Synthesis of experimentally induced glutamine synthetase (glutamotransferase activity) in embryonic chick retina in vitro

DEVELOPMENTAL

BIOLOGY,

Synthesis

8,

341-357

(1963)

of Experimentally

Induced

Synthetase (Glutamotransferase Embryonic Chick Retina DAVID

L.

KIRK

\J7ith the technical lkputiment

of Zoolog!y,

MD

Accepted

October

Activity) in

in

Vitro1

A. A. MOSCOPJA

assistance

University

Glutamine

of Nilda

of Chicago,

Saenz

Chicago,

Illinois

9, 1963

INTRODUCTIOiX

Detailed studies on mechanisms controlling differentiation in embryonic cells and tissues require, in our opinion, experimental systems in which the appearance and activity of characteristic enzyme patterns can be manipulated and effectively modified. A particularly desirable situation would be one in which it would be possible to cause a precocious appearance in a tissue of an enzyme system that is normally associated with the onset of functional differentiation at a later stage of development. It has been recently found (Moscona and Hubby, 1963) that when the neural retina of the early chick embryo is isolated and cultivated in vitro there is a precocious appearance and a very rapid increase of glutamotransferase activity in this tissue. The data suggested that this striking increase in enzyme activity, days in advance of normal ontogeny, was not a nonspecific response to tissue transplantation, but that it represented a modification or acceleration of an aspect of the developmental pattern typical to the retina. The possibilities of a precocious induction, derepression, or stimulation of the enzyme-forming system at the level of genomic or cytoplasmic controls were raised, but the information available did not suffice for further consideration of the mechanisms that might be involved. Since this experimental system appeared highly suitable (both as a specific case and possibly as a model of more general significance) for detailed 1 Supported by grants from the National Science Foundation (C-23852), National Cancer Institute (C-4272)) and the Dr. Wallace C. and Clara A. Abbott Fund of the University of Chicago. 34 1

342

KIRK

AND

MOSCONA

investigation of mechanisms involved in controlling tissue-specific enzymatic patterns, it has been further studied with particular reference to: (1) functional identity of the enzyme, (2) whether the experimentally induced increase in its activity represented new synthesis or an activation of preexisting enzyme, independent of biosynthetic processes, and (3) factors affecting level of enzyme activity in cultured tissue. It will be noted that the enzyme assay method outlined here is a modification of previous ones; detailed discussion of these modifications will be made elsewhere together with a report of some further improvements which have subsequently been made (Kirk, 1963). Some of the advantages of chick neural retina for studies on the molecular aspects of differentiation were listed previously (Moscona and Hubby, 1963). It can be isolated readily and cleanly in relatively large quantities from embryos of different ages and lends itself well to studies in vitro at both tissue and cellular levels. In the embryo, active cell proliferation in this tissue is greatly reduced past the tenth day of incubation (Coulombre, 1961)) and thus the phenomena that accompany further growth and differentiation are not as complicated by extensive cell replication as in some other embryonic systems. In addition to its homogeneous developmental origin, the retina appears also to be relatively homogeneous with respect to presence of glutamotransferase in its different layers (Rudnick, 1963); thus for purposes of studying this enzymatic activity it can be treated, tentatively, as a uniform cell population. A more satisfactory definition of the actual functional identity of the retinal enzyme detectable by its glutamotransferase activity was sought, and the evidence suggests that it is a glutamine synthetase. Although the precise metabolic role of retinal glutamotransferase (or glutamine synthetase) is uncertain, its relevance to retinal function can be inferred from the following lines of information. The in V~UO and accumulation of retinal glutamotransferase in the appearance last few days of embryonic life is temporally correlated with functional maturation of the retina as indicated by appearance of both visual pigments and the electroretinogram (Rudnick and Waelsch, 1955; Wald and Zussman, 1938). Secondly, neural tissues in general are characterized by high levels of glutamine synthetase (Meister, 1962; Wu, 1963) and by highly active metabolic pools of glutamate, glutamine, and associated metabolites (Garfinkel, 1962); neural retina is

SYXTHESIS

Ok

EXPERIMEST.-lLLY

INDUCED

GLUTAMINE

SYSTHETASE

343

no exception to this pattern (Pirie, 1956). Last, it appears that the retina depends upon synthesis of gl~~tamine for maintenance of electrolyte balance, a phenomenon as yet incompletely explained (Pirie, 1956). All these facts contributed to our interest in this system and stimulated a series of improvements in both the culture and assay procedures previously used (Moscona and Hubby, 1963) resulting in increased precision of the system and, thus, increased usefulness as a model for studying this sort of enzymatic differentiation. The previous report (Moscona and Hubby, 1963) discussed only the appearance of enzymatic ~~~~~~~ and offered no information concerning synthesis of enzyme molecules. This communication deals more directly with this problem. Since no quantitative technique for isolation of this enzyme yet exists (Meister, 1962; Pamiljans et al., 1962), direct evidence of c/c rzo2;o synthesis was not feasible and less direct methods were used: a systematic search for enzyme activators or inhibitors at various developmental stages and a determination of the sensitivity of the appearance of activity in vitro to the inhibitor of protein synthesis, puromycin. Furthermore, the recent demonstration (Davidson ct al., 1963) that continued synthesis of a product characteristic of a differentiated cell line was dependent upon an actinomycin D-sensitive process (presumably DNA-primed synthesis of messenger RNA) raised the question to what extent appearance and maintenance of retinal glutamotransferase activity was under control of an actinomycin-sensitive mechanism, Finally, investigation was also made of the influence of the substrate and end product of the enzyme (i.e., glutamate and glLitamine), glucose, other ocufar tissues and extracts of young embryos upon the in vitro appearance of glutamotransferase activity. hlATEHIAL

AND

METHODS

Retinul Cultu7cs Organ cultures of embryonic chick neural retina were established as previously described (Moscona and Hubby, 1963). Two retinas, each cut in two pieces, were suspended in 24 ml of c&Ire medium in a 125-ml Erlertmeyer f&k; the flasks were then gassed with a mixture of 5% CO, in air, sealed and placed on a gyratory shaker rotating at 85 rpm (diameter of rotation gi inch) at 38°C for 12, 24, or 48 hours. Random variability was minimized by distributing pieces of

344

KIRK

AND

MOSCONA

tissue from each pair of retinas through control and experimental flasks. Thus, while each culture contained the equivalent of two retinas, in no case was all the tissue in one culture flask derived from a single embryo. Unless otherwise mentioned all studies were performed on retinas from embryos of 10 days’ incubation. A minimal maintenance medium was routinely used; it consisted of 100 parts Tyrode’s solution, 10 parts horse serum, and 1 part of a penicillinstreptomycin mixture (Microbiological Associates). Additions to the medium were made in sterile, neutral, Tyrode’s-based solutions at the expense of the basal Tyrode’s solution. Whenever experimental protocol called for a change of medium, part of the control cultures were simultaneously changed to fresh medium. A thorough investigation of the effect of light upon retinal cultures indicated a slightly higher rate of enzymatic growth in the dark, so that in the routine procedure retinas were isolated under normal illumination but cultured in a darkened incubation room. At the conclusion of the culture period, tissues were sampled for routine histological examination, harvested, washed quickly three times in cold Tyrode’s solution by decantation, collected by mild centrifugation, and lyophilized immediately in the plastic centrifuge tubes. Just prior to analysis, tissues were suspended in 2.5 ml of cold phosphate buffer (0.01 M, pH 7.1), packed in ice, and submitted to about three 5-second bursts of ultrasound (20 Kcps) from the probe of a Branson model 75 sonifier tuned to maximum output. Such ultrasonic treatment yielded a lightly opalescent, homogeneous suspension and released significantly more activity than Potter-Elvejhem glass grinding.

Glutamotransferase was determined by the following modification (Kirk, 1963) of standard procedures (Moscona and Hubby, 1963; Rudnick and Waelsch, 1955). To 0.35 ml of cold sonicate (diluted with phosphate buffer when necessary) was added 0.50 ml of a fresh pH 5.4 solution containing: L-glutamine, 120 pmoles; acetate buffer, 50 pmoles; NaH,PO,, 5 pmoles; ATP, 0.05 pmole. This mixture was preincubated 10 minutes at 38”C, whereupon 0.15 ml of a solution (pH 5.4) containing 30 poles hydroxylamine and 5 pmoles MnCl, was added to start the reaction. Each assay was run in tripiicate, one tube receiving all but the hydroxylamine and serving as the

SYNTHESIS

OF

EXPERIMENTALLY

INDUCED

GLUTAMLNE

SYSTHETASE

345

blank. The reaction was stopped with the standard ferric chloride reagent (Moscona and Hubby, 1963) after an incubation time estimated to produce approximately 0.3-0.6 ,.mole of product. Absorbance was determined on a Zeiss Ph4QII spectrophotometer at 500 rnp, and related to a succinohydroxamate standard and a biologic standard (see Kirk, 1963). Glutamine synthetase was determined by the method of Levintow et nl. ( 1955). Protein was determined by the method of Lowry et aE. ( 1951)) and specific activity was defined as micromoles of glutamohydroxamate formed per hour per milligram of protein. Materials

Puromycin, adenine nucleotides, and all amino acids employed (except hydroxylysine and methionine sulfoximine) were obtained from Nutritional Riochemicals Corporation. nL-nlEa-S-hydroxylysine was obtained from Sigma Chemical Company. nL-methionine-&-sulfoximine was obtained from California Biochemical Corporation. Actinomycin D was generously supplied by Merck, Sharp, & Dohmcx Inc. RESULTS

At least two discrete enzymes with glutamotransferase activity are known: glutamine synthetase and glutaminase ( Meister, 1962). The enzyme most studied in neural tissue is glutamine synthetase. Among its characteristic properties are an absolute divalent cation requirement (Mn++ effective at lower levels than Mg+‘), an inverse shift in pH optim~im with Mn’+ ~o~~~entratiol~ (for the transferase reaction at least), a ratio of transferase to synthetase between 2 and 15 depending on tissue source and preparative methods (cf. hleister, 1962; Pamiljans et al., 1962), a noncompetitive inhibition by nL-allo-6-hydroxylysine (WI, 1963), and methionine-insensitive inhibition by methioninc sulfoximine (Tower, 1960). Using these properties, preparations of retinal tissue from 20-day embryos and/or retinal cultures were tested to determine whether embryonic retinal glutamotransferase activity is due to a glutamine synthetase (Table 1) . It was found that in all cases there was indeed an absolute requirement for a divalent cation: in the absence of added Mn++ ion the transferase activity was less than 1%that obtained

346

KIRK

AND

MOSCONA

TABLE PROPERTIES

OF Assay

EMBRYONIC

RETINAL

conditions

Standard (see text) Decreased Mn++ 2.5 fimoles 1 pmole 0 Hydroxylysine added 5 ltmoles 50 #moles Methionine sulfoximine added 2.5 wmoles 5 rmoles 5 pmoles plus 10 pmoles methionine Synthetase (by the method of Levintow et al., 1955)

1

GLUTAMOTRANSFERASE Relative

(20-DAY activity

EMBRYOS) pII Optimum

100

5.4

92 87
6.3 6.8 ?

39 I1

-

31

-

18

-

13 20-35

-

in the presence of 5 pmoles/ml. Furthermore, as the Mn++ level was lowered from 5 to 2.5 to 1 pmole/ml, the pH optimum of the transferase reaction catalyzed by embryonic retina shifted from 5.4 to 6.3 to 6.8. Preliminary assays of synthetase activity of crude sonicates yielded transferase: synthetase ratios between 3: 1 and 5: 1. DL-allo~-Hydroxylysine added to the assay mixture at the level of 5 pmoles/ml inhibited the transferase activity of 20-day retina by 61%; at 50 pmoles the degree of inhibition was about 90%. n~“methio~line-~z-sL~lfoximine also was a definite inhibitor of the reaction: 2.5 pmoles/ml gave 70% inhibition, 5.0 pmoles/ml resulted in 80%inhibition. The effect of the sulfoximine could not be reversed by metl~ionine. It is particularly important that by no test yet performed has it been possible to differentiate (qualitatively) between the properties of the enzyme which develops in the retina of late embryos and that which can be caused experimentally to develop several days precociously in the retina in culture. On the basis of these data and in the absence of any data to the contrary, it must be assumed (1) that the enzyme being investigated here is a glutamine synthetase; (2) that it is identical in retina in the embryo and in culture; (3) that its precocious appearance under the experimental conditions described represents, indeed, an acceleration of a developmental trait characteristic of retinal differentiation.

SYNTHESlS

OF

EXP’ERIMENTALLY

INDUCED

GLUTAMINE

347

SYNTHETASE

Evidence for Synthesis of Enzyme 1. Abseme of detectable inhibitors OT activators. As noted above, the previous report discussed only changes in specific activity of the retina and made no attempt to answer the question of de novo synthesis versus activation of preexisting molecules or removal of a specific enzyme inhibitor (Moscona and Hubby, 1963). According to preliminary data (Hubby, Moscona, and Saenz, unpublished) the activities of retinas from early and late embryos were additive, suggesting the absence of any change in concentration of either a competitive inhibitor or a hitherto-unidentified activator. Such mixed assays have been repeated and some representative data are given in Table 2. The precisely additive nature of these results indicates the TABLE I'RODUCTION OF (:LUT.~MOH~.DR~X.\M.\TE REwN 1s OF I~FFERENT .1a~3s, .kX.\YED

2 (GHA)

FROM SEP.~R.\TEl>Y

i 10

0. :35 0 .35

1.7 1.1

0.061 0.280

18 i 18

0.20 0.25 0, 10

0.276

10

0 ,25

18

0. 10

0. 10 I .2 0.08 0.X) 0.08 t

GLUT.\MINE BY ANI) COMBINEU

-

0.181

0.18”

0. :M)

0.338

absence of detectable change in concentration of any activators or competitive inhibitors during this period (from 7 to 18 days of incubation) when retina is undergoing marked escalation of glutamotransferase activity. Similar additive data have been obtained in mixed assays of cultured and freshly isolated retinas. The second classical approach to this problem-varying dilution and incubation time inversely-has been applied to many stages of in ovo and in vitro retinal development. In all cases there is a slight departure (10-l%) from first-order kinetics with respect to enzyme (cf. Kirk, 1963). This appears, however, to be a result of inherent instability of the enzyme

348

KIRK

AND

MOSCONA

at prolonged incubation times since it is more dependent upon time of incubation than upon degree of dilution, In any case, no significant difference has yet been observed in this departure from linearity with age or history of the retina. 2. ~n~~~~~~~ by ~~~~rn~c~~~, of the Norman ~~u~~rn~~e ~~~~~~~~ increase. While the above studies preclude a major participation of variation in competitive inhibitors or activators in the apparent enzymatic growth of the retina, they do not exclude the possibility that removal of n~~~~i~~~e inhibitors or modification of inactive but preexisting enzyme molecules is involved. In an attempt to answer this question, puromycin, an inhibitor of protein synthesis, was used. It was applied in varying concentrations to lo-day retinas, either at the time of explantation into the medium or after a precultivation period of 12 or 24 hours (by which time the tissues had developed readily detectable levels of enzymatic activity). As can be seen from Fig. IA, there is a definite dose-dependent depression exerted by puromycin on the normal rate of increase in ghrtamotransferase

u 2.0 LL 2 8 1.0 -

l.O7/mi.

24

48 HOURS

24 IN

FIG. 1. The effect of puromycin upon transferase activity) of lo-day chick neural

48

CULTURE

the glutamine synthetase (glutamoretinal cultures. Specific activity is defined as micromoles of glutamohydroxamic acid produced per hour per milligram of protein. (A) Concentration-dependent effect of puromycin. Puromycin was supplied to retinas precultured for 24 honrs in control medium, and the cultures were maintained for an additional 24 hours. (B) Partial reversibility of the puromycin effect. Puromycin was added to E&hour cultures and left in 12 hours. The cultures were then washed and transferred to fresh puromycin-free medium for the remainder of the cultivation period.

SYNTHESIS

OF

EXl’ERlhlENTALLY

ISDUCED

GLUTAMITE

SYNTHETASE

349

activity. Puromycin at a level of 1.0 /kg per milliliter of culture medium prevented, consistently and completely, any increase in specific activity of the cultures for as long as it was present, whether added after zero, 19, or 24 hours of culture. Levels as high as 10 pg/ml caused no statistically significant decrease in enzymatic activity from the values existing prior to treatment (at least during the following 24 hours). That the retinal cells had not been irreversibly damaged by such puromycin treatment is demonstrated bv the reversibility data plotted in Fig. 1B. The cultures from which tlie data in Fig. 1B were obtained were washed three times in Tyrode’s solution, cultured 20 minutes in puromycin-free medium, and then transferred to fresh medium, where they remained for the rest of the cultivation period. The degree of reversibility was very variable-sometimes nil-but with vigorous washing, after exposure to puromycin, reversibility could be demonstrated. Histologic examination of puromycin-treated retinas confirmed the opinion that levels of puromycin which completely blocked enzymatic growth were not grossly cytotoxic. From these data it can be concluded that at least one step in the appearance of glutamotransferasc activity in z;itro is puromycin sensitive; in the absence of any contrary data, this can bc taken as strong presumptive evidence for tie nota synthesis of the enzyme under culture conditions.

3. Inhibition by actinomycin of the normal glutamine sgnthetnse inCWCI.W. Since the experimentally induced appearance of glutamine synthetase involves protein synthesis, it is of interest to determine to what extent this synthetic process is under continuous nuclear control. As an initial approach to this problem retinal cultures were briefly exposed to low concentrations of actinomycin D, an inhibitor of DNAdependent RNA synthesis (Hurwitz et al., 1962). As seen in Fig. 2, a 20-minute exposure to 1.0 pg of this antibiotic per milliliter was sufficient to block completely and irreversibly further increase in the enzyme in cultures previouslv undergoing a high rate of enzymatic growth. This finding, coupled with the puromycin effect, indicates that the increase in the appearance of glutamine synthetase in cultured retina depends upon biosynthetic activities which appear to be under rather direct and continuous control by DNA. Furthermore, attention is drawn to the fact that both puromycin and actinomycin at the appropriate levels completely blocked further increase in enzymatic activity, without ever causing any significant reduction from preexisting enzymatic levels even when administered at tenfold blocking concen-

KIRK

AND

MOSCONA

24

Hours

48

in Culture

FIG. 2. The effect of a brief exposure to actinomycin D on the glutamine synthetase (glutamotransferase activity) of organ cultures of neural retinas of lo-day chick embryos. Actinomycin D (1 pg/ml) was added to the cultures at A; at B (20 minutes) the cultures were washed and transferred to fresh medium for the remainder of the cultivation period.

trations; this clearly suggests that the enzyme itself is relatively stable, but that the RNA involved in its production is, under the conditions of these experiments, of low stability. Proof of slrch suppositions rests, of OOUTSC’, upon demonstration that these metabolic inhibitors arc exerting their generally accepted effects upon the retina; hence, studies of the synthetic and turnover rates of retinal proteins and nucleic acids (via labeled precursors) have been commenced at this writing. Absence of Detectable Contml Factors in Ch~h untl Embrfp Extrncts

Tissues

Since the enhancement in rate of enzyme production commences practically irn~~~e~liatel~~ upon isolation of ‘the retina from the embryo, the possible role of systemic suppressors in controlling the level of enzyme in the retina in sitrc must be considered. The possibility was tested by culturing IO-day neural retina in the presence of pigmented epithelium (tapeturn) or vitreous humor from the same embryos, and by culturing retina in medium supplemented with saline extracts of 7- or IO-day whole chick embryos. In none of these cultures was there any significant effect upon the rate of enzyme synthesis in the explanted retinas. 1Vhile these results do not preclude the existence of such systemic control factors, thev indicate that if such occur their demonstration will require a more subtle approach. Such work is in progress. The Partid ~~i~~r~s~~~~Effect of G~~~t~l?li~l~ ccntl Ghrtmmt~~ IIaving tentatively established the occurrence of tie nom synthesis of glutamine synthetase in the explanted retina, a prime consideration is the mechanism whereby this synthesis is normally repressed for several additional davs in the embryo and derepressecl or stimulated in culture. In the light of current theories of control over gene expression (Jacob and Monad, 1961), the reports that in I-IeLa cells ( DeMars, 1958) and L cells (Paul and Fottrcll, 1963) glutamotransferase activity is depressed when glutamine is added to the culture medium were of particular interest, In this context it was postulated that perhaps isolation to culture effected rapid enzymatic growth by exposing the retina to sub-repressing levels of glutamine or inducing levels of glutamate. Earlier data demonstrating a partial repressing effect of glutamine in culture (Hubby, Moscona, and Saenz, unpublished) had supported this possibilitv. Thus it was of obvious

352

KIRK

AND

MOSCONA

interest to determine whether, and to exactly what extent, the glutamine synthetase level of explanted retinas could be controlled by varying the concentrations of glutamine or gI~~tamate in the culture medium. Retinas from lo-day embryos were isolated and grown for 48 hours in the standard Tyrode’s-horse serum culture medium supplemented with glutamine or glutamate at various concentrations. The results are presented graphically in Fig. 3. It can be seen that botlt glutamine control activity-experimental control activity

activity

x 100

and glutamate were effective in partially depressing enzymatic growth in the explanted retina. Whether this partial repressing effect of glutamate represents a significant difference between the response of this tissue and that of HeLa (DeMars, 1958) and L cells (Paul and Fottrell, 1963) is as yet unknown, since the effect of glutamate was not tested with either of those cell lines. However, it is quite clear that under our experimental conditions, neither glutamate nor glutamine depressed enzymatic growth completely: the glutamate curve plateaus at less than 20% repression and the glutamine curve appears to be plateauing at something less than 70% repression. In an attempt to further examine the specificity and significance of these inhibitory effects, analogous studies were made employing r-aminobutyric acid, glutathione ( both oxidized and reduced), asparagine, and aspartic acid. These substances were chosen for their known metabolic and chemical similarity to glutamate and glutamine. Although y-aminobutyric acid proved to be more effective than either glutamine or glutamate (Fig. 3), even this curve, if extrapolated at constant slope, would not reach 100%repression until the concentration reached approximately 100 ~moles/ml--a grossly unphysiologic level. Even the cultures which were grown in the presence of 16 pmoles of y-aminobutyric acid per milliliter and which demonstrated 80% repression relative to control cultures were synthesizing new enzyme at ten times the rate of retinas of similar chronologic age in. situ. Neither glutathione (reduced or oxidized), asparagine, nor aspartate had any detectable effect upon the enzymatic growth. These data suggest that glutamate, glutamine, and y-aminobutyrate may have a rather specific role in quantitatively modifying the amount of glutamine synthetase produced by this tissue; however, this is far from the qualitative sort of

SYP\THESIS

OF

EXYERIMESTALLI-

INDUCED

GLUTAMINE

SYNTHETASE

G$BA

Concentration

in Medium (JJ moles/ml.)

353

354

KIRK

AND

MOSCONA

of the Jacob-Monod model to this aspect of retinal differentiation, it does indicate that the mechanism controlling glutamine synthetase synthesis in the differentiating retina is not by simple and reversible endproduct inhibition or substrate stimulation. Whether the three amino acids which partially suppress formation of glutamine synthetase in retinal cultures function strictly by a simple negative-feedback control on the already derepressed enzyme-forming system or whether they are converted very slowly and at different rates to a common metabolite which functions as a repressor substance is as yet undetermined.

The Absence

of

an Effect

of

Elevated

Glucose

The results obtained with y-aminobutyrate prompted an examination of the effect of glucose content of the medium, since the role of this amino acid in energy metabolism of neural tissue is well established (Albers, 1960; Tower, 1960). It was postulated that the effect of explantation upon glutamotransferase activity of the retina might be part of a general compensatory stimulation of glutamate metabolism resulting from an energy deficiency of the Tyrode’s-horse serum medium. If this were true, not only would the observed effects of glutamate, glutamine, and y-aminobutyrate be expected, but glucose enrichment of the medium should markedly repress formation of the enzyme. Experimental enrichment of the medium up to eight times the control level, however, yielded no statistically significant effect upon glutamine synthetase production. DISCUSSION

The findings presented in this paper raise a number of points for discussion; however, the somewhat exploratory and novel nature of some of the findings reported calls for postponement of detailed consideration and discussion of their implications. The present results justify further interest in embryonic retinal glutamine synthetase as a system for studying mechanisms that control the appearance and activity of tissue-characteristic enzymatic patterns. The demonstration that the glutamotransferase activity of the retina both in the embryo and in culture is attributable to a glutamine synthetase places the phenomenon in a more meaningful physiologic context. By all available indications, de novo synthesis of the enzyme appears to be involved in both the normal development and the precocious development in culture; the evidence appears to exclude the possibility that

SYSTHESIS

Ok’

EXPEHIJZEITALLY

INDUCED

GLUTAMINE

SYKTHETASE

355

the precocious increase in enzyme activity of the cultured retina is due to purely trivial causes such as dilution of an enzyme inhibitor. The suggestion, from studies with actinomycin, that genomic control is exerted directly and continuously over the synthesis of the enzyme during its early ontogenesis broadens the relevance of the problem since it may ultimately provide insight into the nature of in differentiating vertebrate cells. It differential gene expressions should be restated here that in this system an actinomycin-sensitive process (presumably nuclear synthesis of RNA) appears eswntial to new synthesis of the enzyme, but not to maintenance of preexisting activity. This suggests that the enzyme protein is relatively stable, Ijut that its synthesis is dependent on a relatively labile RNA messenger. If tracer studies substantiate this impression, the immediacy of trnclear control over retinal glutamine synthetase will be demonstrated to be intermediate to that proposed by Davidson et al. (1963) for control of polysaccharide (AMPS) synthesis in cultured fibroblasts and that demonstrated by Reich ct al. (1962) for control of hemoglobin synthesis. In the former case an actinomyciI1-sensitive process appears to be essential for maintenance of preexisting levels of enzvme activity (suggesting lability of both the enzyme and the enzymeforming svstem); in the latter case de wL;o synthesis of the tissliei specific protein proceeds uninterrupted in the presence of actinomycin (demonstrating the stability of the corresponding messenger). It is conceivable that an entire spectrum of such relationships will occur, wherein the apparent immediacy of nllclear control over differentiated processes will be predicated bv tllc stabilitv of the characteristic protein and the corresponding l&A.

The pllenomerioii of prwocious appearance and rapid enhancement of glutamotransferase activity of embryonic chick neural retina in response to explanation iI1 vitro has been subjected to more detailed scrutiny. On the basis of cofactor requirements, pH optima, response to specific inhibitors, and ratio of transferase to synthetase activity, the enzyme undergoing change was classified as a glutamine synthctase. Tests for the presence of enzyme activators and/or inhibitors widergoing change during increase in enzyme activity were negative, suggesting that increase in activitv i was due to synthesis of new enzvme. This was substantiated by thr finding that low levels of puromycin

356

KIRK

AND

MOSCONA

blocked completely and reversibly increases in enzyme activity under culture conditions; and that actinomycin D similarly blocked such increases irreversibly. Preexisting levels of activity appeared stable in the presence of either inhibitor. These data were interpreted to mean that enhancement of glutamotransferase activity which occurs in culture is due to synthesis of new enzyme; it also suggested a lability of the RNA involved and consequently a rather direct genomic control over the rate of synthesis of the enzyme in early ontogenesis. Pigmented epithelium of embryonic eye, vitreous humor, and embryo extract showed no effect on the increase in glutamine synthetase activity in the explanted retina; this was interpreted as diminishing but not excluding the possibility that a stable, diffusible systemic factor is responsible for control of early ontogenesis of this enzyme in the embryo. Glutamine, glutamate, and y-aminobutyrate were found to lower synthesis of the enzyme in culture when added to the medium, but none of these amino acids appeared capable of total repression at concentrations approaching physiological values, Glutathione (oxidized and reduced), asparagine, aspartate, and gIucose all had no demonstrable affect upon the growth of the enzyme in culture. REFERENCES R. W. (1960). ~~~nlnl~l-arnino butyric acid, 114 “The ~eur~~~lle~~~istry of Nucleotides and Amino Acids” (R. 0. Brady and U. B. Tower, eds.), pp. 146158. Wiley, New York. GOULOhlUHE, A. J. (1961). Cytology of the developing eye. Intern. Rcu. C;@. 11, 161-194. DAVIIISON, E. H., ALLFHEY, V. G., and MIWSKY, A. E. (1963). Gene expression in differentiated cells. Proc. N&l. Acud. Sci. 17,s. 49, 53-60. DEMAAS, R. ( 1958). The inhibition by glutamine of glutamyl transferase formation in cultures of human cells. Biochim. Bi~phy~. Actu 27, 435-436. GAWFINKEL, D. ( 1962). Computer simulation of steady state ghrtamate metabolism in rat brain. J. TAeoret. B&Z. 3, 412-422. HUWWITZ, J., FG'RTH, J. J., MALANY, ht., and ALEXANDER, hf. ( 1962). The role of C~eo~yribonucIeic acid in ribonucleic acid synthesis. III. The inhibitjon of the enzymatic synthesis of ribonucleic acid and ~leoxyribonucleic acid by actinomy&n D and proflavin. Proc. N&E. Acud. Sci. U.S. 48, 1222-1229. JACOB, F., and MONOD, J. ( 1961). Genetic regulatory mechanisms in the synthesis of proteins. J. Mall. BioE. 3, 318-356. Krmc, D. L. ( 1963). In preparation. LEVIN,~OW, L., MEISTER, A., KUFF, E., amI HOGEBOOW G. H. (1955). Studies on the relationship between the enzymatic synthesis of glutamine and the glutamyl transfer reaction. .1. Am, Chem. SOC. 77, 53044308. ALBENS,

SYNTIIESIS

OF

EXI’ERIhIENTALLY

ISDUCEI~

GLIJTAXZINE

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