Alkaline ribonuclease from the insect Ceratitis capitata

Biochimica et Biophysica Acta 826 (1985) 129-136

129

Elsevier BBA 91515

Alkaline ribonuclease f r o m the insect C e r a t i t i s c a p i t a t a J u a n M. G a r c i a - S e g u r a , Jesi~s M. F o m i n a y a , M. M a r O r o z c o a n d Jos6 G. G a v i l a n e s Departamento de BioquJmica, Faeultadde Ciencias, Universidad Complutense, 28040 Madrid (Spain)

(ReceivedJuly3rd, 1985)

Keywords: Ribonuclease;Insect;(C. capitata)

An alkaline ribonuclease has been purified from Ceratitis capitata larvae. The enzyme preparation gives a single band on SDS-polyacrylamide gel electrophoresis using both silver-stain and ribonudeolytic activity detection. This enzyme has been characterized as a cyclizing endonuclease with optimum pH value at 8.0-8.3. The enzyme preparation does not exhibit phosphatase activity. Poly(C) is the only homopolyribonucleotide degraded under standard assay conditions. Neither native nor denatured DNA is hydrolyzed by this enzyme. The protein is a single polypeptide chain of about 18000 molecular weight; its secondary structure is composed of 19% a-helix, 10% t-structure, 71% aperiodic conformation with an average number of residues per helical segment of 9. The amino acid composition of this alkaline RNAase is also reported. Latent alkaline ribonuclease has been observed in crude insect homogenates. The insect enzyme is inhibited by the RNAase inhibitor from human placenta.

Introduction The non-secretory ribonucleases are a wide group of enzymes whose molecular properties are scarcely known, probably due to the small levels found in the tissues studied. Their enzymic properties, subcellular distribution and possible functions have been reviewed in a number of articles [1-4]. However, the biological significance of their RNA-degrading action is still uncertain. Among these ribonucleases, alkaline RNAases (RNAases I) [4] are a group of intracellular enzymes for which a regulatory role in protein biosynthesis has been proposed [1,5]. Such a hypothesis is based on the existence of specific inhibitor proteins for these alkaline activities [6]. These RNAase inhibitors are free-thiol-dependent proteins and they have been detected in mammalian tissues [1,7-13] as well as in other organisms [14-16]. The inhibitor-RNAase I system might determine the rate of RNA catabolism and, in turn, the rate of protein synthe-

sis [17]; thus, the ratio inhibitor-to-RNAase I is increased in tissues characterized by high rates of RNA synthesis [5,17-20] whereas this ratio decreases when the catabolic activity is dominant [20-22]. Further, the inactive RNAase-inhibitor complex, so-called latent RNAase, may be activated, depending on the physiological state of a particular cell [16]. Holometabolous insects constitute an interesting model in elucidating the functionality of the RNAase-inhibitor system because their metabolism is divided into very distinctive phases. Stages characterized by high biosynthetic activity (larval development) are followed by others characterized by either massive decomposition and remodelling of the accumulated materials (metamorphosis) or high catabolic activity (adult stage). However, the RNA-degrading system is poorly defined in insects. The presence of ribonuclease similar to mammalian alkaline RNAase I has never been directly demonstrated. Activation by thiol-specific

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130 reagents of latent RNAase in crude extracts from the flesh fly S. peregrina has been reported [16]; however, its molecular weight would be twice that of the mammalian system. Early studies on yellow fever mosquito evidenced the lack of inhibited alkaline RNAase in crude extracts [23]. Thus, the results obtained for both holometabolous insects are contradictory and further characterization is required. On the other hand, previous studies on RNAdegrading activities have shown the existence of alkaline ribonucleolytic activity during the development of the holometabolous insect Ceratitis capitata [24]. This activity has been related to the variations of the poly(A)-mRNA levels during the development of this insect [25]; thus, this enzyme may be involved in the control of intracellular R N A levels. Taking all these facts into consideration, we thought of interest the purification of the alkaline ribonuclease from C. capitata, in order to elucidate its possible participation in the control of intracellular R N A levels, as well as to gain new insights into the RNAase-inhibitor system. Materials and Methods

Rearing of insects The insect C. capitata was cultured under controlled conditions of diet, temperature and humidity as previously described [26]. The alkaline ribonuclease was isolated from 4-5-day insect larvae based on the enzyme distribution pattern during the development of C. capitata [24]. Cultured material was maintained at - 4 0 ° C after freezing in liquid nitrogen until required. This storage does not modify the specific activity of the purified protein. Enzyme assay Enzyme activity was determined by measuring the degradation of yeast RNA. The ribonucleic acid was purified by saline precipitation with 3 M sodium acetate, p H 5.5, cold-ethanol precipitation [27] and dialysis against 25 mM EDTA, further against 0.15 M NaC1, and finally against glass-distilled water [9]. The assay mixture was composed of 0.4 ml of 0.25 M Tris-HC1, pH 8.3, containing 3 mM EDTA and 0.2% ( w / v ) horse serum albumin, and 0.1 ml of the sample. This mixture was

incubated at 37°C for 10 min. The reaction was started by adding 0.5 ml of 0.5% (w/v) purified yeast RNA in glass-distilled water. Hydrolysis was carried out for 1 h at 37°C for standard analysis, The RNA degradation was stopped by immersing the reaction mixture in an ice bath for 5 min and further addition of 1 ml ice-cold 10% perchloric acid, containing 0.25% (w/v) uranyl acetate. The suspension was maintained for 30 min in the ice bath and then centrifuged at 3000 x g for 20 min. The supernatant was diluted 10-fold with glassdistilled water and its .,'1::60 w a s measured in a Beckman DU-7 spectrophotometer. Blanks without sample were considered for all assays. One unit of ribonuclease activity was defined as the amount required to produce a A26(I value of 1.0 under the assay conditions.

Purification of the alkaline ribonuclease The amount of starting material for a standard purification procedure was 100 g. All the operations described below were performed at 2-4°C. The insect larvae were homogenized in 300 ml of 50 mM Tris-acetate buffer, pH 5.5, containing 5 mM EDTA, 0.2 g / l chloramphenicol and 1% (v/v) of 20 mM phenylmethylsulfonyl fluoride in isopropanol, using a Teflon-glass homogenizer. The homogenate was centrifuged at 30000 x g for 45 rain. The supernatant was maintained at pH 5.5 for 4 h and then centrifuged under the above conditions. The resulting supernatant was incubated at 50°C for 10 min in a water bath. The suspension was clarified by centrifugation at 30000 × g for 45 rain after cooling at 2-4°C. The supernatant was loaded on an Amberlite CG-50 (200-400 mesh) column (1.4 x 15 cm) equilibrated with 50 mM Tris-acetate buffer, pH 5.5. The column was washed with 200 ml of the equilibration buffer and further with an identical volume of the same buffer containing 0.1 M NaC1. No ribonuclease activity was eluted under these conditions. The enzyme eluted with 50 mM Trisacetate buffer, pH 5.5, containing 0.7 M NaCI. The flow rate was 25 ml/h. Fractions containing alkaline ribonuclease activity were combined; the enzyme was precipitated by adding ammonium sulphate up to 80% saturation. The resulting sediment was dissolved in 10 mM Tris-HCl buffer, pH 7.5, containing 0.1 M NaCI and 0.02% (w/v)

131 sodium azide, and applied to an Ultrogel AcA-44 column (2.6 x 90 cm) equilibrated with the same buffer. The flow rate was maintained at 40 ml/h. Fractions containing ribonuclease activity were pooled and adjusted to pH 5.5 by adding 1 M acetic acid. The clear solution was loaded on a column of Phosphocellulose P-32 (1.6 × 18 cm) equilibrated with 50 mM Tris-acetate buffer, pH 5.5, containing 0.1 M NaC1. The column was eluted with the same buffer until the A28o of the effluent was lower than 0.1. A linear gradient (2 X 100 ml, Tris-acetate buffer, 0.1-1.0 M NaC1) was then applied to the column. The flow rate was maintained at 20 ml/h. Fractions were analyzed for alkaline ribonuclease activity.

Analysis of intermediates on the RNAase catalysis This was studied by using poly(C) instead of yeast RNA in the assay. In this case, the assay buffer was 0.1 M ammonium acetate, pH 8.3, and the incubation time was increased to 24 h. The resulting mixture was dried-down in a Speed-Vac (Savant) and the residue was twice washed with glass-distilled water. The residue was then chromatographed on (20 × 20 cm) silica gel 60 F254 plates using 0.1 M HaBO3/25% (v/v) NH 4OH/isopropanol (3:1:6, v/v) as the chromatographic system. Suitable reference nucleotides were simultaneously chromatographed. The plates were visualized under ultraviolet (254 nm) light. The exo- or endonuclease character of the enzyme was studied by analysis of the molecular size of the degradation products at different incubation times, first considering the differential precipitation of high molecular weight oligonucleotides by uranyl acetate [28,29] and, second, by gel-filtration chromatography of the reaction mixture [30,31]. These determinations were performed with purified yeast RNA as substrate. Analytical procedures Protein concentration was determined according to Ref. 32. Electrophoresis on 12% polyacrylamide slab gels in the presence of 0.1% (w/v) SDS was performed as described [33]; suitable marker proteins were used as reference for molecular weight determination. Gels were prepared containing 6 mg/ml purified yeast RNA (Mr> 300000, Ref. 1) when staining for ribonuclease

activity was required [34]. The hydrodynamic radius of the insect ribonuclease was determined by chromatography on an Ultrogel AcA-54 column calibrated with reference proteins [35]. To determine the amino acid composition, protein samples, about 10 #g, were hydrolyzed in evacuated and sealed tubes with 1 ml of constant boiling HC1 for 24 h at 108°C. Protein hydrolysates were analyzed on a Durrum D-500 amino acid analyzer. The circular dichroism spectrum of the enzyme was obtained on a Jobin Yvon Mark III dichrograph, at 0.5 n m / s scanning speed. E1lipticity values are expressed in units of degree. cm2. dmol-1 per residue considering 108 as mean residue weight for this protein (see amino acid composition). Results and Discussion

Purification of the enzyme A summary of the purification procedure is given in Table I. The elution profiles of the chromatographic steps on Ultrogel AcA-54 and phosphocellulose P-32 are given in Figs. 1 and 2, respectively. The purified enzyme is obtained in a 14% yield; a typical isolation procedure renders about 60 /xg of enzyme from 100 g of biological material. These data indicate that the levels are quite low, as occurs for most intracellular RNAases. The specific activity of the purified enzyme is 4700 units/mg. This value is about 10-times lower than that of bovine pancreatic RNAase in the same assay conditions, but it is similar to that of intracellular mammalian RNAases [1]. After the last purification step, the enzyme appears as a single band on SDS-polycarylamide gel electrophoresis by both silver and ribonucleolytic activity staining methods (Fig. 3). Thus, the purified alkaline ribonuclease is considered as a homogeneous preparation. Enzymic characterization The influence of pH and ionic strength on the activity of the alkaline ribonuclease from insect is given in Fig. 4. The optimum pH value for the degradation of yeast RNA is 8.0-8.3, based on which this enzyme is considered alkaline ribonuclease. The enzyme exhibits maximum activ-

132 TABLE I P U R I F I C A T I O N O F A L K A L I N E R I B O N U C L E A S E F R O M C. C A P I T A T A Step

Units

Protein (mg)

Spec. act. (units/mg)

Purification (-fold)

Yield (%)

Homogenization Acid and heat treatments Amberlite CG-50 Ultrogel AcA-44 Phosphocellulose

1980 1800 1200 900 280

5 400 2 700 250 25 6.10 2

0.37 0.67 4.80 36.00 4700.00

1 2 13 97 12700

100 91 61 45 14

ity at 0.05 M NaC1 when the pH of the assay mixture is 8.3. This optimum value is almost coincident with that observed for pancreatic RNAase A [35] as well as for intracellular RNAases from mammalian tissues [1]. No activity is observed at 0.55 M ionic strength. The activity of the insect ribonuclease is not modified by the presence of EDTA up to 5 mM concentration. Thus, the enzyme would be non-dependent on divalent cations. In fact, Ca 2+, Mg 2+, Mn 2+, Cu 2+ and Zn z+ do not modify the enzyme activity up to 1 mM concentration. However, 5 mM Cu 2+ or Zn 2+ completely inhibits the insect RNAase. This fact has also been observed for many RNAases [4]. The effect of Ca 2+, Mg 2+ and Mn 2+ at 5 mM concentration is a partial inhibition of the enzyme. This could be interpreted in terms

(u/ml)

A21o

l

1.6

1.2

0.8

25

of conformational changes on the RNA substrate, resulting in a structure resistant to the enzymic attack [36]. The exo- or endonuclease character of the alkaline RNAase has been studied by two methods. The first one is based on the differential precipitation of oligonucleotides by uranyl acetate [28,29]; the other is a chromatographic analysis of the molecular size of the degradation products at different reaction times [30,31]. According to both procedures, the alkaline ribonuclease from insect is shown to be an endo- enzyme. The hydrolysis of yeast RNA occurs through oligonucleotides as intermediates, yielding limited-size fragments. The specificity of the enzyme has been studied by using homopolyribonucleotides as substrates. When poly(A), poly(G), poly(U) and poly(C) are

(u/ml)

A2~o 0.15

10 20

,"L

1 o (M)

f.j 15

.]0.8 -]0.6

005 / I

/

6

4

flO.4 5

0.4

__J

,,'5., ',,.J...,

2 "t0.2

, 10

20

30

40

50 FRACTION

10

60 No.

Fig. 1. Elution profile of the chromatography on Ultrogel AcA-44. Fractions were collected after 100 ml were eluted from the column. - - , absorbance at 280 nm; e, enzyme activity in u n i t s / m l . Shaded zone corresponds to the pooled fractions containing alkaline ribonuclease activity.

20

30

40

50

60

FRACTION

No.

Fig. 2. Elution profile of the chromatography on phosphocellulose P-32. - - , absorbance at 230 nm; e, enzyme activity in u n i t s / m l ; . . . . . , NaCI linear gradient. Shaded zone corresponds to the pooled fractions containing alkaline ribonuclease activity.

133 0.1

used, only the last one is hydrolyzed under the standard assay conditions. The m a x i m u m hydrolysis rate for poly(C) is 1.30°times that of yeast RNA. However, base specificity is not an absolute concept, since by increasing the reaction time and enzyme amount such specificity can be modified; for instance, this occurs for RNAase A [37,38]. It also occurs for the alkaline ribonuclease from insect; in fact, increasing both the reaction time and the enzyme amount present in the assay up to 20 h and 20-fold the values of the standard assay, poly(U) is also degraded. Poly(G) and poly(A) are not degraded under these extreme conditions. Thus, the alkaline ribonuclease from insect exhibits preferentiality towards pyrimidine bases. The sugar specificity of the insect enzyme has been studied by considering calf thymus D N A as substrate. The enzyme hydrolyzes neither native nor thermally denatured DNA. Thin-layer chromatography of the end products of the insect enzyme action reveals that this is a cyclizing RNAase. In fact, the products obtained for the hydrolysis of poly(C) at long periods of

I

0.2

0.3

0.4

0.5

I

I

I

I

M

8O I'-

_> 6 0 - I

I

i

< ~

40

20

,

5

6

7

8

9

pH

Fig. 4. Influence of pH ( .... ) and iomc strength (M NaC1 at pH 8.3) (-- -- --) on the activityof purified alkaline RNAase from C. capitata. Values are expressed as percentage of the activity under standard assay conditions and they represent the averages of three different determinations. When the influence of pH was studied, the ionic strength was adjusted to 0.1 M.

reaction are 2',Y-cyclic CMP as well as the corresponding Y-nucleotide. Finally, no phosphatase activity has been observed for the insect alkaline RNAase. According to these observations, the alkaline ribonuclease from insect may be considered as RNAase I [4].

Fig. 3. Polyacrylamide gel electrophoresis in the presence of SDS. (A) Silver-stain [43]: 1, reference proteins (a, aqactalbumin, Mr 14400; b, soybean trypsin inhibitor, 20100; c, carbonic anhydrase, 30000; d, ovalbumin, 43000; e, serum albumin, 67000; f, phosphorylase b, 94000); 2, bovine pancreatic RNAase A, and 3, insect alkaline RNAase, both reduced with 2-mercaptoethanol. (B) Ribonucleolytic activity detection [34]: 1, 100 pg RNAase A; 2, insect alkaline RNAase with activity equivalent to 50 pg RNAase A; 3, 50 pg RNAase A. For this last detection procedure, samples were not reduced.

Molecular characterization The molecular weight of the insect RNAase determined by chromatography on calibrated colu m n of Ultrogel AcA-54 is 17200. When this determination is performed by SDS-polyacrylamide gel electrophoresis of the reduced protein, the value obtained is 18700. Thus, the alkaline ribonuclease from C. capitata is a single-polypeptide-chain protein. The calculated hydrodynamic radius is 20.9 +_ 1.5 ,~ and the diffusion coefficient is 10.3.10 -7 c m 2 . s -t. The partial specific volume determined from the amino acid composition of the protein (see below) [39] is 0.710 cm3/g. Accordingly, the calculated frictional ratio for the native protein is 1.20. The circular dichroism spectrum of the protein in the peptide-bond region is given in Fig. 5. Analysis of this spectrum according to the method

*

134

of Chen et al. [40] indicates that the secondary structure of the protein is composed of 19% a-helix, 10% fl-structure, 71% aperiodic conformation with an average number of residues per helical segment of 9. Analysis of the secondary structure of RNAase A according to the above procedure [40] yields 20% t~-helix, 35% fl-structure, and an average number of residues per helical segment of 15. The amino acid composition of the insect enzyme is given in Table II. Although a large number of RNAases I have been studied in terms of their amino acid composition, they mainly correspond to pancreatic RNAases [6], not to intracellular RNAases. The amino acid composition of the intracellular RNAase I from bovine brain has been determined [41] and it is also given in Table II for comparison. Latent alkaline ribonuclease activity The ribonuclease from the insect C. capitata 210

220

230

2 4 0 nm

T A B L E II AMINO ACID COMPOSITION R I B O N U C L E A S E F R O M INSECT

OF

ALKALINE

N.D., not determined.

Asx Thr Ser Glx Pro Gly Ala Cys a Val Met lie Leu Tyr Phe His Lys Arg Trp

Molar (%)

Residues b

Bovine brain c

9.82 7.28 10.40 11.29 6.44 8.35 7.78 4.72 5.82 2.22 2.99 3.79 4.21 2.56 2.52 5.99 3.80 ND

17 13 18 20 11 15 14 8 10 4 5 7 7 4 4 10 7 -

10.1 6.6 9.9 8.8 8.2 4.8 5.8 3.0 6.3 2.3 2.1 4.0 3.9 2.4 4.5 9.7 6.4 1.2

a Determined as cysteic acid after performic acid oxidation of the protein [42]. h Values calculated based on a molecular weight of 18700 and referred to the nearest integral value. ~ Expressed as residues per 100 [41].

0~16 3 -2

--4

-6 i

--8' I -10

can be clearly considered as an RNAase I according to the above-described characterization. The similarities between this protein and other RNAases I can be observed even considering the reduced number of intracellular RNAases I fairly

T A B L E III A C T I V A T I O N O F L A T E N T RNAase BY -SH R E A G E N T S

-12

Fig. 5. Circular dichroism spectrum in the 200-250 n m wavelength range of the alkaline ribonuclease from insect, e, ellipticity values calculated with the reference parameters of Chen et al. [40] for a secondary structure composed of 19% a-helix, 10% fl-structure, 71% aperiodic conformation with an average n u m ber of residues per helical segment of 9. The protein sample was dissolved in 20 m M Tris-HC1, pH 7.4, containing 0.1 M NaCI, and it was filtered through Millipore (0.5 /tm pore diameter) prior to the spectroscopic study.

GSH, reduced glutathione; GSSG, oxidized glutathione; pHMB, p-hydroxymercuribenzoate. Values are expressed as percentages _+S.D. of the activity in the absence of the modifying agent.

$2082 $20 8 Cu 2+ Cu 2+ pHMB GSSG/GSH

Concentration (mM)

Percent activity

0.9 4.5 0.9 4.5 0.5 0.34/0.17

305 + 92 310 + 102 410_+ 38 1 210_+293 267+ 121 244_+ 34

135

80

60

,o

20

I O.5

I 1.0

I 1.5

INHIBITOR UNITS Fig. 6. Inhibition of alkaline RNAase from insect by RNAase inhibitor from human placenta. Purified alkaline RNAase (0.5 units) was incubated with different amounts of RNAase inhibitor from human placenta (Sigma). One unit of inhibitor is defined as the amount required to produce 50% inhibition of 5 ng RNAase A in the standard assay conditions. Values are referred to the enzyme activity in the absence of inhibitor ±S.D. characterized. The existence of a specific R N A a s e inhibitor has been described for m a n y of these enzymes. This observation is based on a structural property of these inhibitors; they are proteins containing essential free -SH groups and their inactivation by -SH reagents revealed the existence of latent R N A a s e activity [1,7-16]. Taking these facts into consideration, insect homogenates have been investigated in order to determine the existence of latent RNAase. M a n y reagents of -SH groups have been studied. The results obtained are given in Table III. $208z- , Cu 2÷, glutathione and p - h y d r o x y m e r c u r i b e n z o a t e have been d e m o n strated to be effective in producing an increase in the total R N A a s e activity present in the insect homogenates. Taking into account the fact that these agents do not modify the activity of the purified alkaline RNAase, the above results reveal the existence in insect homogenates of alkaline R N A a s e in a latent form. This m a y be interpreted in terms of the presence of an inhibitor dependent on free -SH groups. Addition of purified alkaline R N A a s e to insect homogenates does not result in any inhibition.

This suggests that the a m o u n t of free inhibitor at the studied insect stage of development ( 4 - 5 - d a y larvae) is quite low. Finally, addition of commercial R N A a s e inhibitor from h u m a n placenta to purified insect alkaline ribonuclease results in inhibition of the enzyme (Fig. 6). This fact is additional p r o o f of the similarity between alkaline R N A a s e from insect and R N A a s e s I from mammalian tissues. Results herein reported constitute the first characterization of an alkaline R N A a s e from insects. But it is much more important that its properties and its susceptibility to the action of m a m m a l i a n R N A a s e inhibitors, as well as the existence of endogenous inhibitors, indicate a widespread distribution of the RNAase-inhibitor system in the animal kingdom. Acknowledgments Authors are indebted to Professor A.M. Municio for his interest and help at all times. This work was supported by grant 6 3 7 / 8 1 from the C A I C Y T (Spanish Government). References

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