Enzymes of chitin metabolism in the decapod, Hemigrapsus nudus

Comp. Biochem. Physiol., 1971, Vol. 40B, pp. 757 to 779. Pergamon Press. Printed in Great Britain

ENZYMES OF C H I T I N METABOLISM IN T H E DECAPOD, HEMIGRAPSUS NUDUS LYLE A. HOHNKE* University of Oregon, Eugene, Oregon (Received 22 March 1971) Maximal enzyme activity profiles for glycogen phosphorylase, glycogen synthetase and chitin synthetase have been obtained from tissue extracts of Hemigrapsus nudus and correlated with the molt cycle. 2. Glycogen phosphorylase activity was maximum in early intermolt (C1) in the muscle and integument and in early postmolt in the hepatopancreas. Peak glycogen synthetase occurred in late intermolt (C,) in muscle. 3. Chitin synthetase activity increased in early premolt (D 1) and early postmolt (A). An apparent K,n value of 1.7 x 10-* M was determined. 4. These data support a correlation between glycogen and chitin metabolism and relate metabolic events to molting in H. nudus.

Abstract--1.

INTRODUCTION CARBOHYDRATEmetabolism in crustaceans is typically cyclical with turnover related primarily to the intermolt cycle. The enzymes concerned in synthesis of chitin, the distinctive polysaccharide of arthropod integuments, have been studied in fungi (Glaser & Brown, 1955; Camargo et al., 1967) and insects (Candy & Kilby, 1962; Porter & Jaworski, 1965), but not in crustaceans. Considerable evidence exists for the incorporation of glucose into chitin (Scheer & Scheer, 1951; Hu, 1958; Meenakshi & Scheer, 1961 ; Hornung & Stevenson, 1969), but the proposed role of glycogen as a precursor (Renaud, 1949; Schwabe et al., 1952; Zaluska, 1959; Barnes, 1965) rests only on the coincidence of decreases in glycogen content with formation of chitin. Fat has been suggested as a possible source, in addition to glycogen, of carbon for chitin synthesis (Zaluska, 1959; Bade, 1962; Barnes, 1965; Porter & Jaworski, 1965). The present study reports assays of glycogen phosphorylase, glycogen synthetase, UDP-N-acetylglucosamine transferase (chitin synthetase) and acetyl coenzyme A in the major tissues of the crab, Hemigrapsus nudus, in relation to the intermolt cycle, with some observations on chitin synthetase in the crab, Cancer magister. The results are interpreted on the basis of possible relations among synthesis and breakdown of glycogen and synthesis of chitin. * Present address: Department of Physiology, University of California, School of Medicine, Los Angeles, California 90024.

757

758

LYLE A. HOHNKE

MATERIALS AND METHODS A. Animals Hemigrapsus nudus, the purple shore crab, was collected from the North Cove of Cape Arago or collected by the Oregon Institute of Marine Biology and transported to Eugene, Oregon for study. Collections were made frequently to avoid maintaining animals for extended periods under artificial conditions. Animals were maintained in a refrigerated room at 10°C in 2-3 in. of well aerated sea water which was changed weekly. B. Chemicals and supplies T h e following materials were used in this study: sodium bicarbonate-t4C, uridine diphosphate-N-acetylglucosamine 1-14C (UDP-14C-GlcNAc). E. coli B, ¼ log cells were purchased from Grain Processing Corporation in Muscatine, Iowa. Acetyl-CoA was generously provided by Dr. Allan Larrabee, Department of Chemistry, University of Oregon. C. Methods 1. Animal staging. This study utilized animals at all stages of the intermolt cycle as determined by the morphological criteria outlined by Drach (1939) and Kincaid & Scheer (1952). Premolt animals were staged by examination of the transparent cuticle of the epipodite of the first maxilliped under a low power microscope. 2. Glycogen phosphorylase assay. Phosphorylase activity was measured in the direction of glycogen synthesis by colorimetric determination of inorganic phosphate released from glucose 1-phosphate. The assay was described by Okuno et al. (1964). Inorganic phosphate determinations were made according to Fiske & Subbarow (1925). 3. UDPG-glycogen transglucosylase assay. The method of Leloir & Goldemberg (1960), with modifications by Wang & Scheer (1963), was used to determine glycogen synthetase activity. 4. Chitin extraction. Chitin was isolated from the dorsal carapace by a technique adapted from Hornung & Stevenson (personal communication). The carapace was first digested in 10% N a O H at 70°C for 18-24 hr. T h e deproteinized shell was then washed with distilled water and decalcified with about 15 ml of 90-100% formic acid for 1 hr. The shell was then transferred to 10% formic acid for 12-18 hr. The chitin was then washed several times with 95 % ethanol and finally with anhydrous ether. T h e ether was removed under reduced pressure in a desiccator. 5. UDP-t4C-GlcNAc transferase assay a. Tissue extracts. Extracts were made of the dorsal integument by rapidly removing the tissue and homogenizing in 2 ml of 0"60 m M Tris buffer, p H 7"7 containing 1-5 m M MgCI~ and 1"5 m M E D T A . The crude homogenate was centrifuged at 3000g for 15 rain to sediment cell debris. T h e resulting supernatant was centrifuged for 30 rain at 12,000 g. T h e pellet fraction was then carefully suspended in approximately 0"1--0-5 ml of I'0 M Tris buffer, p H 8'0 containing 0"1 M MgCI~ and stored on ice for assay. b. Assay. A representative incubation mixture contained 170 m M Tris, p H 8"0; 17 m M MgCI~; 17 m M GlcNAc; dextrins, 2"3 mg]ml; 2'0 m M UDP-a4C-GIcNAc (1.6 x 106 dis/ rain per/~mole) in a final volume of 0"03 ml. Reactions were started by the addition of homogenate and were termined by the transfer to a boiling water-bath for 3-4 rain. T h e chitodextrins used in the assay were prepared by desalting fraction I I obtained by fractional precipitation of chitin hydrolysates (Zechmeister & Toth, 1931), on a Sephadex G-10 column. Appropriate fractions were pooled and rechromatographed on a Sephadex G-25 column. Ten-ml fractions were collected and the conductivity and dry weight of each fraction was measured. Gel chromatography of the Zechmeister & Toth Fraction I I revealed two well-separated peaks. T h e first peak, containing the higher molecular weight

ENZYMES OF C H I T I N METABOLISM I N THE DECAPOD

759

dextrins, was examined further with descending paper chromatography. (Solvent: redistilled b u t a n o l - p y r i d i n e - w a t e r / 6 : 4 : 3). Subsequent visualization of the peak I mixture with chlorine gas and o-tolidine (Stahl, 1962) revealed two components corresponding to a pentasaccharide and trisaccharide (R1 = 0"10 and 0-24 respectively) cf. Barker et al. (1957). No further purification of this peak I was attempted; tubes containing this peak were pooled and lyophilized for later use. Subsequent to the assay, 0.02 ml of the reaction mixture was spotted on Whatman No. 1 paper and chromatographed (descending) overnight. (Solvent: isobutyric acid-1 N ammonium hydroxide/5 : 3.) Chromatography was performed in a hood at room temperature. This procedure separated products and reactants allowing further quantification. An autoradiogram scanner was used to locate radioactive spots. These were then cut out, glued to planchets and counted in a gas flow planchet counter (counting efficiency = 7 per cent). Total recovery of counts from the chromatograms was 45-55 per cent of a predicted 67 per cent. Chromatograms were also visualized with a u.v. lamp and the following sprays. a. Silver nitrate-sodium hydroxide. Reducing substances turn black immediately and non-reducing substances appear more slowly (Trevelyan et al., 1950). b. Ninhydrin. Amino sugars turn red (Payne et al., 1954). c. Acetylacetone-p-dimethylaminobenzaldehyde. Free hexose amines give cherry red spots; N-acetylglucosamine turns purple; 1,6 and 1,3 disaccharides with an N-acetylamino sugar reducing unit turn purple; similar 1,4 compounds do not react (Watkins, 1958). d. Benzidine-TCA. Reducing sugars turn brown; disaceharides give different colors depending on the position of the glycosidic link (Watkins, 1958). e. Aniline-diphenylamine. Aldohexoses, grey to brown; pentoses, green-blue; oligosaccharides give similar colors except 1,4 linked reducing aldohexoses, blue (Bailey et al., 1960; Schwimmer et al., 1956). 6. Chitinase digestion Completed reaction mixtures of UDP-X4C-GlcNAe transferase were incubated at 37°C for 6 hr with a commercial grade of fungal chitinase (2.0 mg/ml). T h e digestion was stopped in a boiling water-bath and 25 bd. of the resulting mixture was chromatographed as described. 7. Acetyl CoA pool estimations Estimates of the free acetyl CoA pool in the integument made use of the principle that acetyl CoA is carboxylated by CO ~, in the presence of A T P , M n 2+ and acetyl CoA carboxylase, to form malonyl CoA, the first intermediate of fatty acid synthesis: enzyme A T P + H C O s - + acetyl CoA ~ A D P + Pi + malonyl CoA. MnZ+ a. Tissue extracts. Extracts were prepared by rapidly removing the dorsal integument, blotting on moistened filter paper, weighing and homogenizing in 1 vol. (weight : volttme) of 55 m M imidazole-HCl buffer, p H 6"5. The homogenized tissue was rapidly decanted to thin-walled culture tubes and transferred to a boiling water-bath for 5 rain. Tubes were then cooled and centrifuged for 20 rain at 3000 g. T h e resulting supernatant was used for the assay. b. Enzyme preparation. Acetyl CoA carboxylase was prepared according to a modified procedure of Alberts & Vagelos (1968). Approximately 200 g of E. coli B calls were homogenized in a Manton-Gaulin submicron disperser. Cellular debris was removed b y centrifugation (3000 g, 30 min) and nucleic acids were precipitated by the addition of 0.05 vol. of 1 M MnCI8 to the supernatant. Nucleic acids were removed by centrifugation and the enzyme was precipitated with solid ammonium sulfate between 25-45% saturation at 4°C. T h e precipitate containing the enzyme was centrifuged at 37,000 g, 30 min, and the pellet was dissolved in a minimum volume of imidazole-HCl buffer. After dialysis overnight against 3 1. of the same buffer, the enzyme was diluted to 30-40 mg/ml and stored at 0°C.

760

LYLE A. HOHNKE

Dialysis was performed at 4°C, the enzyme was diluted with buffer and samples of the enzyme were quick-frozen in liquid nitrogen prior to storage at 0°C. c. Assay. Assays were conducted with the following reaction mixtures: 55 mM imidazole-HC1 buffer, pH 6"7; 0"44 mM MnCI~; 0.44 mM ATP; 14 mM NaHI*CO3 (1"0 #ci/ /zmole); tissue extract; and enzymes in a final volume of 0"09 ml. Incubation was 10-15 min at 33°C and the reaction was stopped with 0"01 ml of 2 N HC1. Aliquots (0"05 ml) of the reaction were dried on 2"2 cm Whatman No. 1 filter paper discs under a heat lamp. Discs were placed in scintillation vials containing 0"5 ml of water and 10 ml of Bray's solution (Bray, 1960) then counted in a liquid scintillation spectrometer. A standard curve for acetyl CoA was run with each pool determination. The counting rate was proportional to concentration over the range 3-778/zmoles acetyl CoA. Sample values were interpreted from the standard curve.

8. Protein determinations The method of Lowry et al. (195t) was used throughout this study. 9. Statistical evaluation Significance of results was evaluated in terms of the confidence limits of the means, based on Student's t-tests. RESULTS

1. Chitin analysis of dorsal carapace Table 1 lists the chitin extracted for six stages of the molt cycle as a percentage of the carapace dry weight. Values rise slightly in premolt then decline sharply as exuviation approaches. Postmolt animals have an elevated proportion of chitin TABLE 1--CHITIN CONTENTOF DORSALCARAPACE Stage of molt cycle

Chitin content (% dry wt.)

A B C1 C, D E

14"3 + 3'7" 9"5___0"5 9"5 + 0"7 10"7 + 0"02 12"7 _+0"4 4"9+2"7

* 95 per cent confidence limits. which declines to a relatively constant level in stages B-C1. T h e values reflect changes in calcification also and do not directly define the stages of the molt cycle when chitin deposition is occurring. T h e values do underline the dynamic changes occurring in the cuticle, all of which are related to the stage of molt cycle.

2. Glycogen phosphorylase (E.C. 2.4.1.1) Total phosphorylase activity was measured throughout the molt cycle for three different tissues--hepatopancreas, integument and muscle. T h e catalytically active forms of the enzyme which exist in most organisms, total a + b and active a, were measured by assay with and without added Y - A M P . T h e assay showed a

ENZYMES OF CHITIN METABOLISMIN THE DECAPOD

761

linear relation to incubation time and protein concentration for all tissues. Activity profiles for the hepatopancreas, integument and muscle are summarized in Figs. 1-3 respectively. GLYCOGEN PHOSPHORYLASE ACTIVITY-HEPATOPANCREAS J045-

i'

0,40 -

E ~. 35u~ ~ 300 :~25-

I V#

:t20 _

L

V~

~-15> F-Io-

// //

A

ct

B

C4

STAGE OF MOLT CYCLE

FIG. 1. Glycogen phosphorylase activity--hepatopancreas. Activity is expressed as /Lmoles of phosphate released per m g fresh weight of tissue. Shaded areas represent total activity (a + b); open areas represent activity of the active a form. Vertical bars are 95 per cent confidence limits.

GLYCOGEN PHOSPHORYLASE-INTEGUMENT 500

~. 4 0 0 bJ .J

300 ::L >-

> 200

<

I00

A

B

C~

C4

D

STAGE OF MOLT CYCLE

FIO. 2. Glycogen phosphorylase activity--integument. See Fig. 1 for a complete explanation of the figure symbols.

a. Hepatopancreas. Maximum activity was exhibited in early postmok (A and B). Early intermolt (C1) animals showed a significant decline in total activity followed by an increase in late intermolt. The mean activity in premolt animals (D) was lower than late intermolt (C4); the change of activity was significant (P = 0.1) although the range of values was also greater.

762

LYLEA.

HOHNKE

GLYCOGEN PHOSPHORYLASE-MUSCLE 3ooo

2500~2000 to bJ T

d Jsoo! ~ ,ooo ; .~ 500 A

B

CI

C4

D

STAGE OF MOLT CYCLE

FIG. 3. Glycogen phosphorylase activity--muscle. See Fig. 1 for a complete explanation of the figure symbols.

The percentage of enzyme in the active a form showed no significant change throughout the molt cycle, however, total activity (a + b) did change significantly (P = 0-05) from early postmolt (A and B), early intermolt (C1) and late intermolt (C4). The hepatopancreas showed the lowest total activity of the three tissues examined. b. Integument. Phosphorylase activity in the integument reached a maximum in early intermolt (C1) and declined in both premolt and postmolt animals (Fig. 2). Specific activity ranged from approximately six to twenty-five times that found in the hepatopancreas for corresponding stages of the molt cycle. Both a and b forms of the enzyme were found in the integumentary tissue but neither total activity nor the percentage of enzyme in the active a form changed significantly through the molt cycle. c. Muscle. Total glycogen phosphorylase in muscle tissue (Fig. 3) showed maximum activity in early intermolt and fell dramatically thereafter. Total activity, a + b, showed significant changes (P = 0.05) in stages A-C4; the changes in activity of the a form were also significant (P = 0.01) for A-B and C1-C 4. The percentage of enzyme in the a form also changed significantly in these stages. Total specific activity of glycogen phosphorylase in the muscle ranged from two to five times that found in the integument for corresponding stages of the molt cycle. The differences observed in total specific activity among the tissues were significant (P = 0.05) at all stages of the molt cycle except in premoh.

3. Glycogen synthetase (E.C. 2.4.1.11) Glycogen synthetase activity was measured in vitro for the hepatopancreas, integument and muscle. Values were obtained for nearly all stages of the molt cycle except C 4 of intermoh. This stage was not available at the time of experimentation. The C 4 value for muscle tissue (Fig. 6) is from Wang (1963) and is included for

763

ENZYMES OF CHITIN METABOLISM I N THE DECAPOD

purposes of illustration. Because activities were often low, most experiments were run with tissue pooled from two to four animals of the same stage. a. Hepatopancreas. Glycogen synthetase activity was present in both the I (glucose 6-phosphate independent) and D (glucose 6-phosphate dependent) forms as shown in Fig. 4. Assayed with glucose 6-phosphate present, total activity equals I + D. Maximum activity of the D form occurred in early C for the stages examined. This stage also gave the only indication of a catalytically active I form of the enzyme. Total activity of the enzyme was lower in this tissue than in integument and muscle for all stages examined. ~' 8 GLYCOGEN SYNTHETASE-HEPATOPANCREAS 22,

E

i

>" 6 F-

/// //,

p-

~4

//

'//

o LL bJ r, (/)

2

~ B

///

CI

C4

D

STAGE OF MOLT CYCLE

FIG. 4. Glycogen synthetase activity--hepatopancreas. Specific activity is expressed as milliunits of U D P released per mg protein. One unit equals the/maoles of U D P formed per min. Shaded areas represent total activity I + D; open areas represent the activity of the active I form. Values represent mean values of two or three experiments using tissues pooled from three to five animals. 2o

GLYCOGEN S Y N T H E T A S E - I N T E G U M E N T

E

/// /// F-

u._ 5 U3

...... A

B

/// C~

C4

O

STAGE OF MOLT CYCLE

FIG. 5. Glycogen synthetase activity--integument. See Fig. 4 for a complete explanation of figure symbols.

b. Integument. The pattern of synthetase activity in this tissue (Fig. 5) closely reflects that of the hepatopancreas (Fig. 4). Early intermolt (C1) showed greatest activity of the D form. There was no evidence of an active I form in any of the stages examined. Synthetase values for stages B and C1 are about double the corresponding values, in the hepatopancreas.

764

LYLE A. HOHNKE

c. Muscle. Synthetase activity was greatest in C 4 and was greater than corresponding values for integument and hepatopancreas. Both I and D forms of the enzyme were demonstable and the activity of the I form was always less than the D form. This was also true for the hepatopancreas and integument and is consistent with observations in the more widely studied mammalian systems. 4. UDP-I°C-GlcNAc transferase No insoluble radioactive material was formed when preparations of H. nu&ts extract were treated according to the published procedures of Glaser & Brown (1955) and Carey (1965). The procedure adapted did demonstrate the activity of an enzyme which catalyzes the transfer of 14C-GlcNAc from uridine diphosphate acetylglucosamine into a chitin-like product. Enzyme activity was correlated with most stages of the molt cycle and some properties of the enzyme were investigated in vitro. 40

GLYCOGEN SYNTHETASE-MUSCLE

E

~ 20

//// B

STAGE

CI

C4

D

OF MOLT CYCLE

FIG. 6. Glycogen synthetase activity--muscle. See Fig. 4 for a complete explanation of figure symbols. Partition paper chromatography of transferase reaction products in H. nudus did not correspond exactly with the distribution of chitin synthetase products in fungal systems (Camargo et al., 1967). In particular the chitin-like product had a different mobility under similar experimental conditions. Partial characterization of the chitin-like product suggests that UDpj4C-GlcNAc transferase in H. nudus can be identified with chitin synthetase in other systems. a. Transferase activity from different sources. A C. magister preparation produced tracings (Fig. 7a) nearly identical to those of fungal systems. The largest peak (A) is an untransformed substrate. The chromatographically immobile peak (B 1) has been identified in fungal systems as the chitin of at least six residues length. A small third peak (C) was identified by marker and sprays (a,b,c,d,e) as acetylglucosamine (see Table 2). Preparations of integument from H. nudus produced a different pattern (Fig. 7b). Peak B was of considerable interest because it exhibited a mobility different than that of C. magister and fungal systems and could not be demonstrated in other

+

+++

U.v.

0.61 0.72

+ +

0"32

0"28

++ -

0"15

RI

+++

Radioactive

N = n e g a t i v e reaction. P = p o s i t i v e reaction. X = positive reaction, significance u n k n o w n .

Compound No.

TABLE 2--QUALITATIVE

No

Yes

Yes

No

Yes

Identified by marker

P

P

P

X

N

a

X

P

N

N

N

b

X

P

N

N

N

c

P

P

N

N

N

d

Spray No.

S U M M A R Y OF C H R O M A T O G R A M A N A L Y S I S

X

P

N

N

N

e

Diacetylchitobiose

GIcNAc

UMP

Chitin

UDP-14C-GIeNAc

Suggested identity

Ln

o

t~ ¢3

r~

0

¢3

O

z

t~

766

L'CLEA. HOHNKE

preparations. Only a substrate peak (A) was evident in controls (Fig. 7c) containing no homogenate or boiled homogenate; this pattern was also observed in whole animal preparations of juvenile crickets (dchaeta domesticus) and muscle tissue of

H. nudus. o.g 0.8 07

o6

!----+--~

0.4 0.3 0.2

0

FIG. 7a. Chromatogram scanner tracing---C, magister. F = solvent front; O= origin. See text for an explanation of scanner peaks. 1.0 0(3 0.8 Ogg 0.6 0.5 O.n 0.3

i

- -

0.2 OA 0

FIG. 7b. Chromatogram scanner tracing--H, nudus. See Fig. 7a and the text for an explanation of the symbols. Digestion of completed assay mixtures with fungal chitinase resulted in an increased level of radioactivity in peak C (Fig. 7d). Peak C, containing N-acetylglucosamine, did not show elevated levels of radioactivity when controls containing water, lysozyme or s-amylase were incubated under similar experimental conditions. b, Further characterization of chromatographically separable substances. Reaction products and unreacted substrates were located and identified on the chromatography strips by their reaction with sprays, detectability with u.v. light and comparison with markers. Strips viewed under u.v. light showed two dark spots corresponding to UDP-14C-GlcNAe (R I = 0.15) and U M P (Rj = 0.32). Both compounds were eo-chromatographed with markers for more positive identification. U D P had a mobility identical to that of UDP-14C-GIcNAc in this solvent system. Strips sprayed with the silver nitrate method (1) revealed another substance of high chromatographic mobility (R t = 0.72). Slight amounts of radioactivity were

767

ENZYMES OF CHITIN METABOLISM IN THE DECAPOD

detectable with this spot when strips were sectioned and counted on a gas flow planchet counter but were obscured in scanner tracings. Activity was so low relative to background that reliable counting was not possible. The high mobility and low radioactivity suggests diacetylchitobiose, a chitin intermediate in fungal systems. 1.0 0,9 08 0.7 0.6 0.5 0.4 0,3

A L~.

0.2 0.1

rv~o

G

FIc. 7c. Chromatogram scanner tracing--control. See Fig. 7a and the text for an explanation of the symbols.

/

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

J

0.1 0

'/

J, \F "',-" X~r v

O

F

FIG. 7d. Chromatogram scanner tracing. Six-hr chitinase digest of H. nudus assay mixture. See Fig. 7a and the text for an explanation of the symbols.

A suitable marker for this compound was not available and its identity must remain speculative. In Table 2 a qualitative summary of the chromatogram analysis is given for the five substances found. The compounds are listed (Table 2) in ascending order of R t values. Compound 2 corresponds to peak B (Fig. 7b). The chitin-like product was not detectable by u.v. light and had high levels of 14C-activity. Since the label was in the hexose ring hydrolysis of a labeled acetyl moiety could be discounted. Failure of peak B to fluoresce under u.v. light indicated cleavage of the esterified

768

LYLE A. HOHNKE TABLE 3--THE EFFECT OF DELETING SUBSTRATEB ON ENZYME ACTIVITY

Component deleted

Activity (dis/min)

None Homogenate Acetylglucosamine Dextrins

Percentage of standard reaction

9321 396 8564 14,854

-4 92 160

aldose from UDP and its low chromatographic mobility with respect to GlcNAc further suggested incorporation into an oligosaccharide. A slight amount of UDPJ4C-GlcNAc degradation did occur as evidenced by the radioactivity of compound 4 (Table 2). The dextrin primer used in the assay, when chromatographed alone in isobutyric acid-ammonium hydroxide (5:3), revealed only one spot (R! = 0.42). Amounts larger than those used in the assay had to be spotted, however, and no general comment can be made concerning the chromatographic behavior of the dextrin under standard reaction conditions. c. Effect of deleting various substrates on enzyme activity. Activity was stimulated by the addition of acetylglucosamine and slowed by the addition of dextrins. The increased activity of dextrin-free incubations does not correspond to observations on fungal systems (Glaser & Brown, 1955 ; Camargo et al., 1967). 10 ~- CHITIN SYNTHETASE

8k

/-

j/

DPM (×I03)

2L

/

/

I //

¥

4 PNOTEtN(mg/ml)

6

FIG. 8. Chitin synthetase activity--H, nudus. The graph depicts an experimental plot, each point representing the mean value of a triplicate determination.

d. Incorporation of 14C-GlcNAc into peak B. Incorporation of radioactivity into peak B (Fig. 7b) was proportional to protein content up to 3.5 mg/ml final concentration (Fig. 8) and to time for at least 90 rain (Fig. 9). All assays of maximum transferase activity were conducted within the limits of these parameters. When the substrate (UDpj4C-GlcNAc) was made limiting and the reciprocal reaction velocity plotted against the recriprocal of substrate concentration, an apparent

769

ENZYMES OF CHITIN METABOLISM IN THE DECAPOD

K,,, value of 1.7 x 10 -4 M was obtained (Fig. 10). This is approximately an order of magnitude lower than the reported values in fungal systems (Glaser & Brown, 1955 ; Camargo et al., 1967).

25 CHITINSYNTHETASE 20 15

DPM (x103110 5

. . . . . . . .

3'o

do TIME(rain)

FIG. 9. C h i t i n synthetase activity--H, nudus. The graph depicts a regression plot

of activity vs. incubation time. Samples were run in triplicate.

f CHITIN SYNTHETASE

I/V XlO-4 3 2 I

I/S FIC. 10. Chitin synthetase activity--H, nudus. The graph represents a Lineweaver-

Burke regression plot of chitin synthetase activity vs. UDP-14C-GlcNAc concentration.

e. UDP-z4C-GlcNAc transferase activity vs. stage of molt cycle. Assays of transferase activity were run on four stages of the molt cycle (Fig. 11). Activity was maximal in early postmolt (A) and declined steadily through Ct. Values for C4, not measured in these experiments, might be expected to be very low. With the onset of the premolt condition transferase activity begins to increase and the mean value is nearly equal to stage B animals. Because of the limited number of experiments for each stage of the molt cycle, statistical treatment of the data was not feasible. The values (Fig. 11) are mean

770

LYLE A. HOHNKE

values obtained in two or three experiments using pooled integuments of two to five animals of the same stage for each experiment. 8 CHITIN SYNTHETASE

nMOLES mg

4! 2

A

B

Ct

64.

D

STAGE OF MOLT CYCLE FIc. 11. Chitin synthetase activity--H, nudus. Activity is expressed as n-moles of product formed per mg protein. Histograms represent mean values; see text for an explanation.

5. Acetyl CoA estimations Changes in the concentration of acetyl CoA in the integument were measured enzymatically. Since acetyl CoA is a universal metabolite an enzyme involved in naturally occurring metabolic transformations was used for its detection. The linear relation of acetyl CoA carboxylase activity to increasing concentrations of purified acetyl CoA is shown in Fig. 12. This relation was true for the final enzyme concentration used: 1-3.5 mg/ml. Since it was desirable to measure acetyl CoA 12001ACETYL-CoA CARBOXYLASE ///

900!

//

/

//

DPM

I

600}

// -t

/

I0

20

•

//

30 40 50 60 ACETYL- CoA (riM)

70

80

90

I00

FIG. 12. Acetyl CoA carboxylase activity. The graph depicts a regression plot of enzyme activity vs. acetyl CoA concentration.

concentrations in extracts of H. nudus integument, experiments were performed to test the validity of the assay on this material.

E N Z Y M E S O F CHITIN M E T A B O L I S M

771

IN T H E D E C A P O D

a. Preliminary tests of assay. T h e effect of increasing doses of integumentary extract was a linear increase in 14C incorporation (Fig. 13). T h e curve, extrapolated to the zero added extract, intersects the y-axis near zero suggesting a m i n i m u m amount of interference. T h i s was not true for extracts not thoroughly heated. T h i s suggests that other reactions are competing for the available acetyl CoA and that thorough inactivation of these reactions is critical. 500

Acetyl- CoAcarboxyMse /

e

40C E 0.

'1o

3oc ~ * 20C I00 - -

/

0

~,e 5

I0

15

/.Lt extract

20

25

added

FIG. 13. Acetyl CoA earboxylase activity--dose: Response plot. Solid line is an experimental plot of activity vs. amount of boiled extract added. The extrapolated portion of the curve is represented by a broken line. Samples were run in triplicate, W h e n extracts were added to standard amounts of purified acetyl CoA (Table 4), the results were not additive for volumes in excess of 10/~I. Doubling the a m o u n t of extract f r o m 10 to 20 #1. reduced the predicted activity nearly 50 per cent; addition of 30/~1. extract increased predicted values slightly. T h e s e results TABLE

d----EFFECT O F A D D E D E X T R A C T T O A C E T Y L - C o A

Volume added (#1.)

Activity (counts/min)

Extract

Standard

10 20 30

- -

- -

---

---

Predicted

Obtained

15

--

- -

2 5

- -

31 389 621

10 20 30

25 15 5*

706 500 165

665 215 96

5 *

* Diluted standard.

- -

%

85 111 134

--

- -

26

STANDARDS

94 43 58

772

LYLE A. HOHNKE TABLE 5 - - F R E E ACETYL C o A IN H . nudus INTEGUMENT

Stage of molt cycle Intermolt

Postmolt

Acetyl CoA (n-moles/mg protein) 96 (5) 52 (5) 63 (5) 78 (5)

72

93 (4) 69 (5) 67 (5)

76

suggest that increasing the amount of extract also increases the rate of side reactions in the extract. b. Acetyl CoA in intermolt stages The concentration of acetyl CoA in the integument was determined in the boiled extract for intermolt (C4) and postmolt (B) animals (Table 5). It was necessary to pool the integuments of several animals to obtain sufficient extract. The slight increase in mean acetyl CoA concentration shown between intermolt and postmolt animals was not significant. DISCUSSION Many observations on carbohydrate metabolism in crustaceans stress the variation in metabolism with the nutritional state and stage of the molt cycle (see reviews, Waterman, 1960; Huggins & Munday, 1968; Hohnke & Scheer, 1970). Considerable interest has been focused on the role of glycogen as an energy store since glycogen in crustaceans shows an unusual stability in relation to nutritional status, and undergoes cyclic accumulation-depletion related to molting (Renaud, 1949; Travis, 1955; Martin, 1965; Adiyodi, 1969). Physiologically, some crustaceans also show regular changes in blood sugar levels which correlate with molting. Most studies have demonstrated a premolt increase in blood sugar (Telford, 1968; Parvathy, 1970) with a subsequent decline following the molt. It has been suggested that this pattern may be related to the utilization of blood sugars in the cuticular synthesis of chitin. This idea is supported by tracer studies in Panulirusjaponicus, P. penicillatus (Scheer & Scheer, 1951), H. nudus (Hu, 1958; Bergreen et al., 1961) and Orconectes obscurus (Hornung & Stevenson, 1969) which demonstrated 14Cglucose incorporation into chitin. This pattern may have exceptions. McWhinnie & Scheer (1958) did not find augmented blood glucose levels in premolt H. nudus; rather maximum values appeared in early intermolt (C1). Hu (1957) demonstrated 14C-glucose incorporation into chitin, however, in newly molted H. nudus (presumably A or B stage).

ENZYMES OF C H I T I N M E T A B O L I S M I N THE DECAPOD

773

The physiological fluctuations in blood sugar levels throughout the molt cycle have been related to possible endocrine effects. Wang & Scheer (1962, 1963), in addition to establishing the presence of the UDPG-glycogen transglucosylase system in crustaceans, found that eyestalk extracts from H. nudus inhibited transglucosylase in muscle from the same organism and also in a number of tissues from animals of other phyla, including molluscs and vertebrates. Partial purification of extracts (Ramamurthi et al., 1968) has led to a factor which inhibits highly purified transglucosylase preparations from rat liver. Ramamurthi et al. (1968) also showed that eyestalk ablation, a procedure which removes a significant portion of the neuroendocrine tissue, is followed by increased transglucosylase activity in muscle of H. nudus and C. magister. Glycogen synthesis in the hepatopancreas is also stimulated. Injection of the partly purified factor restores both transglucosylase activity and glycogen synthesis to near normal levels. Phosphorylase activity has been shown not to be affected by this factor (Ramamurthi et al., 1968). However, Keller (1965) reported partial inactivation of phosphorylase after eyestalk removal from Cambarus affinus Say and reversal of this effect by the injection of eyestalk extract. The available evidence suggests that endocrine factors exist in crustaceans capable of controlling both glycogenesis and glycogenolysis. Within this physiological framework the variations in phosphorylase activity throughout the molt cycle and among the three tissues are interesting for several reasons. In nearly every activity profile (Figs. 1-6) the total enzyme activity is lowest at premolt and early postmolt with subsequent increases in early and late intermolt. One interpretation would be that the increasing titer of molting hormones is acting as a brake on the animal's metabolism of glycogen. The glycogen phosphorylase and glycogen synthetase results (Figs. 4-6) suggest this may occur in at least two ways: (1) lowering of maximal enzyme activities during the ecdysial period and (2) lowering the percentage of enzyme in the active form. The complexity of the phosphorylase system must be recognized in the second suggestion. Phosphorylase exists mainly in two interconvertible forms, a and b. Which form predominates at any given time depends on the relative activities of two converting enzymes believed to be the focal point of actual regulatory control by hormone mediated cyclic AMP (Fischer et al., 1968; Krebs et al., 1968; Sutherland et al., 1969; Rasmussen, 1970). The sequence of events leading to glycogen degradation, now well documented in mammals (Cori et al., 1956; Posner et al., 1965), is that an increased level of cyclic AMP, resulting from hormone action on adenyl cyclase, activates phosphorylase kinase. A conversion of phosphorylase b to a follows with concomitant glycogenolysis. The presence of at least part of this system in crustaceans (Cowgill, 1959a, b; Ramamurthi et al., 1968; Sagardia, 1969) gives the variation in phosphorylase activity observed throughout the molt cycle a physiological importance. Chemical evidence is presented for both an a and b form of phosphorylase in two additional tissues--hepatopancreas and integument. The low activity of muscle phosphorylase a in early postmolt followed by significant increases in the proportion of a to b suggest the presence of controlling factors analogous to vertebrate systems. This regulation may be used physiologically

774

LYLEA. HOHNKE

by the organism as a gating mechanism responsive to energy and synthetic needs. The presence and need of a regulatory mechanism is also suggested by the observation that in muscle, maximum glycogen phosphorylase and synthetase activity (Figs. 3 and 6 respectively) do not coincide. Although C I and C 4 are not far removed graphically, there is a large time factor involved. C i lasts days in H. nudus while C 4 is prolonged over a period of several months. This is economically important to the organism because it limits useless recycling by setting up a unidirectional flow of substrate. An even finer control than staggering the rates of maximal activity of phosphorylase and synthetase is possible because glycogen synthetase is also present in two interconvertible forms. This system is important biologically in mammalian muscle (Lamer et al., 1968) although its significance in liver is questionable (Hers & DeWulf, 1958). The presence in crustaceans of phosphorylase and synthetase and their analogy to mammalian systems has physiological implications. Cyclic AMP, a positive effector on the enzyme phosphorylase, is a negative effector on synthetase (Lamer et al., 1968). This inverse polarity is a further safeguard against recycling of substrate. Extending and summing the glycogen phosphorylase and synthetase results, I would therefore predict the possible presence in crustaceans of the adenyl cyclase system (Butcher et al., 1968) which mediates and co-ordinates the action of molting hormones. The peak glycogen synthetase activity in C 4 also needs some explanation since physiological studies (Renaud, 1949; Scheer, 1959, 1960; Scheer & Scheer, 19S9) have indicated maximum glycogen accumulation during premolt, not intermoh. Two possible interpretations would appear to reconcile this difference. One possibility is that biochemical events associated with molting precede distinguishable morphological stages by a substantial period of time. What is not obvious in this interpretation is the amount of time needed for biochemical changes to bring about their associated morphological modifications. A second interpretation is that H. nudus represents an atypical situation and there is some evidence from blood sugar observations on the molt cycle that support this view. Many characteristics of UDP-14C-GIcNAc transferase suggest an identity with chitin synthetase. The ability of chitinase to effect partial degradation of the product is convincing evidence that a chitin-like oligosaccharide is being produced. The chromatographic mobility suggests that if peak B (Fig. 7b) is chitin, H. nudus may make a structurally different variety (e.g. shorter). The increased incorporation of 14C-GIcNAc into product by deletion of dextrins is a departure from similar studies on fungal systems. It is possible that the length of primer used is very critical and that by adding a modified primer a dilution of naturally occurring primer in the homogenate occurred. The reaction with a variety of sprays and the presence of a reducing substance with high chromatographic mobility (R I = 0.72) are analogous to findings of in vitro studies on fungal systems. The general ability of the highly mobile substance to react with a variety of sprays is consistent with the findings of Camargo et al. (1967) who reported an R / = 0.89 for diacetylchitobiose in the solvent system used. The rather weak ability of the product in peak B (Fig.

ENZYMES OF C H I T I N M E T A B O L I S M I N THE DECAPOD

775

7b) to react with indicators of reducing sugars, except silver nitrate-sodium hydroxide, aldoses and hexosamines strongly suggests an oligosaccharide of undetermined length. The activity of the UDP-14C-GlcNAc transferase showed a profile consistent with t4C-glucose incorporation studies (Meenakshi & Scheer, 1961 ; Hornung & Stevenson, 1969). The lack of correlation with glycogen phosphorylase profile suggests that naturally occurring chitinases may provide the bulk of substrate for new chitin production in H. nudus. The chitinolytic systems of Cancer and Maja continually synthesize chitinase and in premolt a marked increase of chitobiose is correlated with the impending ecdysis (Jeuniaux, 1965). An alternative fate of UDP-14C-GlcNAc, if chitin is discounted as being the product is mucopolysaccharide. Meenakshi & Scheer (1959) have found an acid mucopolysaccharide in the cuticle and digestive gland of H. nudus. Hydrolysis of the mucopolysaccharide yielded fucose, galactose and glucose. These sugars were not detectable in the assay controls or in chromatographic analysis of crude integumentary extracts in the present study. This would not rule out their presence but would suggest a very low concentration. The experiments related to measurements of the acetyl CoA concentration in the integument do not support Renaud's general suggestion that the acetyl group for chitin synthesis may be furnished by acetyl CoA. The hypothesis being tested in these experiments was not Renaud's directly but a related one. Renaud's general suggestion might predict a shift in acetyl CoA concentration. This shift could be an increase or decrease depending on the relative rates of fatty acid oxidation and chitin biosynthesis. Any imbalance in these two processes might reflect a change in acetyl CoA concentration if at least two assumptions are made: (1) chitin biosynthesis occurs in the integument and (2) substrate flow in all other input/output channels to the acetyl CoA pool remain constant. That no significant change did occur suggests that a re-evaluation of the assumptions and techniques used might be profitable. The first assumption has been validated in Callinectes sapidus (Carey, 1965) and probably applies throughout arthropods generally. The second assumption, not easily tested, requires experimental verification since both glycogen phosphorylase and synthetase data demonstrate a change in the level of metabolic intensity throughout the molt cycle. The experimental limitations are real and problems were presented in the results to show that the measurements must be regarded as estimates. There is, for example, no convenient way to measure the amount of acetyl CoA that is bound to various cellular subfractions (A. R. Larrabee, personal communication) and is therefore not measured by this method. Another technical limitation is that estimates of acetyl CoA in homogenates require a disruption of the cell integrity. Localized subcellular concentrations might therefore never be detected by this method. These would seem to be the major limitations. A partial compensation for the disadvantages is the specificity of the technique. If the assumptions and limitations are accepted, however, at least three interpretations of the results are possible: (1) the stages of the molt cycle examined were not coincident with peak changes in acetyl CoA concentration, (2) the concentration is carefully regulated and the ability to replace acetyl CoA in the

776

LYLE A. HOHNKE

universal pool greatly exceeds any capacity for utilization and (3) protein concentration of the extracts is also shifting in the same direction as pool size and absolute changes are obscured. SUMMARY Activity profiles for enzymes controlling glycogen metabolism have been obtained throughout the molt cycle for H. nudus. T h e s e data show a correlation with the molt cycle and suggest the presence of control mechanisms which coordinate their physiological function. An enzyme capable of transferring 14C-GlcNAc f r o m U D p - 1 4 C - G l c N A c to a dextrin-like acceptor has been found in H. nudus integument. M a n y properties of the enzyme suggest an identity with chitin synthetase. In vitro, the activity of the enzyme was enhanced by added glucosamine and decreased slightly by chitodextrins. An activity profile for the enzyme throughout the molt cycle agrees with knowledge obtained from tracer studies regarding the timing of chitin synthesis. Estimations of the acetyl CoA concentration in H. nudus integument have also been made for intermolt and postmolt animals. T h e s e studies do not support the hypothesis that acetyl CoA concentrations m a y shift in postmolt. T h e possible involvement of acetyl CoA in chitin biosynthesis and the implications of the experiments are discussed. Acknowledgements--This investigation was supported (in part) by a National Institutes of Health Fellowship (1 F01 GM41854-01) from the General Medical Sciences Institute. I wish to thank Dr. Bradley T. Scheer for his support and guidance of this study and Mr. Marus "Bill" Mumbach for his technical assistance. REFERENCES ADIYODI R. G. (1969) On the storage and mobilization of organic resources in the hepatopancreas of a crab (Paratelphusa hydrodromus). Experientia 25, 43-44. ALBERTSA. W. • VAGELOSP. R. (1968) Acetyl CoA carboxylase--I. Requirement for two protein fractions. Proc. natl Acad. Sci. U.S.A. 59, 561-568. BADE M. L. (1962) Metabolic conversions during pupation of the cecropia silkworm. Biochem. J. 83, 478-482. BAILEY R. W. & BOURNEE. J. (1960) Color reactions given by sugars and diphenylamineaniline spray reagents on paper chromatograms, ft. chromat. 4, 206-211. BARKER S. A., FOSTERA. B., STOREYM. & WEBBERJ. M. (1957) Isolation of a homologous series of oligosaccharides from chitin. Chemy Ind., Lond. 208-209. BARNESH. (1965) Studies in the biochemistry of cirripede eggs..7, mar. Biol. Ass. U.K. 45, 321-329. BERGREEN P. W., MEENAKSHIV. R. & SCHEER B. T. (1961) The oxidation of glucose by crustaceans. Comp. Biochem. Physiol. 2, 218-220. BRAY G. A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. 1, 279-285. BUTCHER R. W., ROBISONG. A., HARDMANJ. G. & SUTHERLANDE. W. (1968) The role of cyclic AMP in hormone actions. Adv. Enz. Reg. 6, 357-389. CAMARGOE. P., DIETRICHC. P., SONNEBORND. & STROMINGERJ. L. (1967) Biosynthesis of chitin in spores and growing cells of Blastocladiella emersonii..7, biol. Chem. 242, 31213128.

ENZYMES OF CHITIN METABOLISMIN THE DECAPOD

777

CANDY D. J. & KILBY B. A. (1962) Studies on chitin synthesis in the desert locust. ~. exp. Biol. 39, 129-140. CAREY F. G. (1965) Chitin synthesis in vitro by crustacean enzymes. Comp. Biochem. Physiol. 16, 155-158. CORI G. T. & ILLINGWORTHB. (1956) The effect of epinephrine and other glycogenolytic agents on the phosphorylase A content of muscle. Biochim. biophys. Acta 21, 105-110. COWGILL R. W. (1959a) Lobster muscle phosphorylase: purification and properties. J. biol. Chem. 234, 3146-3153. COWGILL R. W. (1959b) The conversion of lobster muscle phosphorylase a to b and phosphorylase b to a. J. biol. Chem. 234, 3154-3157. DRACH P. (1939) Mue et cycle d'intermue chez les crustac6s d6capodes. Inst. Oceanogr. 19, 103-391. DRACH P. & LAFON M. (1942) ]~tudes Biochimiques sur le squelette t6gumentaire des d~capodes brachyoures (Variations au cours du cycle d'intermue). Arch. Zool. Exptl. Gen. 82, 100-118. FISCHER E. H., HURD S. S., KOH P., SEERY V. L. & TELLER D. C. (1968) In Control of Glycogen Metabolism (Edited by WHELAN W. J.), p. 19. Academic Press, New York. FISKE C. H. & SUBBAROWY. (1925) Colorimetric determination of phosphorus. J. biol. Chem. 66, 375--400. GLASER L. & BROWN D. H. (1957) The synthesis of chitin in cell-free extracts of Neurospora crossa. J. biol. Chem. 228, 729-742. HERS H. G. & DEWuLF H. (1968) The regulation of glycogen synthesis in the liver. In Control of Glycogen Metabolism (Edited by WHELAN W. J.), p. 65. Academic Press, New York. HOHNIa~ L. & SCHEER B. T. (1970) Carbohydrate metabolism in crustaceans. In Chemical Zoology (Edited by FLORKIN M. & SCH~R B. T.), Vol. V, p. 147. Academic Press, New York. HORNUNG D. E. & STm~NSON J. R. (1969) Changes in the rate of chitin synthesis during the crayfish molting cycle. Am. Zool. 9, 1116. H u A. S. L. (1957) Glucose metabolism in the crab, Hemigrapsus nudus. Ph.D. thesis, University of Oregon. H u A. S. L. (1958) Glucose metabolism in the crab, Hemigrapsus nudus. Archs Biochem. Biophys. 75, 387-395. HUCGINS A. K. & MUNDAY K. A. (1968) Crustacean metabolism. Adv. comp. Physiol. Biochem. 3, 271-378. JEUNIAUX C. (1965) Chitine et Phylog6nie: application d'une m6thode enzymatique de dosage de la chitine. Bull. Soc. Chim. Biol. (Paris) 47, 2267-2278. KELLER R. (1965) fiber eine Hormonale Kontrolle des Polysaccharidstoffwechsels beim Flusskrebs Cambarus affinis Say. Z. vergl. Physiol. 51, 49-59. KINCAID F. D. & SCHEER B. T. (1952) Hormonal control of metabolism in crustaceans--IV. Physiol. ZoM. 25, 372-380. KREBS E. G., HUSTON R. B. & HUNKEImR F. L. (1968) Properties of phosphorylase kinase and its control in skeletal muscle. Adv. Enz. Reg. 6, 245-255. LARNER J., VILLAR-PALASIC., GOLDEERC N. D., BISHOP J. S., HUIJING F., WENGER J. I., SASKO H. & BROWN N. B. (1968) Hormonal and non-hormonal control of glycogen synthesis--control of transferase phosphatase and transferase I kinase. In Control of Glycogen Metabolism (Edited by WHELAN W. J.), p. 1. Academic Press, New York. LELOIR L. F. & GOLDEMBERG S. H. (1960) Synthesis of glycogen from uridine diphosphate glucose in liver. J. biol. Chem. 235, 919-923. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275. MARTIN A. L. (1965) The histochemistry of the molting cycle in Gammarus pulex. J. Zool. 147, 185-200.

778

LvLr A. HOHNKE

McWHINNIE M. & SCHEER B. T. (1958) Blood glucose of the crab Hemigrapsus mtdus. Science, 128, 90. MEENAKSHI V. R. & SCHEER B. T. (1959) Acid mucopolysaccharide of the crustacean cuticle. Science, 130, 1189-1190. MEENAKSHI V. R. & SCHEER B. T. (1961) Metabolism of glucose in the crabs Cancer magister and Hemigrapsus nudus. Comp. Biochem. Physiol. 3, 30-41. OKUNO G., PRICE S., GRILLO T. A. & FOA P. P. (1964) Development of phosphorylase and phosphorylase-activating (glucagon like) substances in the rat embryo. Gen. Comp. Endocrin. 4, 446-451. PARVATHY K. (1970) Blood sugars in relation to chitin synthesis during cuticle formation in Emerita asiatica. Mar. Biol. 5, 108-112. PAYNE W. J. & KIEBER R. J. (1954) The chromatographic determination of glucosamine with ninhydrin. Archs Biochem. Biophys. 52, 1-4. PORTER C. A. & JAWORSKI E. G. (1965) Biosynthesis of chitin during various stages in the metamorphosis of Prodenia eridania. J. Insect Physiol. 11, 1151-1160. POSNER J. B., STERN R. & KREBS E. G. (1965) Effects of electrical stimulation and epinephrine on muscle phosphorylase, phosphorylase b kinase and adenosine 3',5'-phosphate. J. biol. Chem. 240, 982-985. RAMAMURTHI R., MUMBACH M. W. & SCHEER B. T. (1968) Endocrine control of glycogen synthesis in crabs. Comp. Biochem. Physiol. 26, 311-319. RASMUSSENH. (1970) Cell communication, calcium ion and cyclic adenosine monophosphate. Science, 170, 404412. RENAUD L. (1949) Le cycle des resdrves organiques chez les crustaces d6capodes. Ann. Inst. Oceanog. (Paris) 24, 259-357. SACARDIA F. (1969) T h e glycogen phosphorylase system from the muscle of the blue crabs Callinectes danae. Comp. Biochem. Physiol. 28, 1377-1385. SCHEER B. T. (1959) The hormonal control of metabolism--IX. Carbohydrate metabolism in the transition from intermolt to premolt in Carcinus maenas. Biol. Bull. 116, 175-183. SCHEER B. T. (1960) Aspects of the intermolt cycle in natantians. Comp. Biochem. Physiol. 1, 3-18. SCHEER B. T. & ScI-mER M. A. R. (1951) The hormonal control of metabolism in crustaceans - - I . Of the hormonal regulation of metabolism in crustaceans. Physiol. Comparata Oecol. 2, 198-209. SCHWABE C. W., SCHEER B. T. & SCHEER M. A. R. (1952) The hormonal regulation of metabolism in crustaceans--II. T h e molt cycle in Panulirus japonicus. Physiol. Comp. Oecol. 2, 310-320. SCHWIMMER S. & BEVENUE A. (1956) Reagent for differentiation of 1,4 and 1,6 linked glucosaccharides. Science, 123, 543-544. STAHL E. (Editor) (1962) Diinnschicht-chromatographie: ein Laboratoriumshandbuch. Springer-Verlag, New York. SUTHERLAND E. W., HARDMANJ. G., BUTCHER R. W. & BROADUSA. E. (1968) T h e biological role of cyclic H M P (some areas of contrast with cyclic G M P ) . In Progress in Endocrinology. Excerpta Medica Foundation, New York. TELFORD M. (1968) Changes in the blood sugar composition during the molt cycle of the lobster, Homarus americanus. Comp. Biochem. Physiol. 26, 917-926. TRAVlS D. F. (1955) T h e molting cycle of the spiny lobster, Panulirus argus L . - - I I . Preecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. 108, 88-102. TREVELYAN W. E., PROCTOR n . P. & HARRISON J. S. (1950) Detection of sugars on paper chromatograms. Nature, Lond. 166, AA.A.A.A.5. WANG D. H. (1963) Effects of eyestalk extracts on U D P G - - g l y c o g e n transglucosylase. Ph.D. thesis. University of Oregon.

ENZYMES OF CHITIN METABOLISM IN THE DECAPOD

779

WANG D. H. & ScHm~a B. T. (1962) Effect of eyestalk extract on UDPG-glycogen transglucosylase in crab muscle. Life Sd. 5, 209-211. WANG D. H. & SCHEER B. T. (1963) UDPG-glycogen transglucosylase and a natural inhibitor in crustacean tissues. Comp. Biochem. Physiol. 9, 263-274. WATERMANT. H. (Editor) (1960) The Physiology ofCrustacea, Vol. I. Academic Press, New York. WATKINS W. M. (1958) Enzymatic synthesis of nitrogen-containing disaccharides by c~galactosyl transfer. Nature, Lond. 181, 117-118. ZAL~3SKAH. (1959) Glycogen and chitin metabolism during development of the silkworm (Bombyx mori L.). Acta Biol. Exptl. Polish Acad. Sci. 19, 339-351. ZECH~mXS~R L. & TOTH G. (1931) Zur Kenntnis der Hydrolyse yon Chitin mit Salzsiiure (I. Mitteil). Bet. Chem. Ges. 64, 2028-2032.

Key Word Index--Hemigrapsus nudus; chitin; glycogen phosphorylase; glycogen synthetase; chitin synthetase; acetyl coenzyme A; intermolt cycle.