was determined as described ahove and the enzyme activity was expressed as micromoles of citrulline formed per gram of protein per hour using citrulline standards.
UREA
CY(!LE
ENZYMES
IS
OC’l’LAK
TISSPES
485
Argininomuxinate synthetase (E.C. 6.3.4.5) and argininosuccinose (E.C. 4.3.2. I). Argininosuccinate synthetase and argininosuccinase were estimated according to the procedure of Levin (1971). For argininosuccinate synthetase assa.y, the substrate mixture (0% ml), consisting of 10 mM L-aspartic acid, 10 mM L-citrulline, 10 mM ATP, 10 mM Mg SO,. 7H,O, 21 units of arginase and 1 unit of argininosuccinase in 50 my phosphate buffer h omogenate for 1 hr at 37°C. The enzyme (pH SO), was incubated with 05 ml of 20% (w/v) activit,y was terminated by the addition of 1.5 ml of 7 y;, (v/v) perchloric acid. A zero time perchloric acid added control was incubated simultaneously with the test. After centrifugation, the clear supernatants were taken for urea determination. Urea was estimated using a-isonitrosopropiophenone. To 0.5 ml of the supernatant. 1.5 ml of double distilled H,O. 1.5 ml of acid mixture (120 ml of 96”+, H&O,+360 ml of 80°, H,PO,+ 120 ml of double distilled H,O) and @I ml of 5O:, ( w r/ ‘L7) isonitrosopropiophenonr in 95 Y,, (v/v) ethanol were added and heated for 60 min at 100°C in a boiling waterbath. After cooling to room temperature, the optical density readings were read on a spectrophotometer at 540 nm. Since the reaction mixture contains citrulline, the urea standards were prepared with added citrulhne. The enzyme activity was expressed as micromoles of urea formed per gram of protein per hour. For argininosuccinase activity, the assay system consisting of 93 ml of 1.0 M phosphate buffer (pH 76), 04 ml of 6 mM argininosuccinate and 10.5 units of arginase was incubated with 0.4 ml of 20 90 (w/v) homogenate for 1 hr at 37°C. The enzyme activity was terminated with the addition of 1.2 ml of 10”;; trichloroacetic acid. Controls received trichloroacetic acid prior to incubation. After centrifugation. the supernatants were taken for urea estimation. Urea was determined by the procedure of Wybenga. UiGiorgio and Pileggi (1971). To 05 ml of the supernatant, 5.0 ml of urea nitrogen reagent and 95 ml of 2q/, (w/v) diacetylmonoxime were added and heated in boiling waterbath at 100°C for 12 min. After cooling to room temperature. the optical densities were measured on spertrophotometer at 540 nm. The enzyme activity was calculated using urea standards and expressed as micromoles of urea formed per gram of protein per hour. ilrgirLase (E.P. 3.5.3. I). Arginase activity was determined as per the method of Herzfeld and Raper (1976). The tissue was homogenized in a medium containing 56 mM imidazole and 56 mM M&l, (pH 7.4). dialyzed overnight against 2 1 of homogenizing buffer and incubated for 15 min at 52°C. Optimum arginase activity was obtained through activation step. The arginase activity was determined in dialyzed and heat-activated homogenates. Enzyme activity was carried out in a total of 03 mf of incubation mixture containing 100 ,umol of L-arginine, 60 pmol of glycine buffer (both adjusted to pH 9.5 with 1.0 M KaOH) and 0.1 ml of 20 “,b (w/v) activated homogenate. After incubation for 20 min at 37”C, the tea&ion was stopped by adding 1 ml of loo,, (w/v) trichloroacetic acid. Control samples receivedtrichloroacrticacidpriortoinc~ubation.Themixture~ascrntrifugedandsupernatants were used for urea estimation. I’rea was determined as described under argininosuccinase assay and the enzyme activity was expressed as micromoles of‘ urea formed per gram of prot,ein per hour. The enzyme activities were found linear to the duration of incubation time and protein eont)ent under the conditions of the assays employed. Protein content was determined bv the method of Lowry, Rosebrough, Parr and Randall (1951) using hovine serum albumin as standard. For K,,, value determinations, the enzyme activities were measured with different, concentrations of the substrate keeping other factors of the assay constant. Appropriate controls for the enzyme activities were used as described above. TAreweaver-Burke plots (1934) were made for Km value determinations and each value in these kinetics is the mean of minimum of three independent experiments. Statistical analysis for enzyme activities in clear and cataractous human lenses was made by Student,‘s t test,
3. Results The activities of the urea cycle enzymes in ocular tissues of calf and rabbit are given in Table I. It is evident from the results that the ocular tissues, i.e. ciliary body-iris, lens and retina. do not possess the intact urea cycle as ornithine transcarbamylasr
UREA
CYCLE
ENZYMES
IN
O(‘VLAK
TISSI’ES
487
activity was not present in these tissues. The activities of carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase in ocular tissues were found linear to protein content of the homogenate and duration of incubation time (data not shown). Dialysis (overnight in the cold room against 2 1 of homogenizing buffer) and heat activation (at 52°C’ for 15 min) of the lens homogenates yielded maximum arginase activity [3.65 and 720,~mol (g lens protein)-l hr-’ before and after dialysis, respectively, Table IV]. The optimum pH of the argininosuccinate synthetase. argininosuccinase and arginase in calf lenticular tissue were 8.5, 8.0 and 96, respectively. Argininosuccinate synthetase act’ivity exhibited broad pH optimum (pH 8+E+5). The K, values for the substrates of argininosuccinate synthetase, argininosuccinase and arginase activities in calf lens are shown in Table III and Figs 1-5. The K, values for L-aspartic acid, L-citrulline and ATE’ in argininosucrinate synthetase activity in calf lens are 1.0, 1.6 and 2.0 mM. respectively. The Km value for argininosuccinate in argininosuccinase activity in calf lens is 023 mM and for arginine in arginase activity is 18.5 mm. From the results (Table I), it is clear that the activities of carbamyl phosphate synthetase I, argininosuccinate synthetase and argininosuccinase in calf and rabbit lenses are only 3&50 9)” of the values in retina and ciliary body-iris of the respective species. In retina. maximum arginase activity [300%1 and 36274 pmol (g prot,ein)-’ hr-’ in calf and
0.6 t-
0.4o-2 -
II.5
(mt.4, L- asportote)
FIG. 1, Lineweavgr-Burke plot for L-aspartic acid in argininosuccinate The cosubstrate concentrations of L-citrulline, ATP and M&SO,. 7H,O pmol urea (g protein)-’ hr-‘: K,,,= 1.0 x 1OF Y.
I/S
(mtd,
synthetase assay from calf lens. are 6.15 mM each. V,,,= 8.33
t.- citrulllne)
FIG. 2. Lineweaver-Burke plot for L-citrulline in argininosuccinate synthetase The cosubstrate concentrations of r,-aspartate. ATP and MgSO,. 7H,O hr-‘; I&, = 1.6 x 10m3 ~1. ~‘rn,, = 1539 pmol urea (g protein)-’
assay from calf lens. are 815 rnM each.
-0.5
0
0.5
I.0 I/S
I.5
2.0
2.;
(mM, ATP)
FIG. 3. Lineweaver-Burke plot for ATP in argininosuccinate cosuhstrate concentrations of L-aspartic acid, L-citrulline hr-‘: R,=?O x lob3 M. ~‘rnax= 1818 pmol urea (g protein)-’
synthetase and MgSO,.
assay from calf lens. The 7H,O are 6.15 rnM each.
O-8
0.6 -
I/S
FIG. 4. Lineweaver-Burke plot V nmx = 548 pmol urea (g protein)-’
-0.06
-0.04
(mht,
L-argininosuccinate)
for L-argininosuccinate hr-’ ; R, = 227 x lo-’
-0.02
0
0.02
in arpininosurcinase M.
0.04
0.06
0.08
assay from
calf lens.
O-10
I /.S (mt.4, L-orginlne)
FIG. 5. Lineweaver-Burke plot for L-arginine (p protein)-’ hr-* ; K, = 18.52 x 10e3 M.
in arginase
assay
from
calf lens. l.,,,,
= 40 pmol
urea
rabbit, respectively], which is 3 and 50 times higher than the values of ciliary body-iris [78*82 and 83.37 pmol (g protein)-’ hr-’ in calf and rabbit, respectively], and lens [632 and 691 kmol (g protein)-’ hr-i in calf and rabbit respectively] respectively, was observed. The distribution of the activities of carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase in calf lens are shown in Table II. Among the capsule-epithelium, cortex and nucleus of calf lens, capsule-
1TREA
Activities
(‘Y(‘I,E
ENZYMES
of urea, cyck enzymes
IS
(‘arbamyl phosphate synthetase* Argininosuccinatr synthrtaset Argininosuccinaset Arginasri
489
‘I’IssI-ES
in differeat
EpitheliumClLpSUle
Enzyme
o(‘I’l,AK
regions
of clear
( ‘ortrx
calf
lens
suclcus
IX~59*4~1-2
(16)
0~7!)*0?27
(4)
14%%+%.74 llGJ+126 r0-52*2oti
(14) (14) (14)
H~ll)+%~l4 (4) -5.71 f 1.70 (4) @14fl44 (4)
oc?!~~o~Irl
(4)
6.12+ 1.16 (4) 4.80 f 0.85 (4) 340 & 0.37 (4)
* Activity is expressed as micromoles of citrulline formed per gram of protein per hour. of protein per hour. t Activity is expressed as micromoles of urea formed per mm ” Values are means of eight drterminations+s.s. Parentheses indicates numbers of lenses used. T.ABI,E
Apparent
Enzyme
Argininosuccinate synt,hetase
Km values (‘osubstratr concentrations L-Citrulline ATP MgSO, .7H,O L-Aspartatc ATP Mg80,. 7H,O L-Aspartatc L-Citrulline M&O,. 7H,O
of urea
III
cycle
enzymes
in clew
lens
Substrate
(m.M)
under
(@15) (615) (6.15) 1 (Q15) (6,15) (6.15) I (6,15) (6.15) (6.15) 1
K,
study
1.0 x lo-‘3 .M
I,-(‘itrullinr
1.6 x lo-”
ATP
“+I x lo-:’ M
I,-Argininosuccinate
Arginase
1:Argininr
TAALE
&rl value
r.-Aspartate
Argininosuccinasr
Arginase
calf
1,
2.27 x lo-1 M 1X.52x
1(1-i’ M
IV
acti,&ty before and ajter dialysis (‘alf lens
A rginase*
Heat-wtivated homogenate Dialyzed and heat-activated homogenate * Enzyme activity is expressed as micromoles Values are means of three determinations Parentheses indicate numbers of lenses used.
of urea formed
per pra.m of protein
per hour.
epithelium revealed maximum activities of carbamyl phosphate synthetase 1, argininosuccinate synthetase, argininosuccinase and arginase [1839, 14.22, 11.69 and 1052 pmol (g protein)-’ hr-‘, respectively]. Further, carbamyl phosphate synthetase I activity, which is purely mitochondrial, was found exclusively in the capsuleepithelium region of the lens where mitochondria is present. Carbamyl phosphate 17
EEH 39
* Activity t Activity Parentheses Values are
Activities
D (68 years) change
C (63 years) change
R (74 years) change
A (68 years) change
lens
phosph,atP
O-072 f 0.005 -3571
@112*Ow7
(5)
(4)
(6)
(5)
(5)
h,uman
o-347 * 0.049 -4813
O.669+0.013
(6)
(4)
(6)
(3)
(4)
lenses
synthetase.
Argininosuccinate synthetaset
Nrgilai,~L~Ru,ccinatP
(!arbamyl phosphate synthetase*
synfhetaar.
V
is expressed as micromoles of citrulline formed per average lens per hour. is expressed as micromoles of urea formed per average lens per hour. indicate numbers of lenses used (individual lenses are analyzed for enzymatic means f S.E. ; n.s. = st,at,ist,irally not significant : average ages of the patients
Type Percentage P Type Percentage P Type Percentage P Type Percentage P
(‘ataract
Clear human (72 years)
of ccrrbamyl
TABLE
activities) are given.
0.118+0~014 -42.16 < 0.05 0~083~0~018 -5931 < 0.01 0.063+0.01% -69.72 10.01 ON2 * 040 - 79.41
0204+ON7
Agrininosuccinasef
*rgirLinosurcitLusr
1 (6)
(6)
(6)
(6)
(5)
ant1
in dear
OT29+0’061 2634 n.s. 1.046*@166 81% tO901 0851+~117 47.49 < 0.05 0846+0.136 4862 < 0.05
+ 0.033
Arginaset
0-557
ctrgi,nase
(7)
(5)
(9)
(5)
(5)
trtd
cattrrtrcto?cs
VREA
CYCLE
EKZYMES
IN
OCYLAR
491
TTSSlTES
synthetase I activity in cortex and nucleus of the lens was found to be very low, as these parts of the lens lack mitochondria [@79 and 0.29 ymol (g protein)-’ hr-’ in cortex and nucleus of the calf lens, respectively]. The activities of carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase in human normal and different types of senile cataractous lenses are shown in Table V. The activities of carbamyl phosphate synthetase I, argininosuccinate synthetase and argininosuccinase were decreased significantly in all the four types of senile cataractous lenses (i.e. A to D grade, Table V) as compared to the values in clear age-matched, non-cataractous human lenses (obtained from the eye bank). On the other hand, a 2681 y/o increase in arginase activity was observed in senile cataracts as compared to the values in age-matched control (Table V). 4. Discussion The functional role of urea cycle enzymes in hepatic tissue is mainly attributed to the excretion of amino nitrogen (Schimke, 1962; Pierson and Brien, 1980). In extrahepatic tissues, such as lactating mammary gland and brain, only the latter half of urea cycle enzymes, namely argininosuccinate synthetase, argininosuccinase and arginase, are present. The physiological significance of the presence of these enzymes in mammary gland and brain is directly related to the production of proline and glutamate through ornithine aminotransferase activities (Morris and Knox, 1972; Buniatian and Davitian, 1966). Our study demonstrates the presence of carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase activities in the ciliary body-iris, lens and retina of calf and rabbit and in clear and cataractous lenses. However, the ornithine transcarbamylase activity, the second starting enzyme in urea cycle, was not found in any one of the ocular tissues studied, indicating that these tissues possess only an incomplete urea cycle. It may be mentioned that our observations are in full contradiction to the previous postulation that lens contains an intact urea cycle (Dardenne and Kirsten, 1962). Recently, Jernigan (1983), using [14C]-bicarbonate, could not find any radioactivity in the urea in cultured bovine and rat lenses which also stands contradictory to Dardenne and Kirsten’s postulation that lens contains complete urea cycle. However, ocular tissues, including lens, possess carbamyl phosphate synthetase I activity. The carbamyl phosphate synthetase I activity in ocular tissues was dependent on the presence of N-acet*ylglutamate in the assay system, and thus it differs from carbamyl phosphate synthetase II activity, which is involved in pyrimidine biosynthesis. The failure of Jernigan (1983) to find radioactivity in urea in bovine and rat lenses cultured in the presence of [14C]-bicarbonate could be due do the absence of ornithine transcarbamylase activity in the lens but not due to the lack of carbamyl phosphate synthetase I. argininosuccinate synthetase, argininosuccinase and arginase activities. The role of the presence of carbamyl phosphate synthetase I activity without ornithine transcarbamylase activity in ocular tissues is not clear and remains to be examined, The optimum pH of argininosuccinate synthetse. argininosuccinase and arginase in calf lens are 85, 8.0 and 9.6, respectively, and agree with optimum pH of 8.5 for argininosuccinate synthetase (Petrack and Ratner. 1958; Rochovansky and Ratner, 1967) 7.6 for argininosuccinase (Nuzum and Snodgrass, 1976), and 95 for arginase livers. The K, values for (Hirsch, Heine, Kolb and Greenberg, 1970) .rn mammalian L-aspartate, L-citrulline and ATP in argininosuccinate synthetase activity of calf lens are 1.0. 1.6 and 2.0 mM, respectively. The K, values for argininosuccinate in I i-2
49%
G. S. It,\0
ANJ)
E. (‘O’I’LIEJI
argininosuccinase activity of calf lens is 0.23 mM, which is twice the value of Ratner in bovine liver (1976), and is four times less than the value of Brown and Cohen in tadpole liver (1959). The h;, value for arginine in arginase activity of calf lens is 18.5 mM, which is three times higher than the value obtained in rat liver preparation (6.8 mM) and is the same as that obtained for rat kidney (Kaysen and Strecker, 1973). The K, value of 26 mM for arginine in lens arginase activity was reported by Jernigan (1983). Among the urea cycle enzymes, arginase activity was found to be more in all the tissues examined (Brown and Cohen, 1959,196O). The lens, however, showed the least activity of arginase as compared with argininosuccinate synthetase and argininosuccinase activities. In lens, heat activation alone did not yield maximum arginase act’ivity. However, dialysis of the lens homogenate followed by heat activation yielded a two-fold increase in arginase activity indicating the presence of a small molecular weight inhibitor in lens. Previously, an inhibitory effect of lysine on arginase activity has been shown by Cittadini, Pietropaolo, DeCristofaro and D’Ayjello-Caracciolo (1964). A considerable amount of lysine in the lens free amino acid pool was reported by Kinoshita, Barber, Merola and Tung (1969). Increased arginase activity in lens homogenates after dialysis may be explained in terms of the loss of lysine and of any low molecular weight arginase inhibitory substances from the lens. The presence of the latter half of the urea cycle enzymes together with low arginase activity in lens may be functionally significant in synthesizing arginine from citrulline and aspartic acid. The activities of carbamyl phosphate synthetase I, argininosuccinate synthetase and argininosuccinase were decreased significantly in senile cataracts as compared to the values of clear non-cataractous human lenses. On the other hand, a 26-81 y0 increase in arginase activity was observed in senile cataracts as compared to the values in age-matched control human lenses. The increase in arginase activity in senile cataracts cannot be explained only in terms of the loss of lens free lysine and of any other low molecular weight arginase inhibitory substances from cataracts, as the same does occur in clear lens homogenate during dialysis. It is also unlikely that the increased arginase activity in cataracts was due to the increase in arginase enzyme content, as protein synthesis decreases in cataracts (Maraini, Carta, Pescatori and Prosperi, 1971). The increase in arginase activity may produce more polyamines in cataracts through ornithine and ornithine decarboxylase activity. Though ornithine decarboxylase activity is yet to be tested, a two- to three-fold increase in the specific activities of histidine and 5-hydroxytryptophan decarboxylases in cataracts was reported by Ono, Hirano, Koizuka and Obara (1976). Further, Kremzner et al. (1983) reported high free polyamine contents in senile cataracts. They also reported increased levels of y-glutamylspermidine and y-glutamylspermine contents in senile cataracts. It was also shown that polyamines cause conformational changes in cellular proteins by covalent binding (Folk et al., 1980). It is likely that the increased arginase activity in senile cataracts may initiate changes in the conformation of lens proteins by producing more polyamines through ornithine and ornithine decarboxylase activity. Elevated spermidine content was reported in red blood cells in uremic condition (Swendseid, Panayua and Kopple, 1980). Carbamylation of lens proteins, particularly y-crystallin, was shown to be one of the possible factors in cataractogenesis (Harding, 1980; Harding and Rixon, 1980). Nicholson et al. (1970) has observed cataracts in humans treated with cyanate to prevent sickling. It was also shown that cyanate produces cataracts in beagles and
UREA
CYCLE
ENZYMES
IN OCULAR
TISSUES
493
the lens was more vulnerable to cyanate effects than other ocular tissues (Kern et al., 1977). Since urea in solution equilibrates with cyanate (Cerami and Manning, 1971; Hagel, Gerding, Fieggen and Bloemendal, 1971), it is reasonable to speculate that the increased activity of arginase in cataracts may facilitate more production of cyanate, which in its turn may carbamylate lens protein. Further, decreases in argininosuccinate synthetase and argininosuccinase activities together with increased arginase activity may cause depletion in arginine content in senile cataracts. The decrease in arginine content may lead to an altered protein synthesis in cataract. The production of arginine deficient cataracts in timber wolves has been clearly established (Vainisi, Edelhauser, Wolf, Cotlier and Resser, 1981). The presence of the latter half of the urea cycle enzymes, i.e. argininosuccinate synthetase, argininosuccinase and arginase, in retina is of considerable importance as in gyrate atrophy condition, an inborn error of metabolism due to deficiency of ornithine b-aminotransferase activity, the retina and choroid are known to degenerate. In retina the presence of argininosuccinate synthetase, argininosuccinase and arginase, together with high activity of ornithine b-aminotransferase (Rao and Cotlier, 1984), may be functionally significant in the production of either proline or glutamate. ACKNOWLEDGMENTS This work was supported by a research grant Bethesda, MD, Grant EY 02490-06.
from the National
Institute
of Health,
REFERENCES Barber, G. W. (1968). Free amino acids in senile cataractous Invest.
Ophthalmol.
lenses: possible osmotic etiology.
7, 564-83.
Brown, G. W. and Cohen, P. P. (1959). Comparative biochemistry of urea synthesis. 1. Methods for the quantitative assay of urea cycle enzymes in liver. J. Biol. Chem. 234. 1769-74. Brown, G. W. and Cohen, P. P. (1960). Comparative biochemistry of urea synthesis. 3. Activities of urea cycle enzymes in various higher and lower vertebrates. Biochem. 1.75, 82-91. Buniatian, H. Ch. and Davitian, M. A. (1966). Urea synthesis in brain. J. Neurochrm. 13. 743-53. Cerami, A. and Manning, J. M. (1971). Potassium cyanate as an inhibitor of the sickling of erythrocytes in vitro. Proc. Natl. Acad. Sci. U.S.A. 68, 11893. Cicchetti, D. V., Sharma, Y. and Cotlier, E. (1982). Assessment of oberserver variability in the classification of human cataracts. Yule J. Biol. Med. 55, 81-8. Cittadini, D., Pietropaolo, C., DeCristofaro, D. and D’Ayjello-Caracciolo, M. (1964). In vivo effect of L-lysine on rat liver arginase. Nature 203, 643-4. Dardenne, U. and Kirsten, G. (1962). Presence and metabolism of amino acids in young and old lenses. Exp. Eye Res. 1, 415-21. Folk, J. E., Park, M. H., Chung, S. I., Schrode, J., Lester, E. P. and Cooper, H. L. (1980). Polyamines as physiological substrates for transglutaminases. J. BioE. Chem. 225, 3695-700.
Hagel, P., Gerding, J. J. T., Fieggen, W. and Bloemendal, H. (1971). Cyanate formation in solutions of urea. 1. Calculation of cyanete concentrations at different temperature and pH. Biochim. Biophys. Acta. 243, 366-73. Harding, J. J. (1980). Possible causes of the unfolding of proteins in cataract and a new hypothesis to explain the high prevalence of cataract in some countries. In Aging of thr Lens (Eds Regnault, F., Hockwin, 0. andcourtois, Y.). Pp. 71-80. Elsevier, Amsterdam. Harding, J. J. and Rixon, K. C. (1980). Carbamylation of lens proteins: a possible factor in cataractogenesis in some tropical countries. Exp. Eye Res. 31, 567-71.
194
t:. N. RAO
I\?;11
E. (‘O’I’1,1Eli
Herzfeld, A. and Raper. S. M. (1976). The heterogeneity ofarginases in rat tissues, Hioche~r. .I. 153, 469-78. Hirsch, K. H.. Heinr. .J. I’.. Kolb, H. J. and Greenberg. 1). M. (1970). C’omparative physicalchemical studies of mammalian arginases. (‘amp. Hiochrm. Physiol. 37, F&k.%%. Jemigan, H. M.. .Jr (1983). I!rea, format,ion in rat,. bovine and human lens. fl:rp. E.yye Res. 37, 551-8. Kaysen, G. A. and Strecker. H. (1973). Purification and properties of arginase of rat kidney. Riochem. J. 133. 779-88. Kern, H. L.. Bellhorn, R. W. and Peterson. c’. M, (1977). Sodium cyanate induced ocular lesions in the beagle. J. Pharmacol. Exp. !Z’hw. 200, l&6. Kinoshita, CJ. H. and Merola. L. 0. (1973). C’yanatr effects on the lens in vitro. J~~~r>e,st. Ophthalmol. 12, .X4-7. Kinoshita, J. H., Barber, G. W., Merola, L. 0. and Tung. B. (1969). Clhanges in the levels of free amino acids and myo-inositol in the galactose-exposed lens. Invest. Ophthalmol. 8. 625-32. Kremzner, I,. T., Roy, D. and Spector. A. (1983). Polyamines in normal and cataractous human lenses: evidence for post-translational modification. Exp. Eye Res. 37. 649-59. Kulhanek, V. and Vojtiskova, V. (1964). On the determination of ornithine-carbamyl transferase activity. Clin. Chim. Acta 9, 95-6. Levin, B. (1971). Hereditary metabolic disorders of the urea cycle. In Advances in C%nicaZ ~‘hemistry, Vol. 14 (Eds Bodansky, 0. and Latnrr. A. I,.). Pp. 65-143. Academic Press. New York. Lineweaver, H. and Burke, D. (1934). The determination of enzyme dissociation constants. J. Am. Chem. Sot. 56, 65&66. Lowry. 0. H., Rosebrough. N. J., Farr. $. L. and Randal1.R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-75. Maraini. G.. Carta. F.. Pescatori, A. and Prosperi. L. (1971). Protein metabolism in human senile cataract. Exp. Eye Res. 11, 83-8. Morris, C. M. V. and Knox, W. E. (1972). Function of arginase in lactating mammary gland. Biochem. ,J. 127, 893-9. Nicholson, D. H., Harkness, D. R., Benson, W. E. and Peterson, C. M. (1976). Cyanateinduced cataracts in patients with sickle-cell hemoglobinopathies. Arch. OphthaZmoZ. 94, 927-30. Nuzum, C. T. and Snodgrass, P. J. (197 1). lirea cycle enzyme adaptation to dietary protein in primates. Science 172, 104223. Nuzum, C. T. and Snodgrass, P. ,J. (1976). Multiple assays of the five urea cycle enzymes in human liver homogenates. In The Urea Cycle (Eds Grisolia. S.. Baguena, R. and Mayor, F.). Pp. 32549. John Wiley & Sons, New York and London. Ono, S., Hirano, H., Koizuka, T. and Obara, Y. (1976). 5-Hydroxytryptophan and histidine decarboxylase activity in the senile cataractous lens. Ophthalmic Res. 8, 464-6. Petrack, B. and Ratner, S. (1958). Biosynthesis of urea. VII. Reversible formation of argininosuccinic acid. J. Riol. Chem. 233, 1494-500. carbamyl phosphate spnthetase I. ./. Hiol. Pierson, D. L. and Brien, ,J. M. (1980). H uman (‘hem. 255, 7891-5. Rao. G. N. and Gotlier, E. (1984). Ornithine S-aminotransferase activity in retina and other tissues. Neurochem. Res. 9, 555-62. Rao. G. N., Swamy, M.. Siva Reddy. P. and Sadasivudu, B. (1981). Studies on arginase and ornithine aminotransferase activities in human lens. IRCS Med. Sci. 9. 3956. Ratner, S. (1976). Enzymes of arginine and urea synthesis. In The I!rea Cycle (Eds Grisolia, S., Baguena, R. and Mayor F.). Pp. 181-219. John Wiley & Sons, New York and London. Rochovansky. 0. and Ratner. S. (1967). Biosynthesis of urea. XII. Further studies on argininosuccinate synthrtase: substrate affinity and mechanism of action. .I. Biol. Chem. 242. 3839-49. Schimke, R. T. (1962). Adaptive characteristics of urea cycle enzymes in the rat. .J. Niol. (‘hem. 237, 459-68. Stewart, P. M. and Walser. M. (1980). Short-term regulation of ureagenesis. J. KioE. (,‘hetn. 255. 5270-80.
UREA
CYCLE
ENZYMES
IK
O(‘I:LAR
TISSI’ES
495
Swendseid, M. E., Panaqua, M. and Kopple, J. D. (1980). Polyamine concentration in red cells and urine of patients with chronic renal failure. Ll(,f Sci. 26, 533-9. Takki, K. and Simell, D. (1976). Gyrate atrophy of the choroid and retina with hyprrornithinemia. Birth Defects Orig. Art. Ser. 12, 373-84, 401-8. Vainisi, S. J., Edelhauser, H. F., Wolf, E. D., Cotlier, E. and Reeser. F. (1981). Nutritional cataracts in timber wolves. J. Am. Vet. Med. Assoc. 179, 117580. Wybenga, D. R., DiGiorgio, J. and Pileggi, V. J. (19’71). Manual and automated methods for urea nitrogen measurement in whole serum. Clin. Chem. 17, 891-5. Zigler. J. S., Jr and Goosey. J. (1981). Aging of protein molecules: lens crystallins as a model svstrm. Trends Biol. Sci. 6. 133-6.
Ey
RP~. (1984)
39, 483&K%
Urea Cycle Enzymes in Retina, Ciliary and Senile Cataracts GADIPARTHI
F:yr
Biochemistry
IU.KAO
Laboratorirs,
Medical
AND
Departmud
C’ollrge. Sew
(Received 2 January
EDWART)
Body-Iris, C'OTLIER
of Ophthdmoloyy.
Cornell
York, ,VY 10081.
1984 and accepted 4 April
Lens
1 TnioPrsity
(‘.S.d. 1984. Xew
York)
(‘arbamylation of lens proteins induces conformational changes and may play a role in the development of cataracts in uremic patients. Thus, the activities of the urea cycle enzymes: carbamyl phosphate synthetase I, ornithine transcarbamylase. argininosuccinate synthetsse, argininosuccinase and arginase. were determined in lens, retina and riliary body-iris of calf and rabbit. No ornithine transcarbamylaae activity was found in ciliary body-iris, lens and retina of calf and rabbit whereas carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase activities in calf lens were 502rtO.21. S%50+0.29. 9.17 +@lS and 632+0.19 &mol (g protein)-’ hr-‘1. respectively. Except arginase. the activities of carbamyl phosphate synthetase I, argininosuccinate synthetase and argininosuccinase in lens were 3&50 “/, of the values in retina or ciliary body-iris. The K,,, for each of the substrates was obtained for argininosuccinate synthetase. argininosuccinase and arginase of calf lens. Activities of carbamyl phosphate synthetase 1, argininosuccinate synthetase. argininosuccinase and arginase in clear human lenses, aged 67-87 years, were 0.11 + WOJ (0.67 + @Ol, @20 f 001 and 03Xf@03 (pmol lens-’ hr-’ ) , res spectively. Two-fold increase in the activity of arginase was found in senile cataracts, but all other enzymes had 38-87 “” decreases in a&vi&s. It is likely that the rise in arginase activity in cataracts could facilitate polyamine synthesis through ornithine and ornithine decarboxylase and additional formation of cyanate. a carbamylating compound, both of which have been implicated in cataract formation. Further. derreased activities of argininosuccinate synthetasr and argininosuwinase together with increased arginase activity could lead to the depletion of arginine in senile cataracts. Key words; arginase. argininosuwinate synthetase. argininosuc~cinase. urea cycle enzymes. retina. lens, ciliary hodyGris. calf. rahhit. human.
1. Introduction Although the Krebs-Hensleit urea cycle plays an import,ant role in the excretion of excessive ammonia from body tissues (Schimke, 1962; Nuzum and Snodgrass, 1971: Pierson and Brien, 1980; Stewart and Walser, 1980), little is known on the presence and functions of this pathway in lens despite its high protein content (Zigler and Goosey, 1981) and the high levels of such amino acids as ornithine and arginine (Barber, 1968). Previously, we have reported increased activities of arginase in senile cataractous lenses (Rao, Swamy, Siva Reddy and Sadasivudu. 1981). Furthermore, high polyamine levels have been found in senile cataracts (Kremzner, Roy and Spector, 1983) and Harding and Rixon (1980) f ound carbamylated lens proteins in cat’aracts and postulated that carbamylation may be a possible factor in cataractogenesis in the uremic condition. Act’ually potassium cyanate at 1 mM concentration induced opacity formation in rabbit lenses in culture (Kinoshita and Merola, 1973). Sodium cyanate intake induced cataracts in humans and dogs (Nicholson, Harkness. Benson and Peterson, 1976; Kern, Bellhorn and Peterson, 1977). In gyrate atrophy. an inborn error of metabolism due to the deficiency of ornithine S-aminotransferase
Address University
correspondence Medical College,
0014-4835/84/040483
to: Gadiparthi K. Rae, PhD.. Sew York, KY 10021. CT.S.A.
+ 13 $03.00/O
Department
@ 1984 Academic
of Ophthalmology,
Press Inc. (London)
Cornell
Limited
-Ml
activity. Ornithine further of the senile
t:. s subcapsular is derived understand urea cycle cataractous
cataract from
I<.\0
fi)rmation
arginasr.
.\SI)
II (‘o’I’I,II~:I:
has been the
last,
ol~rrvrd
enzymatic.
the role of the urea c*yc*le in the eye in the ocaular tissucss of calf ant1 rabbit
(Takki reaction
and
Sirnett.
in t’hts urea
we dett~rminrtl and in human
l!+ifi). (,y(ale.
‘1’0
t#hr enzymes normal ntltl
lenses.
2. Materials
and Methods
r,-Arginine hydrochloride. r,-aspartic acid. 1,.citrulline. r.-ornithine hydrochloride. ATP (disodium salt). N-acetylglutamate, oligomycin, carbamyl phosphate (dilithium salt). argininosuccinate (harium salt), arpinase. argininosuccinate lyase. omithine carhamyltransferase, glycinr, glycylglycine, imidazolr. Tris. a-isonitrosoprof)iophenone, diacetylmonoxime and dimethylglyoxime were purchased frotn Sigma (Ihemical Company. All other reagents used were of analytical grade. Rabbits with 2-3 kg body weight (New Zealand) were used throughout the study. Calf eyes were obtained from slaughterhouse. Normal human lenses were supplied by the Connecticut, Eye Bank & Visual Research Foundation, Inc., New Britain, Connecticut. The lens, ciliary hodg-iris and retina were dissected out from the eyeballs of rabbit and calf carefully. The capsule-epithelium, cortex and nucleus regions of the lens were separated and pooled. The dissected tissues were used either fresh or frozen. C’ataractous lenses were supplied by Yale-New Haven Hospital and categorized according to (licchrtti, Sharma and Cotlier (1982). The tissues were homogenized in cold. double distilled water using a Potter-Elvehjem homogenizer unless otherwise mentioned. Homogenization in cold. double distilled water did not cause any losses in urea cycle enzymf~ activities. l3nzym.e
assays
Carbamyl phosphate synthetasr I (il:.(‘. h’ .3. I. I/?). Carbamyl phosphate synthetase activity was determined as per the method of Stewart and Walser (1980). The assay mixture (1.0 ml. pH 7.2) consisting of 150~mol K(I. lO,~mol NH&I. 20,~mol KHCO,, 20,umol ornithine. 15 pmol MgCI,. 100 pmol Tris, 10 pmol N-acetylglutamate, 5 ,ug ml-’ of oligomycin (added as ethanolic solution), 5 ,amol ATP and excess of ornithine transcarbamylase was incubated with 0.2 ml of 3O%, (w/v) homogenate for 30 min at, 37°C’ in a waterbath. The reaction was terminated by the addition of 1.2 ml of 10”; (w/v) trichloroacetic acid. A zero time trichloroaceticacid added control was maintained always. In the absence of A-acetylglutamatr in the assay mixture. no carbamyl ph0sphat.e qnthetase I act,ivity was observed. After centrifugation, the clear supernatants were used for citrulline estimation. The citrulline was determined according to the procedure of Kulhanek and Vojtiskova (1964). To 0.25 ml of the supernatant. 0.25 ml of 1 ‘:$ (w/v) dimethylglyoxime in 96 9, (v/v) ethanol and 2.5 ml of acid mixture (155 ml of 96”& cont. H,SO,+ 25 ml of 80?, H,PO, + 320 ml of double distilled H,O + 8 g of antipyrine) were added and heated in a boiling waterbath at 100°C for 15 min. The tubes were cooled to room temperature and optical densities were measured at 447 nm on Gilford model 260 spectrophotomrter. Llsing citrulline standards, the enzyme act)ivity was calculated and expressed as micromoles of uitrulline formed per gram of protein per hour. Ornithine carbamyltransjerasr (E.f’. ;?, I, 3.3). Ornithinr carbamyltransferase activity was assayed as per the method described by Levin (1971). The assay mixture consisted of 02 ml of 60 mM ornithine in 60 mM glycylglycine huffer (pH 8.0) and 0.2 ml of lo’:,& (w/v) homogenate. The enzyme reaction was started with the addition of 61 ml of 190 mM freshly prepared carbamyl phosphate solution. After 30 min incubation at 37°C’ the enzyme activity was terminated by the addition of 10 o/C, (w/v) trichloroacetic acid. At the end of incubation in the control tube trichloroacetic acid was added, followed hy homogenate. The cent,rifuged supernatants were taken for citrulline analysis. The c:itrulline was determined as described ahove and the enzyme activity was expressed as micromoles of citrulline formed per gram of protein per hour using citrulline standards.
UREA
CY(!LE
ENZYMES
IS
OC’l’LAK
TISSPES
485
Argininomuxinate synthetase (E.C. 6.3.4.5) and argininosuccinose (E.C. 4.3.2. I). Argininosuccinate synthetase and argininosuccinase were estimated according to the procedure of Levin (1971). For argininosuccinate synthetase assa.y, the substrate mixture (0% ml), consisting of 10 mM L-aspartic acid, 10 mM L-citrulline, 10 mM ATP, 10 mM Mg SO,. 7H,O, 21 units of arginase and 1 unit of argininosuccinase in 50 my phosphate buffer h omogenate for 1 hr at 37°C. The enzyme (pH SO), was incubated with 05 ml of 20% (w/v) activit,y was terminated by the addition of 1.5 ml of 7 y;, (v/v) perchloric acid. A zero time perchloric acid added control was incubated simultaneously with the test. After centrifugation, the clear supernatants were taken for urea determination. Urea was estimated using a-isonitrosopropiophenone. To 0.5 ml of the supernatant. 1.5 ml of double distilled H,O. 1.5 ml of acid mixture (120 ml of 96”+, H&O,+360 ml of 80°, H,PO,+ 120 ml of double distilled H,O) and @I ml of 5O:, ( w r/ ‘L7) isonitrosopropiophenonr in 95 Y,, (v/v) ethanol were added and heated for 60 min at 100°C in a boiling waterbath. After cooling to room temperature, the optical density readings were read on a spectrophotometer at 540 nm. Since the reaction mixture contains citrulline, the urea standards were prepared with added citrulhne. The enzyme activity was expressed as micromoles of urea formed per gram of protein per hour. For argininosuccinase activity, the assay system consisting of 93 ml of 1.0 M phosphate buffer (pH 76), 04 ml of 6 mM argininosuccinate and 10.5 units of arginase was incubated with 0.4 ml of 20 90 (w/v) homogenate for 1 hr at 37°C. The enzyme activity was terminated with the addition of 1.2 ml of 10”;; trichloroacetic acid. Controls received trichloroacetic acid prior to incubation. After centrifugation. the supernatants were taken for urea estimation. Urea was determined by the procedure of Wybenga. UiGiorgio and Pileggi (1971). To 05 ml of the supernatant, 5.0 ml of urea nitrogen reagent and 95 ml of 2q/, (w/v) diacetylmonoxime were added and heated in boiling waterbath at 100°C for 12 min. After cooling to room temperature. the optical densities were measured on spertrophotometer at 540 nm. The enzyme activity was calculated using urea standards and expressed as micromoles of urea formed per gram of protein per hour. ilrgirLase (E.P. 3.5.3. I). Arginase activity was determined as per the method of Herzfeld and Raper (1976). The tissue was homogenized in a medium containing 56 mM imidazole and 56 mM M&l, (pH 7.4). dialyzed overnight against 2 1 of homogenizing buffer and incubated for 15 min at 52°C. Optimum arginase activity was obtained through activation step. The arginase activity was determined in dialyzed and heat-activated homogenates. Enzyme activity was carried out in a total of 03 mf of incubation mixture containing 100 ,umol of L-arginine, 60 pmol of glycine buffer (both adjusted to pH 9.5 with 1.0 M KaOH) and 0.1 ml of 20 “,b (w/v) activated homogenate. After incubation for 20 min at 37”C, the tea&ion was stopped by adding 1 ml of loo,, (w/v) trichloroacetic acid. Control samples receivedtrichloroacrticacidpriortoinc~ubation.Themixture~ascrntrifugedandsupernatants were used for urea estimation. I’rea was determined as described under argininosuccinase assay and the enzyme activity was expressed as micromoles of‘ urea formed per gram of prot,ein per hour. The enzyme activities were found linear to the duration of incubation time and protein eont)ent under the conditions of the assays employed. Protein content was determined bv the method of Lowry, Rosebrough, Parr and Randall (1951) using hovine serum albumin as standard. For K,,, value determinations, the enzyme activities were measured with different, concentrations of the substrate keeping other factors of the assay constant. Appropriate controls for the enzyme activities were used as described above. TAreweaver-Burke plots (1934) were made for Km value determinations and each value in these kinetics is the mean of minimum of three independent experiments. Statistical analysis for enzyme activities in clear and cataractous human lenses was made by Student,‘s t test,
3. Results The activities of the urea cycle enzymes in ocular tissues of calf and rabbit are given in Table I. It is evident from the results that the ocular tissues, i.e. ciliary body-iris, lens and retina. do not possess the intact urea cycle as ornithine transcarbamylasr
UREA
CYCLE
ENZYMES
IN
O(‘VLAK
TISSI’ES
487
activity was not present in these tissues. The activities of carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase in ocular tissues were found linear to protein content of the homogenate and duration of incubation time (data not shown). Dialysis (overnight in the cold room against 2 1 of homogenizing buffer) and heat activation (at 52°C’ for 15 min) of the lens homogenates yielded maximum arginase activity [3.65 and 720,~mol (g lens protein)-l hr-’ before and after dialysis, respectively, Table IV]. The optimum pH of the argininosuccinate synthetase. argininosuccinase and arginase in calf lenticular tissue were 8.5, 8.0 and 96, respectively. Argininosuccinate synthetase act’ivity exhibited broad pH optimum (pH 8+E+5). The K, values for the substrates of argininosuccinate synthetase, argininosuccinase and arginase activities in calf lens are shown in Table III and Figs 1-5. The K, values for L-aspartic acid, L-citrulline and ATE’ in argininosucrinate synthetase activity in calf lens are 1.0, 1.6 and 2.0 mM. respectively. The Km value for argininosuccinate in argininosuccinase activity in calf lens is 023 mM and for arginine in arginase activity is 18.5 mm. From the results (Table I), it is clear that the activities of carbamyl phosphate synthetase I, argininosuccinate synthetase and argininosuccinase in calf and rabbit lenses are only 3&50 9)” of the values in retina and ciliary body-iris of the respective species. In retina. maximum arginase activity [300%1 and 36274 pmol (g prot,ein)-’ hr-’ in calf and
0.6 t-
0.4o-2 -
II.5
(mt.4, L- asportote)
FIG. 1, Lineweavgr-Burke plot for L-aspartic acid in argininosuccinate The cosubstrate concentrations of L-citrulline, ATP and M&SO,. 7H,O pmol urea (g protein)-’ hr-‘: K,,,= 1.0 x 1OF Y.
I/S
(mtd,
synthetase assay from calf lens. are 6.15 mM each. V,,,= 8.33
t.- citrulllne)
FIG. 2. Lineweaver-Burke plot for L-citrulline in argininosuccinate synthetase The cosubstrate concentrations of r,-aspartate. ATP and MgSO,. 7H,O hr-‘; I&, = 1.6 x 10m3 ~1. ~‘rn,, = 1539 pmol urea (g protein)-’
assay from calf lens. are 815 rnM each.
-0.5
0
0.5
I.0 I/S
I.5
2.0
2.;
(mM, ATP)
FIG. 3. Lineweaver-Burke plot for ATP in argininosuccinate cosuhstrate concentrations of L-aspartic acid, L-citrulline hr-‘: R,=?O x lob3 M. ~‘rnax= 1818 pmol urea (g protein)-’
synthetase and MgSO,.
assay from calf lens. The 7H,O are 6.15 rnM each.
O-8
0.6 -
I/S
FIG. 4. Lineweaver-Burke plot V nmx = 548 pmol urea (g protein)-’
-0.06
-0.04
(mht,
L-argininosuccinate)
for L-argininosuccinate hr-’ ; R, = 227 x lo-’
-0.02
0
0.02
in arpininosurcinase M.
0.04
0.06
0.08
assay from
calf lens.
O-10
I /.S (mt.4, L-orginlne)
FIG. 5. Lineweaver-Burke plot for L-arginine (p protein)-’ hr-* ; K, = 18.52 x 10e3 M.
in arginase
assay
from
calf lens. l.,,,,
= 40 pmol
urea
rabbit, respectively], which is 3 and 50 times higher than the values of ciliary body-iris [78*82 and 83.37 pmol (g protein)-’ hr-’ in calf and rabbit, respectively], and lens [632 and 691 kmol (g protein)-’ hr-i in calf and rabbit respectively] respectively, was observed. The distribution of the activities of carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase in calf lens are shown in Table II. Among the capsule-epithelium, cortex and nucleus of calf lens, capsule-
1TREA
Activities
(‘Y(‘I,E
ENZYMES
of urea, cyck enzymes
IS
(‘arbamyl phosphate synthetase* Argininosuccinatr synthrtaset Argininosuccinaset Arginasri
489
‘I’IssI-ES
in differeat
EpitheliumClLpSUle
Enzyme
o(‘I’l,AK
regions
of clear
( ‘ortrx
calf
lens
suclcus
IX~59*4~1-2
(16)
0~7!)*0?27
(4)
14%%+%.74 llGJ+126 r0-52*2oti
(14) (14) (14)
H~ll)+%~l4 (4) -5.71 f 1.70 (4) @14fl44 (4)
oc?!~~o~Irl
(4)
6.12+ 1.16 (4) 4.80 f 0.85 (4) 340 & 0.37 (4)
* Activity is expressed as micromoles of citrulline formed per gram of protein per hour. of protein per hour. t Activity is expressed as micromoles of urea formed per mm ” Values are means of eight drterminations+s.s. Parentheses indicates numbers of lenses used. T.ABI,E
Apparent
Enzyme
Argininosuccinate synt,hetase
Km values (‘osubstratr concentrations L-Citrulline ATP MgSO, .7H,O L-Aspartatc ATP Mg80,. 7H,O L-Aspartatc L-Citrulline M&O,. 7H,O
of urea
III
cycle
enzymes
in clew
lens
Substrate
(m.M)
under
(@15) (615) (6.15) 1 (Q15) (6,15) (6.15) I (6,15) (6.15) (6.15) 1
K,
study
1.0 x lo-‘3 .M
I,-(‘itrullinr
1.6 x lo-”
ATP
“+I x lo-:’ M
I,-Argininosuccinate
Arginase
1:Argininr
TAALE
&rl value
r.-Aspartate
Argininosuccinasr
Arginase
calf
1,
2.27 x lo-1 M 1X.52x
1(1-i’ M
IV
acti,&ty before and ajter dialysis (‘alf lens
A rginase*
Heat-wtivated homogenate Dialyzed and heat-activated homogenate * Enzyme activity is expressed as micromoles Values are means of three determinations Parentheses indicate numbers of lenses used.
of urea formed
per pra.m of protein
per hour.
epithelium revealed maximum activities of carbamyl phosphate synthetase 1, argininosuccinate synthetase, argininosuccinase and arginase [1839, 14.22, 11.69 and 1052 pmol (g protein)-’ hr-‘, respectively]. Further, carbamyl phosphate synthetase I activity, which is purely mitochondrial, was found exclusively in the capsuleepithelium region of the lens where mitochondria is present. Carbamyl phosphate 17
EEH 39
* Activity t Activity Parentheses Values are
Activities
D (68 years) change
C (63 years) change
R (74 years) change
A (68 years) change
lens
phosph,atP
O-072 f 0.005 -3571
@112*Ow7
(5)
(4)
(6)
(5)
(5)
h,uman
o-347 * 0.049 -4813
O.669+0.013
(6)
(4)
(6)
(3)
(4)
lenses
synthetase.
Argininosuccinate synthetaset
Nrgilai,~L~Ru,ccinatP
(!arbamyl phosphate synthetase*
synfhetaar.
V
is expressed as micromoles of citrulline formed per average lens per hour. is expressed as micromoles of urea formed per average lens per hour. indicate numbers of lenses used (individual lenses are analyzed for enzymatic means f S.E. ; n.s. = st,at,ist,irally not significant : average ages of the patients
Type Percentage P Type Percentage P Type Percentage P Type Percentage P
(‘ataract
Clear human (72 years)
of ccrrbamyl
TABLE
activities) are given.
0.118+0~014 -42.16 < 0.05 0~083~0~018 -5931 < 0.01 0.063+0.01% -69.72 10.01 ON2 * 040 - 79.41
0204+ON7
Agrininosuccinasef
*rgirLinosurcitLusr
1 (6)
(6)
(6)
(6)
(5)
ant1
in dear
OT29+0’061 2634 n.s. 1.046*@166 81% tO901 0851+~117 47.49 < 0.05 0846+0.136 4862 < 0.05
+ 0.033
Arginaset
0-557
ctrgi,nase
(7)
(5)
(9)
(5)
(5)
trtd
cattrrtrcto?cs
VREA
CYCLE
EKZYMES
IN
OCYLAR
491
TTSSlTES
synthetase I activity in cortex and nucleus of the lens was found to be very low, as these parts of the lens lack mitochondria [@79 and 0.29 ymol (g protein)-’ hr-’ in cortex and nucleus of the calf lens, respectively]. The activities of carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase in human normal and different types of senile cataractous lenses are shown in Table V. The activities of carbamyl phosphate synthetase I, argininosuccinate synthetase and argininosuccinase were decreased significantly in all the four types of senile cataractous lenses (i.e. A to D grade, Table V) as compared to the values in clear age-matched, non-cataractous human lenses (obtained from the eye bank). On the other hand, a 2681 y/o increase in arginase activity was observed in senile cataracts as compared to the values in age-matched control (Table V). 4. Discussion The functional role of urea cycle enzymes in hepatic tissue is mainly attributed to the excretion of amino nitrogen (Schimke, 1962; Pierson and Brien, 1980). In extrahepatic tissues, such as lactating mammary gland and brain, only the latter half of urea cycle enzymes, namely argininosuccinate synthetase, argininosuccinase and arginase, are present. The physiological significance of the presence of these enzymes in mammary gland and brain is directly related to the production of proline and glutamate through ornithine aminotransferase activities (Morris and Knox, 1972; Buniatian and Davitian, 1966). Our study demonstrates the presence of carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinase and arginase activities in the ciliary body-iris, lens and retina of calf and rabbit and in clear and cataractous lenses. However, the ornithine transcarbamylase activity, the second starting enzyme in urea cycle, was not found in any one of the ocular tissues studied, indicating that these tissues possess only an incomplete urea cycle. It may be mentioned that our observations are in full contradiction to the previous postulation that lens contains an intact urea cycle (Dardenne and Kirsten, 1962). Recently, Jernigan (1983), using [14C]-bicarbonate, could not find any radioactivity in the urea in cultured bovine and rat lenses which also stands contradictory to Dardenne and Kirsten’s postulation that lens contains complete urea cycle. However, ocular tissues, including lens, possess carbamyl phosphate synthetase I activity. The carbamyl phosphate synthetase I activity in ocular tissues was dependent on the presence of N-acet*ylglutamate in the assay system, and thus it differs from carbamyl phosphate synthetase II activity, which is involved in pyrimidine biosynthesis. The failure of Jernigan (1983) to find radioactivity in urea in bovine and rat lenses cultured in the presence of [14C]-bicarbonate could be due do the absence of ornithine transcarbamylase activity in the lens but not due to the lack of carbamyl phosphate synthetase I. argininosuccinate synthetase, argininosuccinase and arginase activities. The role of the presence of carbamyl phosphate synthetase I activity without ornithine transcarbamylase activity in ocular tissues is not clear and remains to be examined, The optimum pH of argininosuccinate synthetse. argininosuccinase and arginase in calf lens are 85, 8.0 and 9.6, respectively, and agree with optimum pH of 8.5 for argininosuccinate synthetase (Petrack and Ratner. 1958; Rochovansky and Ratner, 1967) 7.6 for argininosuccinase (Nuzum and Snodgrass, 1976), and 95 for arginase livers. The K, values for (Hirsch, Heine, Kolb and Greenberg, 1970) .rn mammalian L-aspartate, L-citrulline and ATP in argininosuccinate synthetase activity of calf lens are 1.0. 1.6 and 2.0 mM, respectively. The K, values for argininosuccinate in I i-2
49%
G. S. It,\0
ANJ)
E. (‘O’I’LIEJI
argininosuccinase activity of calf lens is 0.23 mM, which is twice the value of Ratner in bovine liver (1976), and is four times less than the value of Brown and Cohen in tadpole liver (1959). The h;, value for arginine in arginase activity of calf lens is 18.5 mM, which is three times higher than the value obtained in rat liver preparation (6.8 mM) and is the same as that obtained for rat kidney (Kaysen and Strecker, 1973). The K, value of 26 mM for arginine in lens arginase activity was reported by Jernigan (1983). Among the urea cycle enzymes, arginase activity was found to be more in all the tissues examined (Brown and Cohen, 1959,196O). The lens, however, showed the least activity of arginase as compared with argininosuccinate synthetase and argininosuccinase activities. In lens, heat activation alone did not yield maximum arginase act’ivity. However, dialysis of the lens homogenate followed by heat activation yielded a two-fold increase in arginase activity indicating the presence of a small molecular weight inhibitor in lens. Previously, an inhibitory effect of lysine on arginase activity has been shown by Cittadini, Pietropaolo, DeCristofaro and D’Ayjello-Caracciolo (1964). A considerable amount of lysine in the lens free amino acid pool was reported by Kinoshita, Barber, Merola and Tung (1969). Increased arginase activity in lens homogenates after dialysis may be explained in terms of the loss of lysine and of any low molecular weight arginase inhibitory substances from the lens. The presence of the latter half of the urea cycle enzymes together with low arginase activity in lens may be functionally significant in synthesizing arginine from citrulline and aspartic acid. The activities of carbamyl phosphate synthetase I, argininosuccinate synthetase and argininosuccinase were decreased significantly in senile cataracts as compared to the values of clear non-cataractous human lenses. On the other hand, a 26-81 y0 increase in arginase activity was observed in senile cataracts as compared to the values in age-matched control human lenses. The increase in arginase activity in senile cataracts cannot be explained only in terms of the loss of lens free lysine and of any other low molecular weight arginase inhibitory substances from cataracts, as the same does occur in clear lens homogenate during dialysis. It is also unlikely that the increased arginase activity in cataracts was due to the increase in arginase enzyme content, as protein synthesis decreases in cataracts (Maraini, Carta, Pescatori and Prosperi, 1971). The increase in arginase activity may produce more polyamines in cataracts through ornithine and ornithine decarboxylase activity. Though ornithine decarboxylase activity is yet to be tested, a two- to three-fold increase in the specific activities of histidine and 5-hydroxytryptophan decarboxylases in cataracts was reported by Ono, Hirano, Koizuka and Obara (1976). Further, Kremzner et al. (1983) reported high free polyamine contents in senile cataracts. They also reported increased levels of y-glutamylspermidine and y-glutamylspermine contents in senile cataracts. It was also shown that polyamines cause conformational changes in cellular proteins by covalent binding (Folk et al., 1980). It is likely that the increased arginase activity in senile cataracts may initiate changes in the conformation of lens proteins by producing more polyamines through ornithine and ornithine decarboxylase activity. Elevated spermidine content was reported in red blood cells in uremic condition (Swendseid, Panayua and Kopple, 1980). Carbamylation of lens proteins, particularly y-crystallin, was shown to be one of the possible factors in cataractogenesis (Harding, 1980; Harding and Rixon, 1980). Nicholson et al. (1970) has observed cataracts in humans treated with cyanate to prevent sickling. It was also shown that cyanate produces cataracts in beagles and
UREA
CYCLE
ENZYMES
IN OCULAR
TISSUES
493
the lens was more vulnerable to cyanate effects than other ocular tissues (Kern et al., 1977). Since urea in solution equilibrates with cyanate (Cerami and Manning, 1971; Hagel, Gerding, Fieggen and Bloemendal, 1971), it is reasonable to speculate that the increased activity of arginase in cataracts may facilitate more production of cyanate, which in its turn may carbamylate lens protein. Further, decreases in argininosuccinate synthetase and argininosuccinase activities together with increased arginase activity may cause depletion in arginine content in senile cataracts. The decrease in arginine content may lead to an altered protein synthesis in cataract. The production of arginine deficient cataracts in timber wolves has been clearly established (Vainisi, Edelhauser, Wolf, Cotlier and Resser, 1981). The presence of the latter half of the urea cycle enzymes, i.e. argininosuccinate synthetase, argininosuccinase and arginase, in retina is of considerable importance as in gyrate atrophy condition, an inborn error of metabolism due to deficiency of ornithine b-aminotransferase activity, the retina and choroid are known to degenerate. In retina the presence of argininosuccinate synthetase, argininosuccinase and arginase, together with high activity of ornithine b-aminotransferase (Rao and Cotlier, 1984), may be functionally significant in the production of either proline or glutamate. ACKNOWLEDGMENTS This work was supported by a research grant Bethesda, MD, Grant EY 02490-06.
from the National
Institute
of Health,
REFERENCES Barber, G. W. (1968). Free amino acids in senile cataractous Invest.
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Hagel, P., Gerding, J. J. T., Fieggen, W. and Bloemendal, H. (1971). Cyanate formation in solutions of urea. 1. Calculation of cyanete concentrations at different temperature and pH. Biochim. Biophys. Acta. 243, 366-73. Harding, J. J. (1980). Possible causes of the unfolding of proteins in cataract and a new hypothesis to explain the high prevalence of cataract in some countries. In Aging of thr Lens (Eds Regnault, F., Hockwin, 0. andcourtois, Y.). Pp. 71-80. Elsevier, Amsterdam. Harding, J. J. and Rixon, K. C. (1980). Carbamylation of lens proteins: a possible factor in cataractogenesis in some tropical countries. Exp. Eye Res. 31, 567-71.
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Herzfeld, A. and Raper. S. M. (1976). The heterogeneity ofarginases in rat tissues, Hioche~r. .I. 153, 469-78. Hirsch, K. H.. Heinr. .J. I’.. Kolb, H. J. and Greenberg. 1). M. (1970). C’omparative physicalchemical studies of mammalian arginases. (‘amp. Hiochrm. Physiol. 37, F&k.%%. Jemigan, H. M.. .Jr (1983). I!rea, format,ion in rat,. bovine and human lens. fl:rp. E.yye Res. 37, 551-8. Kaysen, G. A. and Strecker. H. (1973). Purification and properties of arginase of rat kidney. Riochem. J. 133. 779-88. Kern, H. L.. Bellhorn, R. W. and Peterson. c’. M, (1977). Sodium cyanate induced ocular lesions in the beagle. J. Pharmacol. Exp. !Z’hw. 200, l&6. Kinoshita, CJ. H. and Merola. L. 0. (1973). C’yanatr effects on the lens in vitro. J~~~r>e,st. Ophthalmol. 12, .X4-7. Kinoshita, J. H., Barber, G. W., Merola, L. 0. and Tung. B. (1969). Clhanges in the levels of free amino acids and myo-inositol in the galactose-exposed lens. Invest. Ophthalmol. 8. 625-32. Kremzner, I,. T., Roy, D. and Spector. A. (1983). Polyamines in normal and cataractous human lenses: evidence for post-translational modification. Exp. Eye Res. 37. 649-59. Kulhanek, V. and Vojtiskova, V. (1964). On the determination of ornithine-carbamyl transferase activity. Clin. Chim. Acta 9, 95-6. Levin, B. (1971). Hereditary metabolic disorders of the urea cycle. In Advances in C%nicaZ ~‘hemistry, Vol. 14 (Eds Bodansky, 0. and Latnrr. A. I,.). Pp. 65-143. Academic Press. New York. Lineweaver, H. and Burke, D. (1934). The determination of enzyme dissociation constants. J. Am. Chem. Sot. 56, 65&66. Lowry. 0. H., Rosebrough. N. J., Farr. $. L. and Randal1.R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-75. Maraini. G.. Carta. F.. Pescatori, A. and Prosperi. L. (1971). Protein metabolism in human senile cataract. Exp. Eye Res. 11, 83-8. Morris, C. M. V. and Knox, W. E. (1972). Function of arginase in lactating mammary gland. Biochem. ,J. 127, 893-9. Nicholson, D. H., Harkness, D. R., Benson, W. E. and Peterson, C. M. (1976). Cyanateinduced cataracts in patients with sickle-cell hemoglobinopathies. Arch. OphthaZmoZ. 94, 927-30. Nuzum, C. T. and Snodgrass, P. J. (197 1). lirea cycle enzyme adaptation to dietary protein in primates. Science 172, 104223. Nuzum, C. T. and Snodgrass, P. ,J. (1976). Multiple assays of the five urea cycle enzymes in human liver homogenates. In The Urea Cycle (Eds Grisolia. S.. Baguena, R. and Mayor, F.). Pp. 32549. John Wiley & Sons, New York and London. Ono, S., Hirano, H., Koizuka, T. and Obara, Y. (1976). 5-Hydroxytryptophan and histidine decarboxylase activity in the senile cataractous lens. Ophthalmic Res. 8, 464-6. Petrack, B. and Ratner, S. (1958). Biosynthesis of urea. VII. Reversible formation of argininosuccinic acid. J. Riol. Chem. 233, 1494-500. carbamyl phosphate spnthetase I. ./. Hiol. Pierson, D. L. and Brien, ,J. M. (1980). H uman (‘hem. 255, 7891-5. Rao. G. N. and Gotlier, E. (1984). Ornithine S-aminotransferase activity in retina and other tissues. Neurochem. Res. 9, 555-62. Rao. G. N., Swamy, M.. Siva Reddy. P. and Sadasivudu, B. (1981). Studies on arginase and ornithine aminotransferase activities in human lens. IRCS Med. Sci. 9. 3956. Ratner, S. (1976). Enzymes of arginine and urea synthesis. In The I!rea Cycle (Eds Grisolia, S., Baguena, R. and Mayor F.). Pp. 181-219. John Wiley & Sons, New York and London. Rochovansky. 0. and Ratner. S. (1967). Biosynthesis of urea. XII. Further studies on argininosuccinate synthrtase: substrate affinity and mechanism of action. .I. Biol. Chem. 242. 3839-49. Schimke, R. T. (1962). Adaptive characteristics of urea cycle enzymes in the rat. .J. Niol. (‘hem. 237, 459-68. Stewart, P. M. and Walser. M. (1980). Short-term regulation of ureagenesis. J. KioE. (,‘hetn. 255. 5270-80.
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ENZYMES
IK
O(‘I:LAR
TISSI’ES
495
Swendseid, M. E., Panaqua, M. and Kopple, J. D. (1980). Polyamine concentration in red cells and urine of patients with chronic renal failure. Ll(,f Sci. 26, 533-9. Takki, K. and Simell, D. (1976). Gyrate atrophy of the choroid and retina with hyprrornithinemia. Birth Defects Orig. Art. Ser. 12, 373-84, 401-8. Vainisi, S. J., Edelhauser, H. F., Wolf, E. D., Cotlier, E. and Reeser. F. (1981). Nutritional cataracts in timber wolves. J. Am. Vet. Med. Assoc. 179, 117580. Wybenga, D. R., DiGiorgio, J. and Pileggi, V. J. (19’71). Manual and automated methods for urea nitrogen measurement in whole serum. Clin. Chem. 17, 891-5. Zigler. J. S., Jr and Goosey. J. (1981). Aging of protein molecules: lens crystallins as a model svstrm. Trends Biol. Sci. 6. 133-6.