DEVELOPMENTAL BIOLOGY 61, 94-103 (1977)

Induction of Multiple Proteases during the Early Larval Development of Artemia salina

CARMEN OSUNA, ASUNCI~N OLALLA, ANTONIO SILLERO, MARIA A. GUNTHER SILLERO,

AND JES~~S SEBASTIAN

Znstituto de Enzimologh de1 CSZC, Facultad de Medicina, Universidad Authoma, Madrid 34, and Departamento de Fisiologin y Bioquimica, Facultad de Medicina,

Universidad de Valladolid, Valladolid, Spain

Received June 1,1977; accepted June 14,1977

Extracts from dormant and developing embryos ofArtemia salina contain almost undetecta- ble levels of protease activity. However, after hatching of the nauplii, there is a high increase in the activity of the extracts on different proteolytic substrates. The activities correspond to four different enzymes, A, B, C, and D, that have a sequential timing of induction during the early larval development. The enzymes show a different elution pattern after chromatography on DEAE-cellulose and a different sensitivity to protease inhibitors. Enzyme A is strongly inhibited by phenylmethylsulfonyl fluoride, while the other enzymes are mostly insensitive. Enzymes A, B, and C are sensitive at low concentrations of the soybean trypsin inhibitor. The temporal induction of these enzymes is an example of the expression, at the biochemical level, of the developmental program of Artemia salina.

INTRODUCTION

The developmental program of the crus- tacean Artemia salina involves a number of well-defined stages (Hentschel and Tata, 1976). Starting with dormant em- bryos, the postgastrular embryonic devel- opment and the early larval development ofArtemia can be studied in the laboratory in fairly good synchronous populations with a high mass of embryos or larvae. This fact makes Artemia salina a good experimental system to study the bio- chemistry and molecular biology of animal development.

Several biochemical events have al- ready been characterized during the devel- opment of Artemia. These events involve among others, the chromatin modification (Cano and Pestafia, 19761, the machinery of transcription (Birndorf et al., 1975; Re- nart and Sebastian, 1976), the machinery of translation (Sierra et al., 1974; Slobin and Moller, 19751, the expression of the hemoglobins (Moens and Kondo, 1976), the mitochondrial biogenesis (Marco and Val-

lejo, 19761, and the metabolism of the yolk platelets (Olalla et al., 1977), nucleotides (Warner and McClean, 1968; Vandenbos and Finamore, 1974; Warner et al., 1974; Vallejo et al., 1974; Renart et al., 19761, and nucleic acids (McClean and Warner, 1971).

An interesting aspect of the studies on the biochemistry of animal development is the characterization of the proteolytic en- zymes present in embryos and larvae. The proteases can play an important role in the morphogenetic changes and structural re- organizations that occur during develop- ment, as well as in the regulation of the protein turnover. On the other hand, the induction of specific proteases are bio- chemical parameters of the differentiation of specific tissues and organs. There are also methodological arguments in favor of the investigation of the proteases in devel- oping organisms. Changes in the levels or appearance of new proteases during devel- opment represent a source of artifacts pro- duced by the in vitro proteolysis of differ-

94 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0012-1606

OSUNA ET AL. Proteases during A. salina Development 95

ent proteins during their extraction and purification. Therefore, it is important to be aware of the proteases present in differ- ent stages of development and the inhibi- tors that can be used to prevent their dele- terious effects.

The early report by Bellini (1957) on the existence of an increase in proteolytic ac- tivity during Artemia development points out the importance of the characterization of the enzymes involved, as well as the study of their role in the different stages of development of Artemia salina.

In this paper we report the sequential induction of multiple proteolytic enzymes during the early larval development and their specificities toward different proteo- lytic inhibitors.

MATERIALS AND METHODS

Chemicals and buffers. The following compounds were obtained from Sigma Chemical Co.: a-N-benzoyl-on-arginine p- nitroanilide; (p-toluenesulfonyl)-L-argi- nine methyl ester; N-carbenzoxy-L-leucine p-nitrophenyl ester; soybean trypsin inhib- itor type I-S; trypsin inhibitor from white egg (ovomucoid) type II-O; phenylmethyl- sulfonyl fluoride; and N-a-p-tosyl-lysine chloromethyl ketone. Casein was pur- chased from F.E.R.O.S.A., azocasein from Calbiochem, dimethyl sulfoxide from BDH, and DEAE-cellulose from Serva. All other chemicals were of analytical grade. Buffer P contains 50 miV Tris-HCl, 0.2 mM EDTA, pH 7.5.

Organism and growth conditions. Ar- temia salina cysts from two sources were used in the experiments without signifi- cant differences. The cysts were first ob- tained from Longlife Fish Food Products, Division of Sternco Industries Inc., Harri- son, N.J. 07029. More recently, the cysts were obtained from San Francisco Bay Brand Inc., Division of Metaframe Co., Menlo Park, Calif. 94025.

Treatment of the dry embryos and growth conditions were as described else- where (Renart and Sebastian, 1976). New-

born nauplii were isolated by means of a separatory funnel, taking advantage of the phototropism of the larvae. This homoge- neous population was incubated in the same medium and was grown synchro- nously at 30°C. Samples were taken at intervals and the animals were collected by filtration on a cloth and were washed with distilled water. The samples were kept at -70°C until use.

Preparation of the extracts and enzy- matic assays. Frozen embryos or nauplii were resuspended in 2 vol of buffer P and were homogenized in a Kontes Dual1 Grinder at 400 rpm for 5 min. The homoge- nate was centrifuged at 30,OOOg for 30 min, and the supernatant at 150,OOOg for 90 min. The resulting clear supernatant was the soluble extract.

The proteolytic activities were assayed using different substrates. The activity on a-N-benzoyl-m-arginine p-nitroanilide (BAPNA) was performed according to the method of Erlanger et al. (1961). The sub- strate, at a concentration of 10 mM, was dissolved in dimethyl sulfoxide. The assay mixture contained in 1 ml: 25 r&f Tris- HCl, pH 7.5, 1 mM BAPNA, and the en- zyme preparation. The amount of p-ni- troanilide liberated was monitored contin- uously in a spectrophotometer at 410 nm. One unit of activity was defined as the amount of enzyme which produces a change of one unit of absorbance in 1 hr at 25°C under the assay conditions.

The activity on (p-toluenesulfonyl)-L-ar- ginine methyl ester (TAME) was mea- sured according to the method of Hummel (1959). A solution of 1 mM TAME was prepared in 40 n&f Tris-HCl, 10 mM CaCl,, pH 8.1. The assay mixture con- tained 1 ml of the substrate solution and the enzyme preparation. The change in absorbance at 247 nm was recorded contin- uously. One unit of activity on TAME was defined as the amount of enzyme that pro- duces a change of one unit of absorbance at 247 nm in 1 hr at 25°C.

The activity on N-carbobenzoxy-L-leu-

96 DEVELOPMENTAL BIOLOGY VOLUME 61. 1977

tine p-nitrophenyl ester (CLNE) was de- brought to 50 ml with water. The assay termined according to Geneste and Bender mixture contained 0.3 ml of azocasein solu- (1969). A solution of 2.5 mM CLNE was tion, the enzyme preparation, and water prepared in dimethyl sulfoxide. The assay up to 1 ml. The incubation was done at mixture contained in 1 ml: 25 mit4 Tris- 3o”C, and the reaction was stopped by ad- HCl, pH 7.5, 0.1 ml of methanol, 0.05 mM dition of 1 ml of 10% cold TCA. The tubes CLNE, and the enzyme preparation. The were centrifuged, and 0.5 ml of 10% NaOH change in absorbance at 410 nm was moni- was added to the supernatants. The ab- tored continuously in a spectrophotometer. sorbance was measured at 440 nm. One One unit of activity was defined as the unit of activity on azocasein was defined as amount of enzyme that produces a change the amount of enzyme that produces one of one unit of absorbance in 1 hr at 25°C. unit of absorbance at 440 nm in 1 hr.

The activity on casein was determined according to the method described by Las- kowski (1955). The substrate was prepared by heating, in a boiling water bath for 15 min, a solution of 1% casein in 0.1 M phos- phate buffer, pH 7.6. The assay mixture contained: 0.5 ml of 50 miV Tris-HCl, pH 7.5, 0.5 ml of casein solution, and the en- zyme preparation. The incubation was done at 3o”C, and the reaction was stopped by addition of 1 ml of 10% trichloroacetic acid (TCA). After 1 h in the cold, the tubes were centrifuged, and the absorbance of the supernatants was measured at 280 nm. One unit of activity on casein was defined as the amount of enzyme that produces one unit of absorbance at 280 nm of TCA-solu- ble material in 1 hr.

Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.

RESULTS

Levels of Proteolytic Activities during A. salina Development

The activity on azocasein was deter- mined according to the method described by Charney and Tomarelly (1947). The substrate solution was prepared as follows: 0.62 g of azocasein was dissolved in 12.5 ml of 2% NaHCO, at 60°C. The pH was ad- justed to 7.2 with HCl, and the volume was

Table 1 shows the activities of the solu- ble extracts from four stages of A. salina early development on five different sub- strates. The activities of dormant and de- veloping embryos on BAPNA, CLNE, and TAME are under the resolution level of the methods. Only on casein and azocasein is it possible to measure a very low activity in agreement with previously reported re- sults (Bellini, 1957). In contrast with the embryos, the proteolytic activities of nau- plii extracts are high on all the substrates. Extracts prepared by mixing embryos and nauplii give the expected levels of proteo- lytic activities corresponding to the nau- plii. This experiment indicates that the undetectable levels in the embryos are not

TABLE 1

LEVELS OF PROTEOLYTIC ACTIVITIES DURING EARLY DEVELOPMENT OF ARTEMIA SALINA~ Developmental stage

Dormant embryos (T,) Developing embryos (T,) Newborn nauplii CT,,) 35h-old naunlii CT,,)

BAPNA”

10.02 co.02

0.05 42.7

Proteolytic substrate

CLNE b TAME *

co.1 <0.2 <O.l co.2

0.3 0.4 62 83

Casein Azocasein

0.08 0.03 0.07 0.04 0.14 0.12 8.1 13.3

dl The proteolytic activities of the soluble extracts from four developmental stages were measured with five proteolytic substrates. The data are given in units per milligram of soluble protein.

b The full names of the substrates are given in Material and Methods.

OSIJNA ET AL. Proteases during A. salina Development 97

due to the presence of some inhibitor in these extracts.

The results shown in Table 1 are com- patible with the induction of proteolytic activities during the early larval develop- ment ofArtemia salina, however, it is not possible to conclude the number of en- zymes responsible for the activities. To in- vestigate this problem a chromatography of nauplii extracts was carried out.

Separation of Multiple Proteolytic En- zymes from Nauplii Extracts by Chro- matography on DEAE-Cellulose

A soluble extract from 35-hr-old nauplii (T5J was subjected to chromatography on DEAE-cellulose, and the fractions were assayed for activity on the five substrates indicated before. Figure 1 shows the re- sults of this experiment. Only the activi-

6 t 2

;

i

4 P 0

ties on CLNE, BAPNA, and casein are shown in order to simplify the figure.

Multiple peaks of activities with differ- ent substrate specificities were obtained after the chromatography. Only one peak of activity was detected using CLNE as substrate. The enzyme responsible for this activity was called enzyme A.

There are two peaks of activity on BAPNA. The fractions from these two peaks also have activity on TAME. The ratio of activities on TAME/BAPNA is the same along the fractions corresponding to each peak, indicating that the activities on both substrates in each peak are due to the same enzyme. The enzyme responsible for the activity on BAPNA and TAME eluting with 0.47 M KC1 was called enzyme B, and the enzyme that elutes with 0.85 M KC1 was called enzyme C. The ratio of activi-

IO 20 30 40 50 60 70 00 90 100 110

i.

1.0

3 8 -

0 Y

.6

Fraction number

FIG. 1. Separation of multiple proteases from extracts of Artemia salina nauplii by chromatography on DEAE-cellulose. Ten milliliters of a soluble extract from 35hr-old nauplii CT,,) was adjusted with KC1 to a concentration of 0.2 M. The extract was subjected to chromatography on a 30-ml of DEAE-cellulose column equilibrated with buffer P containing 0.2 M KCl. After application of the sample the column was washed with 30 ml of buffer P plus 0.2 M KC1 and was eluted with a 90 ml x 2 linear gradient of 0.2-1.2 M KC1 in buffer P. Fractions of 2 ml were collected and assayed for activity withN-carbenzoxy+leucinep-nitrophenyl ester (CLNE) (A); &V-benzoyl-DL-argininep-nitroanilide (BAPNA) (0); and casein (HI. The methods and units are defined in Material and Methods.

98 DEVELOPMENTAL BIOLOGY VOLUME 61, 1977

ties on TAMEIBAPNA was of 2.2 for en- zyme B and 3.5 for enzyme C.

The screening of the proteolytic activity on casein of the fractions from the column shows a peak in the position of enzyme B but not fully coincident with it. The activ- ity on casein has a shoulder at the right side of peak B, suggesting the existence of some enzyme heterogeneity. The assay of the activity of the fractions in the presence and absence of a proteolytic inhibitor, soy- bean trypsin inhibitor, shows the exis- tence of two different enzymes active on casein. Figure 2 shows the results of the assay. There is one peak active on casein, which is sensitive to the inhibitor and coincident with enzyme B. The other en- zyme active on casein is insensitive to the soybean trypsin inhibitor and is called en- zyme D. Enzymes B and D are also active on azocaseine. The ratio of activities on- casein/azocasein is 0.7 for enzyme B and 1.5 for enzyme D.

The ratio of the four enzymes is the same, regardless of the length of time prior to the chromatography, which is an indica-

6

4

2

30 40 50 60 70 so Fraction number

FIG. 2. Identification of two hydrolytic activities on casein in extracts from Artemia nauplii. Frac- tions from the DEAE-cellulose column shown in Fig. 1 with hydrolytic activity on casein were assayed in the absence and presence of 100 pg/ml of soybean trypsin inhibitor (STD. The figure shows the STI- sensitive activity (0) and the STI-insensitive activ- ity (ml.

tion against the in vitro interconversion of the enzymes in the extracts.

The results of chromatography on DEAE-cellulose of soluble extracts from 35-hr-old nauplii indicate the existence of four proteolytic enzymes. Enzyme A is ac- tive on CLNE; enzyme B on BAPNA, TAME, casein and azocasein; enzyme C on BAPNA and TAME; and enzyme D on ca- sein and azocasein. The presence of multi- ple proteases raises the problem of the tim- ing of appearance of each of the enzymes during the larval development ofArtemia.

Timing of Induction of the Proteases dur- ing the Early Development of A. salina

The activities of enzymes A, B, C, and D were measured in extracts from embryos and nauplii at different times during their development.

The level of enzyme A was determined by assay of the activity of the extracts on CLNE. The activities of enzymes B and C were measured using BAPNA as substrate after their separation by chromatography on DEAE-cellulose columns. The level of protease B was also determined by the soybean trypsin inhibitor-sensitive activ- ity of the extracts on casein, corresponding the insensitive activity to the level of pro- tease D.

Figure 3 shows the timing of induction of the four enzymes. The increase in the activities occurs during early larval devel- opment. Proteases B and C are induced shortly after hatching, while enzymes D and A are sequentially induced several hours later.

The low level of activity on casein and azocasein observed in the dormant and de- veloping embryos corresponds to th? en- zyme insensitive to the soybean trypsin inhibitor, protease D.

The steep increase in the proteolytic ac- tivities during larval development is a good indication of the synchronization of the nauplii population and of the existence of a precise timing of induction for differ-

&WNA ET AL. Proteases during A. salina Development 99

I I I 20 30 40 50 60

Hours of development

FIG. 3. Timing of induction of the proteases during the early development ofArtemia salina. Forty grams of dormant embryos was incubated in 8 liter of saline medium with aeration at 30°C. Samples of developing embryos were taken at 8 and 15 h. All nauplii born between 16 and 18 h were collected and reincubated in saline medium under the same conditions. Samples were taken at the indicated times. Extracts were prepared with 3 g wet weight of embryos or nauplii, and the activities on different substrates were measured. The figure shows the specific activities of enzyme A (A) assayed with CLNE; proteases B (0) and C (0) assayed with BAPNA; and protease D (m) measured with casein in the presence of 100 pglml of soybean trypsin inhibitor.

ent hydrolytic enzymes in the develop- mental program of Artemia salina.

Effect of Proteolytic Znhibitors on the Pro- teases from A. salina Nauplii

The studies on the effect of different pro- teolytic inhibitors on the Artemia pro- teases have a double interest. These stud- ies contribute to a further characterization of the properties of the enzymes and also provide information which is necessary in avoiding some potential proteolytic arti- facts.

Several protease inhibitors have been used in these studies. The soybean and ovomucoid trypsin inhibitors (ST1 and OTI) are proteins of different biological origins that interact and inhibit trypsin (Kassell, 1970 a,b). Phenylmethylsulfonyl fluoride (PMSF) is a compound that in- hibits serine proteases by interaction with a serine residue of the active site of the enzyme (Gold, 1967). N-a-p-Tosyl-lysine

chloromethyl ketone (TLCK) inhibits by interaction with a histine residue of the active site of some proteases (Shaw et al., 1965). The Artemia proteases were isolated by chromatography of the nauplii extracts on DEAE-cellulose. The sources of pro- teases B and D were the fractions from the DEAE column without each other’s con- tamination. Enzyme A was assayed using CLNE as substrate, protease B with BAPNA and casein, protease C with BAPNA and TAME, and protease D with casein and azocasein. Figure 4 shows the effect of the inhibitors on the nauplii pro- teases.

Enzymes A, B, and C are inhibited by low concentrations of the soybean trypsin inhibitor, while protease D is insensitive at concentrations up to 1 mg/ml. A similar qualitative pattern of inhibition was found with the ovomucoid trypsin inhibitor, but with the opposite affinity. Enzyme A is the most sensitive to ST1 and least sensitive to

100 DEVELOPMENTAL BIOLOGY VOLUME 61, 1977

OTI, while protease B shows the reverse affinity.

TLCK strongly inhibits proteases B and C, while enzymes A and D are insensitive to high concentrations. A 50% inhibition of proteases B and C is obtained with 10 and 80 @ TLCK, respectively.

Enzyme A is inhibited by PMSF at very low concentrations. PMSF at 1 PM causes 50% inhibition of this enzyme. Proteases B, C, and D are mostly insensitive to PMSF. Only by preincubation of the en- zymes and the inhibitor is it possible to get some inhibition of the proteolytic activi- ties. A concentration of PMSF as high as 2 mM, preincubated with protease B for 30 min, only inhibits about 50% of the activ- ity on BAPNA or casein. The same concen- tration of PMSF inhibits less than 30% of the activity of protease D after 30 min of preincubation.

The results of these experiments show the existence of a different qualitative and quantitative pattern of sensitivity to dif-

ferent inhibitors of the multiple proteases from Artemia nauplii. None of the four compounds tested inhibited the four en- zymes, however, the soybean inhibitor can be used during preparation of the extracts to prevent the undesirable proteolytic ef- fect of proteases A, B, and C in the studies of proteins and enzymes during the larval development of Artemia salina.

DISCUSSION

The sequential induction of multiple proteolytic activities during the early lar- val development of Artemia salina is an example of expression, at the biochemical level, of the developmental program of this crustacean. It is remarkable that there is only a low level of protease activity pres- ent in soluble extracts from dormant and developing embryos. The greatest increase in the proteolytic enzymes occurs after hatching, since newborn nauplii still have very low levels of activities on different substrates. The precise timing of appear-

ti vg STI /ml vg OTI / ml

[TLCK] mM [PMSF] ~1 M

FIG. 4. Effect of inhibitors on the activities of the protease from Artemia nauplii. The enzymes A (A), B (01, C (O), and D (m) were obtained from nauplii extracts by chromatography on DEAE-cellulose. The activities were assayed in the presence of the indicated concentrations of inhibitors. The inhibitors were dissolved in buffer P, except for PMSF which was dissolved in dimethyl sulfoxide.

OSUNA ETAL. Proteases during A. salina Development 101

ante of the proteases makes these enzymes good biochemical markers of the larval de- velopment of Artemia.

It is not possible to elucidate from the reported experiments the molecular mech- anism involved in the induction of the nauplii proteases. Many levels of regula- tion can be implicated in the process, in- cluding control at the level of gene tran- scription, messenger RNA processing, and/or unmasking and activation of previ- ously synthesized enzymes. The high in- crease in the activity of each of the pro- teases and the steep pattern of this in- crease make these enzymes an interesting model system in which to study, at the molecular level, the mechanism involved in the regulation of their temporal expres- sion.

The nauplii extracts contain multiple enzymes with proteolytic activities. The enzymes are different in their chromatog- raphy behavior and their substrate and inhibition specificities. Other differences have been found in the properties of the proteases in studies with partially purified enzymes (Olalla et al., submitted).

Three classes of substrates have been used to detect proteolytic, amidase, and esterase activities in the extracts and to characterize the specificities of the en- zymes separated by chromatography on DEAE-cellulose. Enzyme A should be characterized by its esterase activity on CLNE, a leucine-containing synthetic es- ter, and by its failure to hydrolyze TAME, another ester containing arginine as the amino acid residue. Protease B has hydro- lytic activity on casein as well as amidase and esterase activities on arginine-con- taining synthetic substrates. Protease B is, by its substrate specificity, a trypsin- like enzyme (Walsh, 1970). Enzyme C shows amidase and esterase activities, but does not hydrolyze casein. However, this enzyme has proteolytic activity on the pur- ified Artemia RNA polymerases (Osuna and Sebastian, in preparation). Protease D

has proteolytic activity on casein but is inactive on synthetic esters containing leucine or arginine as well as on BAPNA.

Inhibitors of trypsin from soybean and ovomucoid are active on the enzymes A, B, and C, while protease D is not inhibited by these compounds. Enzyme A is inhibited at very low concentrations of PMSF, but is fully resistant to TLCK. On the contrary, proteases B and C are sensitive to TLCK and resistant to PMSF. The lack of inhibi- tion of the proteases B, C, and D by PMSF is important information from a methodo- logical point of view, since this protease inhibitor is widely used to avoid proteo- lytic artifacts in biochemical studies. In the case of Artemia, ST1 instead of PMSF should be chosen to prevent some of the potential artifacts, although its presence does not prevent the effect of protease D. Therefore, more research must be done to find specific inhibitors of this protease.

The physiological role of the proteases induced during the nauplii development is unknown. It is likely that some of these enzymes are products of the differentiation of the digestive system which occurs dur- ing early larval development (Benesch, 1969). They may also be implicated in the morphogenetic events taking place after hatching as well as in the degradation of reserve proteins. In fact, protease B is ac- tive in vitro in the degradation of the pro- teins from the yolk platelets (Olalla et d., in preparation). Recently, Twardowski et al. (1976) have shown the presence, in nau- plii extracts, of a proteolytic activity that produces in vitro the transformation of the elongation factor I from a heavy to a light form. The change of this elongation factor has been reported to be a developmental event that takes place after hatching of the nauplii (Slobin and Moller, 1975). Finally, it has been found that the naupliar pro- teases are active in the in vitro modifica- tion and degradation of the Artemia RNA polymerases (Sebastian et al., 1976; Osuna and Sebastian, in preparation).

102 DEVELOPMENTAL BIOLOG iY VOLUME 61. 1977

We thank Elvira Dominguez and Francisca de Luchi for their technical assistance. C.O. holds a fellowship from P.F.P.I., Spain. This work was sup- ported in part by a grant from Fundacion Juan March and Fondo National para el desarrollo de la Investigation Cientifica.

REFERENCES

BELLINI, L. (1957). Studio delle dipeptidasi e protei- nasi nello suiluppo di Artemia salina. Atti Accad. Naz. Lincei Roma 22, 340-346.

BENESCH, V. R. (1969). Zur ontogenic und Morpholo- gie von Artemia salina. Zool. Jahrb. Abt. Anat. Ontog. Tiere 86, 307-458.

BIRNDORF, H. C., D’ALESSIO, I., and BAGSHAW, I. C. (1975). DNA dependent RNA polymerases from Artemia embryos. Characterization of polymer- ases I and II from nauplius larvae. Develop. Biol. 45, 34-43.

CANO, A., and PESTAAA, A. (1976). Regulation of histone acetyl transferase activity during devel- opment of Artemia salina. Characterization of an inhibitor in nauplii larvae. Develop. Biol. 54, 276- 287.

CHARNEY, J., and TOMARELLY, R. M. (1947). A color- imetric method for the determination of the prote- olytic activity of duodenal juice. J. Biol. Chem. 171, 501-505.

ERLANGER, B. F., KOKOWSKY, N., and COHEN, W. (1961). The preparation and properties of two new chromogenic substrates of trypsin. Arch. Bio- them. Biophys. 95, 271-278.

GENESTE, P., and BENDER, M. L. (1969). Esterolytic activity of elastase. PFOC. Nat. Acad. Sci. USA 64, 683-685.

GOLD, A. M. (1967). Sulfonylation with sulfonyl hal- ides. In “Methods in Enzymology” (C. H. W. Hirs, ed.), vol. 11, pp. 706-711. Academic Press, New York.

HENTSCHEL, C. C., and TATA, J. R. (1976). The mo- lecular embryology of the brine shrimp. Trends Biochem. Sci. 1, 97-100.

HUMMEL, B. C. W. (1959). A modified espectrophoto- metry determination of chymotrypsin, trypsin and thrombin. Canad. J. Biochem. Physiol. 37, 1393-1399.

KASSELL, B. (1970a). Trypsin and chymotrypsin in- hibitors from soybeans. In “Methods in Enzymol- ogy” (G. E. Perlmann and L. Lorand, eds.), Vol. 19, pp. 853-862. Academic Press, New York.

KASSELL, B. (1970b). Proteinase inhibitors from egg white. In “Methods in Enzymology” (G. E. Perl- mann and L. Lorand, eds.), Vol. 19, pp. 890-906. Academic Press, New York.

LASKOWSKI, M. (1955). Trypsinogen and trypsin. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 2, pp. 26-36. Academic Press, New York.

LOWRY, 0. H., ROSEBROUGH, N. I., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265- 275.

MARCO, R., and VALLEJO, C. G. (1976). Mitochon- drial biogenesis during Artemia salina develop- ment: Storage of precursors in yolk platelets. J. Cell Biol. 70, 321a.

MCCLEAN, D. K., and WARNER, A. H. (1971). As- pects of nucleic acid metabolism during develop- ment ofthe brine shrimpArtemia salina. Develop. Biol. 24, 88-105.

MOENS, L., and KONDO, M. (1976). Structure ofAr- temia salina hemoglobins. Comparative charac- terization of four naupliar and adult hemoglobins. Eur. J. Biochem. 67, 397-402.

OLALLA, A., RENART, J., SILLERO, M. A. G., and SILLERO, A. (1977). Metabolism0 de las plaquetas de vitelo durante el desarrollo de la Artemia sal- ina. In “Advances de la Bioquimica” (L. Cornu- della, C. F. Heredia, and J. Or6 y A. Sols, eds.), pp. 111-120. Editorial Salvat, Barcelona.

RENART, J., and SEBASTIAN, J. (1976). Characteriza- tion and levels of the RNA polymerases during the embryogenesis of Artemia salina. Cell. Differ. 5, 97-107.

RENART, M. F., RENART, J., SILLERO, M. A. G., and SILLERO, A. (1976). Guanosine monophosphate re- ductase from Artemia salina: Inhibition by xan- thosine monophosphate and activation by digu- anosine tetraphosphate. Biochemistry 15, 4962- 4966.

SEBASTIAN, J., OSUNA, C., OLALLA, A., RENART, J., CRUCES, J., and SILLERO, M. A. G. (1976). In vitro proteolytic inactivation of the RNA polymerases during the development of Artemia salina. J. Cell Biol. 70, 340a.

SHAW, E., MARES-GUIA, M., and COHEN, W. (1965). Evidence for an active center histidine in trypsin through use of a specific reagent l-chloro-3-tosy- lamide-7-amino-2-heptatone, the chloromethylke- tone derived from N-a-tosyl+lysine. Biochemis- try 4, 2219-2224.

SIERRA, J. M., MEIER, D., and OCHOA, S. (1974).

Effect of development on the translation of mes- senger RNA in Artemia salina embryos. Proc. Nat. Acad. Sci. USA 71, 2693-2697.

SLOBIN, L. I., and MOLLER, W. (1975). Changes in form of elongation factor during development of Artemia salina. Nature (London) 258, 452-454.

TWARDOWSKI, T., HILL, J. M., and WEISSBACH, H. (1976). Disaggregation of elongation factor I by extracts of Artemia salina. Biochem. Biophys. Res. Commun. 71, 826-833.

VALLEJO, C. G., SILLERO, M. A. G., and SILLERO, A. (1974). Diguanosine tetraphosphate guanylohy- drolase in Artemia salina. Biochim. Biophys. Acta 358, 117-125.

OSUNA ET AL. Proteases during A. salina Development 103

VANDENBOS, G., and FINAMORE, F. J. (1974). Unu- sual pathway for synthesis of adenosine triphos- phate by purine requiring organism Artemia sal- ina. J. Biol. Chem. 249, 2816-2818.

WALSH, K. A. (1970). Trypsinogens and trypsins of WARNER, A. H., and MCCLEAN, D. K. (1968). Stud- various species. In “Methods in Enzymology” (G. ies on the biosynthesis and role of diguanosine E. Perlmann and L. Lorana, eds.), Vol. 19, pp. 41- tetraphosphate during growth and development of 63. Academic Press, New York. Artemia salina. Develop. Biol. 18, 278-293.

WARNER, A. H., BEERS, P. C., and HUANG, F. L. (1974). Biosynthesis of diguanosine nucleotides. I. Purification and properties of an enzyme from yolk platelets of brine shrimp embryos. Canad. J. Biochem. 52, 231-251.