Physicochemical characteristics of glycogen from Anthonomus grandis Boheman

Comp. Biochem. Physiol., 1968, Vol. 24, pp. 163 to 175. Pergamon Press. Printed in Great Britain

PHYSICOCHEMICAL CHARACTERISTICS OF GLYCOGEN FROM A N T H O N O M U S GRANDIS BOHEMAN*t NORMAN L. BETZ,*§ W. C. NETTLES, JR.~ and A. F. NOVAK§ 4115 Gourrier Avenue, Baton Rouge, Louisiana 70808 (Received 12 June 1967)

Abstract--Glycogen was extracted from the boll weevil, Anthonomus grandis Boheman, with 5% trichloroacetic acid (TCA) and with cold chloroformglycine buffer (cold water extraction). Glucose was the only sugar detected in acid hydrolyzates. The average chain lengths, as determined by methylation, hydrolysis, silanization and gas-liquid chromatography, were 11"0, 11"9 and 11"8 glucose units, respectively, for glycogen extracted with cold water from eggs and larvae and glycogen extracted with TCA from larvae. The high percentage (82) of 2,3,6-trimethylglucopyranose obtained indicated that the principal linkage between glucose units was 1,4. Similar i.r. spectra were obtained from glycogens extracted with TCA and cold water. BoU weevil glycogen complexes with iodine-iodide gave absorption spectra similar to a rabbit liver glycogen standard. The optical rotation of glycogen extracted with TCA from larvae was +200.0 °. Measurements of optical rotation could not be made of material extracted with cold water because of high opalescence. INTRODUCTION COMPLEX chemical structures of polysaccharides have long been of interest to biochemists and physiologists. Although glycogen is a major carbohydrate reserve of insects, only brief and fragmentary structural data have been reported for insect glycogens. Von Kemnitz (1916) reported that glycogen from Gasterophilus intestinalis (De Geer) had an optical rotation of + 192.6 °. Loring & Pierce (1943) used trichloroacetic acid (TCA) to extract glycogen from two species of aphids: the samples gave the same rate of hydrolysis with 0.5 N sulfuric acid as did the glycogen standard from rabbit liver; only glucose was identified in the hydrolyzates. Also, the glycogens from both species (Acyrthosiphon pisum and Aphis brassicae) gave the characteristic color with iodine and their water solutions had an optical rotation of * Coleoptera: Curculionidae. In co-operation with the Louisiana Agricultural Experiment Station. Accepted for publication, 26 June 1967. Mention of a trade name does not necessarily imply endorsement of this product by the U.S.D.A. **Entomology Research Division, Agr. Res. Serv., U.S.D.A., 4115 Gourrier Avenue, Baton Rouge, Louisiana 70808. § Department of Food Science and Technology, Louisiana State University, Baton Rouge, Louisiana 70803. 163

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NORMANL. BETZ,W. C. NETTLES,JR. ANDA. F. NOVAK

+ 194 °. Levenbook (1951) found that glycogen of G. intestinalis larvae contained only glucose as the component monosaccharide. An extensive analysis of thirtyseven glycogens from numerous sources was carried out by Abdel-Akher & Smith (1951); included in their work was glycogen isolated from drone larvae of Apis mellifera L., which had an optical rotation of + 191 ° and, by chain length analysis (periodate oxidation of 0.55 g), an average chain length of twelve glucose units. After hydrolysis with N HC1, Chino (1957) found only glucose in glycogen extracted with hot T C A from Bombyx mori (L.). More recently, Bade & Wyatt (1962) found a predominance of glucose in acid hydrolyzates of glycogen from Hyalophora cecropia (L.); the optical rotation of a 0.5% solution of this glycogen was + 184 °. Lindh (1967), using a phenol-water extraction and T C A and La 3+ precipitation, found four types of glucan in Calliphora erythrocephala Meigen pupae. Acid hydrolysis yielded glucose and amino acids; papain digestion indicated that peptides were bound to an inner part of the glycogen molecule. Using electrophoresis as a criterion, Villeneuve & Lemonde (1965) reported that three polysaccharides identical to the main polysaccharides found in plants (two amylose and amylopectin) were present in Tribolium confusum Duval; all three of the polysaccharides were different from rabbit liver and oyster glycogen. Interestingly, glycogen-like materials which are not true glycogen have been isolated from insects (Evans, 1932; Levenbook, 1950; Howden & Kilby, 1960). The object of this study was to determine the physical and chemical properties and the molecular structure of glycogen extracted from the boll weevil, Anthonomus grandis Boheman. METHODS

Rearing Eggs were removed from squares (cotton-flower buds) or bolls, surface sterilized with cupric sulfate (Nettles & Betz, 1966), and implanted onto larval diet by the method of Betz (1966). The implanted dishes were held at 27°C and a photoperiod of 13 hr. The larval diet was essentially that described by Earle et al. (1959).

Source of glycogen Glycogen was extracted with cold chloroform-glycine buffer (cold water extraction) (Bueding & Orrell, 1965) from eggs of various strains obtained from Dr. R. Gast (Boll Weevil Research Laboratory) and stored in either methanol or ethanol at - 70°C. Glycogen was obtained from late last instar larvae by extracting with either 5% trichloroacetic acid (TCA) or cold water; the extraction with TCA was performed on larvae stored at - 2 0 ° C in ethanol and the cold-water extraction was made with unstored larvae. Glycogen was also extracted from 14-day-old reproducing (nondiapausing) adults. The primary standard was "Mann Assayed" rabbit liver glycogen (Mann Research Laboratories, Inc., New York, New York) extracted by the procedure of Somogyi (1934), which consists of prolonged exposure in hot alkali (KOH at 100°C).

CHARACTERISTICSOF GLYCOGENFROMANTHONOMUS GRANDIS

165

T h e source of the glycogen samples and the extraction procedures for each are listed in Table 1. TABLE 1--CODE NUMBERSOF GLYCOGENSAMPLESACCORDINGTO EXTRACTIONPROCEDURE, STORAGEAND DIET Code number and sample 190--14-day-old reproducing adults of Castleberry strain 112--Eggs of Mexican strain 120--Late last instar larvae of Castleberry strain 130--Late last instar larvae of Castleberry strain 133--Eggs of Mexican strain RL---Rabbit liver

Extraction

Storage

Diet

Cold water

Stored in ethanol, 12 months at -20°C

Bolls

Cold water

Stored in methanol, 2 months at -70°C Unstored; extraction done immediately

Semidefined adult*

Cold water

High sucrose t

TCA

Unstored; extraction done immediately

High sucrose t

Cold water

Stored in ethanol, 2 months at -70°C Obtained from Mann Res. Biochemicals

Semidefined adult*

KOH

--

* Vanderzant & Davieh (1961). f Earle et al. (1959); ten times the normal level of sucrose. Extraction and purification of glycogen One gram of glycogen was extracted with 5 % T C A from 1600 boll weevil larvae that had been stored at - 2 0 ° C in ethanol for 1-5 weeks. T h e insects were blended at high speed (45,000 rpm) with a Virtis " 4 5 " homogenizer in 80% ethanol (v/v) and after standing overnight at room temperature, the homogenate was centrifuged at 12,000g for 10 min at 20°C. T h e ethanol precipitate was rehomogenized twice with 30 ml of 5% T C A and centrifuged at 12,000g for 10 min. T h e T C A supernatants were combined and mixed with 5 vol. of 95% ethanol and after standing overnight at room temperature, the mixture was centrifuged at 12,000 g for 10 min. T h e precipitated glycogen was dissolved in water and reprecipitated by mixing with 5 vol. of 95% ethanol. After centrifuging, the precipitated glycogen was re-extracted with water-ethanol ( 1 : 5 / v / v ) . Since the purified glycogen did not always precipitate in the last ethanol extraction, the water--ethanol mixture was sometimes removed under reduced pressure with a rotary evaporator. T h e glycogen was then dried in a vacuum desiccator over calcium chloride, the powder was dissolved in water, and the solution was extracted three times with ethyl ether. T h e aqueous phase was dried under reduced pressure at 30°C. During our preliminary extractions of glycogen with T C A , Bueding & Orrell (1965) reported that a mild extraction caused less degradation of glycogen than

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NORMANL. BETZ,W. C. NETTLES,JR. ANDA. F. NOVAK

extraction with TCA and that glycogen extracted by either hot alkali, TCA or hot water had an average molecular weight ten to fifty times lower than glycogen from the same species when it was extracted by a cold-water procedure. They concluded that although less product may be obtained by cold-water extraction (because of manipulations), the product would not preferentially contain material of either high or low molecular weight. Since anthrone analyses of the boll weevil glycogens extracted with cold water indicated a high degree of purity, additional analyses for non-carbohydrate constituents were not performed on these glycogens. ANALYSES The carbohydrate content of the boll weevil glycogens was determined by the anthrone procedure of Carroll et al. (1956), the sulfuric acid method of Mendel et al. (1954) and by calculation from the amount of glucose (determined by anthrone, glucose oxidase and gas-liquid chromatography (GLC)) yielded on complete hydrolysis. Contaminating protein in the glycogen extracted with TCA was determined by the methods of Moore & Stein (1954) and Koch & McMeekin (1924). Moisture content was determined by daily weighings of duplicate (50 rag) samples that were dried in vacuo over phosphorus pentoxide and ash content was determined with a Muffle furnace held at 600°C for 12 hr. The optical rotation of larval glycogen extracted with TCA was calculated from the specific rotation of an aqueous solution in a Rudolph Polarimeter (Model 70) by using the D-line of sodium. The method of Archibald et al. (1961) was used to determine the iodine absorption power of the boll weevil samples and i.r. spectra were obtained with solid samples (KBr pellets) by using a Beckman IR-5 Spectrophotometer. Samples of glycogen obtained by extraction with TCA and by cold-water extraction of eggs and larvae were subjected to vigorous acid hydrolysis in N sulfuric acid, diluted to about 50 ml with water and neutralized with barium hydroxide. The precipitated barium sulfate was removed by centrifugation, washed once with water and reacted with anthrone reagent to determine the amount of carbohydrate trapped by the precipitate. Ethanol was added to an aliquot of the supernatant to precipitate any unhydrolyzed glycogen. The remaining supernatant was evaporated to dryness under reduced pressure at 30°C, and the residue was redissolved in water for thin-layer chromatography (TLC) and glucose oxidase assay. A portion of the solution was taken to dryness, dissolved in pyridine, reacted with trimethylchlorosilane and hexanlethyldisilazane and subjected to gas-liquid chromatography (GLC) by the method of Sweeley et al. (1963). Gas chromatographic analyses of the hydrolyzates were made by using a Micro-Tek unit with a single hydrogen flame detector. Retention data were obtained with authentic s-glucose, fl-glucose and mutarotated ~- and fl-glucose. Glucose in the hydrolyzates was also determined with glucose oxidase (Glucostat Special) as described in the data sheet furnished by the manufacturer (Worthington Biochemical Corporation). For TLC, silica-gel H plates were prepared with 0.1 N boric acid. Solvent systems used were: n-butanol-acetone-water (4 : 4 : 1), n-butanol-acetic acid-water

CHARACTERISTICS OF GLYCOGEN FROM .4NTHONOMUS GRANDI,.~

167

(4 : 1 : 1) and ethyl acetate-i-proponol-water (3 : 3 : 1). The plates were sprayed with aniline, diphenylamine and phosphoric acid (Harris & MacWilliam, 1954). T h e average chain length of the boll weevil glycogens was determined nonenzymically with G L C of the hydrolytic products after exhaustive methylation, according to the method of Betz, Nettles & Novak (unpublished data). Also, glycogen samples were methylated by a modification (increased time of reaction from 12 hr to 5 days at 30°C) of the procedure of Kuhn et al. (1955). Acid hydrolysis of the methylated glycogen was then carried out with 90% formic-0.1 N sulfuric acids at 75°C, a modification of the method of Bouveng & Lindberg (1965). Identification of isomers of the methylated glucosides released from methylated glycogen by acid hydrolysis and silanization (after consideration of mutarotation) provided us with data indicative of the interior linkages from which they were obtained. T h e ~- and fl-isomers were identified by their retention times with a non-polar liquid phase (SE-30) by co-chromatography of authentic standards, and by chromatography of the hydrolytic products from methylated glycogen. RESULTS Carbohydrate content

Since much glycogen is present in last instar larvae of the boll weevil (Nettles & Betz, 1965), most of the glycogen was extracted from this stage. T h e repeated analyses using the anthrone reagent of Caroll et al. (1956) of the glycogen extracted from larvae with T C A yielded a maximum carbohydrate content of 67.5 per cent with an average value of 66.3 + 0"8 per cent. Calculation of the glycogen content of the material extracted with T C A by using the amount of glucose obtained from complete acid hydrolysis gave a total carbohydrate content of 54.9 per cent when glucose oxidase was used and 64.0 per cent when the sulfuric acid test of Mendel et al. (1954) was used. The composition of glycogen obtained from larvae by extraction with T C A and by cold-water extraction and the glycogen standard (rabbit liver) is shown in Table 2. Using the anthrone method, the glycogen content of larvae extracted with T C A T A B L E 2 - - P U R I T Y OF GLYCOGEN OBTAINED BY THREE IE~TRACTION PROCEDURES

Percentage isolated by extraction procedure Component of extracted glycogen Carbohydrate (anthrone reaction) Protein Moisture Ash

Cold water*

5% TCA*

KOHJ"

99"2

66"3

87.6

5"8 10"4 7"7

0'0 10"4 0"0

-

-

---

* Glycogen from boll weevil larvae. Commercial; glycogen from rabbit liver.

168

NORMAN L. BETZ, W. C. NETTLES, JR. AND A. F. NOV~K

and eggs and larvae extracted with cold water and the glycogen standard was 66.3, 99-2 and 87.6 per cent, respectively. The lowest value obtained for any glycogen extracted with cold water was considerably higher than the value obtained for either the standard glycogen or the glycogen extracted with TCA. Purity of these glycogens was also checked by calculating the amount of glucose obtained by complete acid hydrolysis with N sulfuric acid at 100°C for 2.5 hr; values were generally lower than those obtained by using the direct anthrone analysis. The protein content (5.8%) of the glycogen extracted from larvae with TCA, as determined by two methods, was in close agreement. This value is higher than that reported for any other glycogens (literature values are less than 1 per cent (Good et al., 1933)). Moisture content of glycogen extracted from boll weevil larvae with TCA and the standard (rabbit liver) glycogen was 10.4 per cent, an indication that the moisture probably resulted, at least in part, from handling. Ash content of the sample extracted with TCA was 7.7 per cent and the rabbit liver sample, similarly treated, was ash-free. We suspect that some ash in the sample extracted with TCA resulted from the large volumes of undistilled reagents used in its purification. Because of the purity of the glycogens obtained by cold-water extraction (99.2 per cent by direct anthrone analysis), no additional analyses were performed.

Optical rotation The optical rotation of the glycogens extracted by TCA and the standard glycogens was determined, but the specific rotation of the glycogens obtained by cold-water extraction could not be determined because of the opalescence of the material. The optical rotation of the standard glycogen (literature value = + 178 °) was + 174.0 °; for boll weevil glycogen it was + 200.0 °. A sample of maltose (Mann Research Laboratories, specific rotation = + 131.7 °) used as an additional standard for calibration of the polarimeter had an observed value of + 131.3 °.

Iodine complexes The iodine complexes of glycogen in a solution of 0.02% iodine in 0.2% KI obtained with the recording spectrophotometer are presented in Fig. 1. Rabbit liver glycogen and the samples of boll weevil glycogens were similar in that maximum absorption occurred in the 375415 m/~ range.

LR. Spectra I.R. absorption spectra for glycogens extracted with TCA and cold water were determined by analyzing potassium bromide pellets with a Beckman IR-5 spectrophotometer. The regions of absorption were characteristic of glycogen (~-glucopyranoses and hydroxy compounds) and were identical to the absorption areas of the standard. The characteristic i.r. absorption bands corresponded to primary secondary and tertiary alchohol structures, and to the bands characteristic of o~-D-glucopyranose structures. Also, the i.r. spectra of boll weevil glycogens were

CHARACTERISTICSOF GLYCOGENFROMANTHONOMUSGRAND1S

169

identical to those of the standard rabbit liver glycogen based on the Sadtler code obtained from the Sadtler Index (Anonymous, 1959).

112

.g_

120

109

130

390 430

510

590

670

Wavelength

FIG. 1. Absorption spectra of the iodine complexes of boll weevil and standard glycogens. No. 109: reproducing adults of Castleberry strain, extracted with cold water; No. 112: eggs of Mexican strain, stored in methanol, extracted with cold water; No. 120 : last instar larvae of Castleberry strain, extracted immediately with cold water; No. 130: last instar larvae of Castleberry strain, extracted immediately with TCA; RL: Rabbit liver glycogen, extracted with KOH, obtained from Mann Research Biochemicals.

Thin-layer chromatography of acid hydrolyzates When the sulfuric acid hydrolyzates of glycogen were neutralized with barium hydroxide, total losses of glucose and glycogen (including unhydrolyzed glycogen) were 29 Fg/mg of glycogen. The hydrolyzates were chromatographed on thin layers of silica-gel H prepared with 0.1 N boric acid. The solvent systems were: nbutanol-acetone-water (4 : 4 : 1), n-butanol-acetic acid-water (4 : 4 : 1) and ethyl aeetate-i-propanol-water (3 : 3 : 1). The plates were sprayed with aniline-diphenylamine-phosphoric acid (Harris & MacWilliam, 1954). In all three solvent systems, only glucose was released after complete acid hydrolysis. Glucose in the hydrolyzates was determined quantitatively with glucose oxidase. The samples obtained by extracting larvae with TCA averaged 54-8 per cent glucose; average content of the samples obtained by cold-water extraction was 91.6 per cent.

NORMANL. BETZ,W. C. NETTLF_S,Ja. ANDA. F. NOVAK

170

Analysis by gas-liquid chromatography The hydrolyzates were simultaneously analyzed qualitatively and quantitatively by G L C of the trimethylsilyl derivatives (TMS) by using polar (20% diethylene glycol succinate, EGS) and nonpolar (3% General Electric SE-30) liquid phases. Qualitative analysis of the peaks was based on the retention times (tR) for the T M S derivatives of dextrose (s-glucose), fl-glucose and the mutarotated forms of these sugars. Chain length (CL) was determined by a new technique which involved methylation, hydrolysis, silanization and G L C of the methylated-silanized products (Betz, Nettles & Novak, unpublished data.) A part of the methylated hydrolyzate was removed and chromatographed on thin layers of silica-gel G by using a modification of the paper chromatographic method of Hirst et al. (1949). The results of T L C in n-butanol-ethanol-water-ammonia (40 : 10 : 49 : 1) are presented in Table 3 for the hydrolyzed methylated glycogens. The ~- and fl-isomers of the methylated hexoses were not resolved by TLC. TABLE

3--R! VALt~S

FROM THIN-LAYER CHROMATOGRAPHY OF THE HYDROLYTIC PRODUCTS OF METHYLATED GLYCOGENS*

Rj of products of boll weevil glycogens (by code number)t Sugar (glucopyranose)

No. 112

No. 133

No. 120

No. 130

Standard

o~-Tetramethyl + fl-Trimethyl et+ fl-Dimethyl

0"72 0.64 0"60

0.71 0.65 0"58

0"73 0.67 0"59

0"72 0.66 0"61

0-72 0"66 0'61

* On silica-gel n-butanol-ethanol-water-ammonia (40 : 10 : 49 : 1) sprayed with 1% p-anisidine in 10 ml methanol diluted to 100 ml with butanol (Hough et al., 1950). t No. 112: eggs of Mexican strain, stored in methanol, extracted with cold water; No. 133 : eggs of Mexican strain, stored in ethanol, extracted with cold water; No. 120: larvae of Castleberry strain, extracted with cold water; No. 130: larvae of Castleberry strain, extracted with TCA.

Following T L C and identification of the methylated glucose derivatives, the remaining solutions were transferred to small glass vials and the methylated sugars were silanized by the method of Sweeley et al. (1963). Identification of the hydrolytic products of methylated boll weevil glycogen was based primarily on relative retention times (with respect to ~-tetramethylglucopyranose) after GLC. Therefore, the GLC analyses were made on the individual methylated glucose standards before and after mutarotation. Freshly prepared, mutarotated trimethylsilyl-2,3,4,6-tetra-O-methyl-~-Dglucopyranoside was found to have a major peak with a retention time (tR) of 10"0 min and a trace (less than 1 per cent) of the fi-isomer; apparently, there was little mutarotation from the s-form. After a water solution of 2,3,6-tri-O-methyl~-D-glueopyranose was allowed to reach equilibrium, the T M S derivative

171

CHARACTERISTICS OF GLYCOGEN FROM ..4NTHONOMUS GRtINDIS

was prepared and analyzed by GLC. Although they were not well resolved by gas chromatography, the n- and /3-isomers of this sugar were easily distinguished, that is, a double peak appeared with peak heights nearly 1 min apart. The tR's for the n- and /3-isomers were 16.8 and 17.5 min, respectively. The equilibrium mixture of trimethylsilyl-2,3-di-O-methyl-4,6-di-O-trimethylsilylD-glucopyranoside amounted to 38 per cent n and 62 per cent/3, with retention times of 22.0 and 28.0 min, respectively. The ratio of tetra : tri : dimethylglucopyranose (TMS derivatives) obtained for the methylated samples of glycogen is presented in Table 4. The gas-liquid chromatograms are shown in Figs. 2 and 3. The internal standard (trimethylsilyl2,3,4,6-tetra-O-methyl-n-D-glucopyranoside) had a retention time of 10.0 min. The three dimethyl peaks (Fig. 3) resulted from storage of the T M S derivatives of hydrolyzed, methylated rabbit liver glycogen (Betz, unpublished data).

T A B L E 4----AVERAGE CHAIN LENGTHS DETERMINED BY THE RELATIVE AMOUNTS OF T E T R A : T R I : DIMETHYLGLUCOPYRANOSIDES ( T M S DERIVATIVES) OBTAINED BY GAS--LIQUID CHROMATOGRAPHY OF THE HYDROLYTIC PRODUCTS FROM METHYLATED BOLL WEEVIL AND STANDARD GLYCOGENS

Boll weevil glycogen* Sugar (glucopyranose)

No. 112

No. 133

a-Tetramethyl + fl-Trimethyl a + fl-Dimethyl

1"3 8"7 1"0

1'2 10"1 1"0

11.0

12.3

No. 120

No. 130

1"3 1"4 9"6 9"3 1"0 1"0 Average chain length 11"9

11"7

Rabbit liver (standard) 1"7 8"3 1"0 11.0

* No. 112: eggs of Mexican strain stored in methanol, extracted with cold water; No. 133 : eggs of Mexican strain, stored in ethanol, extracted with cold water; No. 120: larvae of Castleberry strain, extracted with cold water; No. 130: larvae of Castleberry strain, extracted with TCA.

lnternal glucose branching Hydrolysis produced about 82 per cent of 2,3,6-tri-O-methyl-D-glucopyranose, an indication that at least 82 per cent of the linkages are 1,4. About 13 per cent of the molecular structure yielded n-2,3,4,6-tetra-O-methyl-D-glucopyranose, which proved that all outer glucose units (attached only at the anomeric carbon atom) were of the a-configuration. Although 2,3-dimethylglucopyranose would freely mutarotate in an aqueous medium, no configurational assignment could be made for this triply branched glucose unit. However, the branched glucose unit in boll weevil glycogen was a 1,4,6-1inked moiety.

172

NORMAN L.

BETZ,W. C. NETTLES,JR. AND A. F. NOVAK

I00-

Methanol ( no. 112) 50-

n."

100-

50-

Ethanol (no. 133)

o

I00-

50Extracted with cold water

I00-

50Extracted with TCA

0

2

4

6

8

I0 12 14 16 18 20 22 24 26 28 30 32 54 Min

Fio. 2. Gas-liquid chromatograms o£ the methylated, hydrolyzed and silanized derivatives of boll weevil glycogen from eggs and larvae extracted with cold water and from larvae extracted with T C A . No. 112: eggs of Mexican strain, stored in methanol, extracted with cold water; No. 133: eggs of Mexican strain, stored in ethanol, extracted with cold water.

CHARACTERISTICS OF GLYCOGEN FROM A N T H O N O M U S

173

GRANDIS

I00-~

5c

nr

0

4

8

12

16

20

24

28

Min

F I G . 3.

Gas-liquid chromatogram of the methylated, hydrolyzed and silanized derivatives of rabbit liver glycogen extracted with KOH.

DISCUSSION Quantitatively, glycogen has been studied extensively in many insects, but except for a few analyses that indicate that several insect glycogens are composed of glucose and for the work of Villeneuve & L e m o n d e (1965) and L i n d h (1967), this is the first structural study of insect glycogen. Most physical and chemical properties of boll weevil glycogen were similar to those of mammalian glycogen: glucose was the component monosaccharide, the average chain length was 11 +_ 1 units, the triply branched unit was 1,4,6-1inked and the optical rotation was only slightly higher than that of other insect glycogens (Table 5). T h e 6 ° difference probably was not significant. T A B L E 5 - - O P T I C A L ROTATION AND HYDROLYTIC PRODUCTS OF INSECT AND MAMMALIAN GLYCOGEN

Organism A. A. C. G. B. H. A. A.

brassicae pisum erythrocephala intestinalis mori cecropia grandis mellifera

Rabbit liver

Optical rotation + 194° + 194 ° -+ 192-6° -+ 184° + 200° + 191 o + 178°

Sugar obtained from acid hydrolysis Glucose Glucose Glucose Glucose Glucose Glucose + maltose ? Glucose -Glucose

Reference Loring & Pierce (1943) Loring & Pierce (1943) Lindh (1967) yon Kemnitz (1916) Chino (1957) Bade & Wyatt (1962) Abdel-Akher & Smith (1951)

174

NORMANL. B~TZ, W. C. NETTLES,JR. AND A. F. NOVAK

Boll weevil glycogen differed from mammalian glycogens in that the fl-amylolysis limit dextrins were the smallest defined to date (Betz et al., 1966a) and the molecular weight of egg glycogen was exceedingly high (Betz et al. 1966b). Boll weevil glycogens were degraded 51-62 per cent by r-amylase; and rabbit liver glycogen, similarly treated, was digested 50 per cent (Betz et al., 1966a). These results together with the methylation data indicated that the exterior chains of boll weevil glycogen were longer than those of mammalian glycogen. Archibald et al. (1961) and Manners (1957) reported that the iodine absorption powers of polysaccharides were indicative of their respective interior chain lengths: polysaccarides with maximum absorption around 400 m/~ have an average interior chain length of less than five glucose units, and polysaccharides with maximum absorption greater than 500 m/~ have an average interior chain length greater than seven glucose units. The results of interior chain length determinations on boll weevil glycogen and the standard support this theory since the interior chain averaged 3"0-3"5 glucose units (Betz et al., 1966a, b).

REFERENCES

ABDEL-AKHERM. ~ SMITH F. (1951) The repeating unit of glycogen. J. Am. Chem. Soc. 73, 994-996. Anonymous (1959) Sadtler Standard Spectra, No. 8671. Sadtler Research Laboratories, Philadelphia. ARCHIBALDA. R., FLEMINOI. D., LIDDI~ A. M., MANNERSD. J., MERCERG. A. & WRIGHTA. (1961) tv-l,4-Glucosans. Part XI. The absorption spectra of glycogen and amylopectiniodine complexes. J. Chem. Soc. 1183-1190. BADE M. L. & WYATTG. R. (1962) Metabolic conversions during pupation of the cecropia silkwormNdeposition and utilization of nutrient reserves. Biochem. ~. 83, 470-478. BETZ N. L. (1966) Improved laboratory methods for rearing the boll weevil. J. econ. Ent. 59, 374-376. BE'rz N. L., NETTLESW. C., JR. & NovaK A. F. (1966a) Unpublished data. B~rz N. L., ORRELLS. A., JR., BUEDINGE., NETTLESW. C., JR. & I~OVAKA. F. (1966b) Unpublished data. BouvxNo H. O. & LINDBEROB. (1965) Hydrolysis of methylated polysaccharides. Methods in Carbohydrate Chemistry (Edited by WHISTLERR. L.) Vol. 5, pp. 296-298. Academic Press, New York. BUEDINO E. & ORRELL S. A. (1965) A mild procedure for the isolation of polydisperse glycogen from animal tissues. ~. biol. Chem. 239, 4018--4020. CAROLLN. V., LONaLEYR. W. & ROE J. H. (1956) The determination of glycogen in liver and muscle by use of the anthrone reagent. J. biol. Chem. 220, 583-593. CHINO H. (1957) Carbohydrate metabolism in diapause eggs of the silk-worm, Bombyx raori--I. Diapause and the change of glycogen content. Embryologia 3, 295-316. EARLEN. W., GAINESR. C. & ROUSSELJ. S. (1959) A larval diet for the boll weevil containing an acetone powder of cotton squares. ~. econ. Ent. 52, 710-712. EVANSA. C. (1932) Some aspects of chemical changes during insect metamorphosis. J. exp. BioL 9, 314-321. GOOD C. A., KRAMERH. and SOMOOYIM. (1933) The determination of glycogen, ft. biol. Chem. 100, 485-491. HAmus G. & MAcWILLIAMI. C. (1954) A dipping technique for revealing sugars on paper chromatograms. Chem. Ind., Lond. 73, 249.

CHARACTERISTICS OF GLYCOGEN FROM .4NTHONOMUS GRANDIS

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