Glycosidases of marine invertebrates from Posiet Bay, Sea of Japan

,

ppl

463

to

470.

Pergamon Press. Printed in

MARINE INVERTEBRA' BAY, SEA OF JAPAN

)M

G. VAFINA, A. KIM, E. V. ?YUKOV and Y. G. B L I N O V

DVA,

ic Chemistry, Far East Science Cent ~cmnces ot the U.S.S.R. U.~5.~.1-¢,.,Vladivostok-22, Vladivostok=22: U.S.S.R

of

(Received 10 August 1973)

Alwstraet--1. N-acetyl-]3-D-glucosarninidase, N-acetyl yl=~-D-gala( /g-glucosidase, a-glucosidase, ]~-galactosidase, cx-gala( alactosidase, a-xylosidase, ~-mannosidase, cx-mannosidase, and N-acetyl=c~=D=gh N-ace1 have been found in a variety of Invertebrates. 2. The distribution of these glycosidases is described describ( in sele( Coelenterata, Annelida, Arthropoda, Mollusca, Echinoderma Echin( ar

e)

>f

INTRODUCTION ,VtPLEX biopolymers have recently been subjected to t,. . . . . .a. . . .rathe ................. j. study. w, while the examination of relatively simple low-molecular Io~ substances or horn( nopolymers may involve conventional, i.e. more or less complicated chemical procedures cedures, the study of complex biopolymers can in revolve only mild, generally biochemical chemical techniques. The most promising of these thes are enzymatic methods. Cart•bohydrases are widely used in the study of natural natur biopolymers containing carbohydrates. Today this is virtually the sole means of strictly selective seh splitting of glycoside bonds tds with definite configurations, e.g. a- and fl-ghlucosidases and a- and /3galactosidases, etc. Each of the said carbohydrases specifically hydrolyses a definite monosaccharide linked nked by a specific c~- or/}-bond with the other residues. Among the first to be studi, lied were carbohydrases of almond emulsin and of snail digestive juice. At present, ;nt, cultural media of different micro-organisms are generally used as carbohydrase rase sources. This is because organisms have so far been insufficiently studied with respect to carbohydrase content. Thus, marine organisms have been examined only very little; however, they are beginning to receive greater attention on the part lrt of biochemists. The purpose of this work )rk is to investigate the distribution of certain important carbohydrases in marine invertel nvertebrates. MATER] ERIA I[ATERIALS AND METHODS Substrates p-Nitrophenyl-2-deoxy-2-acetami tmid -acetamido-]~-D-glucopyranoside, m.p. 215°C, [C¢]D~°--26"4° (c 0"2; H20); p-nitrophenyl-2-deo :leox] ~rl-2-deoxy-2-acetamido-/~-D-galactopyranoside, m.p. 214°C,

463

N. V. MOLODTSOVet al. obtained by a method described p -deoxy-2-acetamido-a-D-glucopyranc obtained by melting glucosamine uent glycoside acetate purification of the latter, p-Nitropheny-c~-D-gh 120); p-nitrophenyl-/~-D-glucopyran nitrophenyl-a-D-galactopyranoside, : yl-/3-D-galactopyranoside, m.p. 179°( noside, m.p. 158°C, [~]i) 2° +213 ° n.p. 161°C, laiD 2° --56 ° (c 1; H~O) riD2° + 1 6 9 ° (c x 1; H20); and p-nitr( ained from moside, m.p. 205°C, [ec]D~° +45 ° (c 0"45 ; H~O) were obtaJ ates by melting them with p-nitrophenol in the preseence of to subse lferich, 1944) or zinc chloride (Westphal & Feier, 1956) with ,~ oside acetates by means of chromatography on silica gel and desace ~ ' ~ . t " d

. . . . . . . . .

,

....

r

.

.

.

.

.

7

I,.---,,I .

.

.

.

.

.

.

.

•

urabjan 274°C, p-nitroJgraphy e, m.p. 152°C, 6° ( c 2 ; aol) ; pxyl-c~-Dmannolg sugar lic acid ation of Lelatter.

yme sources

)tember 1972 ir Marine animals were collected in August and Septemb~ al in. Only intact, fresh organisms were used in the work. Tissues ' e animals and fully ground small species were used for extracts. e~

Sea of rgans of

~aration of enzyme solution

u~t~

sample The work was performed in the cold within the shortest shorte., possibh led was lied was thoroughly ground with pure sand, and the homogenat, h, centriacted with a fivefold volume of 0"05 M phosphate buffer, pH ] 7"5 ; tk being instantly used to tc .~d for 10-15 m i n at 9000 rev/min (5000 g), the s u pmrnatant em ;rmine the enzymatic activity and protein content.

Determination of enzyme activity ~henylglycoside (0"1 ml), dilutedI crude crudc AA mixture of an 0"1% aqueous solution ofp-nitrophenylgl ml) act (0"1 ml) and 0"2 M citrate-phosphate buffer with 1 M sodium chloride (0"1 ml), extract M pH 4"0, was incubated for 45 min at 37°C. The reaction w~¢as terminated by adding 1 IV] at spectrophotometrically al COs (2 ml), and the liberated p-nitrophenol was determined determi Na2COs 440 nm. Protein concentration was estimated by the Lowry et al. (1951) procedure usin~~g as the quantity of p.)ine serum albumin as a standard. Specific activity was calculated c: bovine standard conditions. liberated in 1 m i n / l mg of protein under nitroophenol/xmoles ?

rESULTS AND DISCUSSION RESI.

,,tabolic at of carbohydrases is required to ensure metabolic A considerable amount ebrates. This is due to the wide variety of their feedin~g processes in marine inverter reeds ,~oo- and phytoplankton, green, brown and red seaweed., sources, such as detritus, zoormes and numerous constituent carbohydrates. The identification of different enzyme., has been insufficiently studied. Works have recentl3Y in marine invertebrates has 'avorov ebrate laminarinase (Sova et al., 1970), alginase (Favoro~ appeared on marine invertek zetyl-fl-D-glucosaminidase (Molodtsov & Vafina, 1972):), & Vaskovsky, 1971), N-acetyl-t }rate 1972); there is also a paper on invertebrate cellulase and chitinase (El~lyakova, 3 :t to feeding habits (Kristensen, 1972). However, this carbohydrases with respect ,ne ~qake general conclusions. Moreover, even within one evidence is still insufficient: to make mak

~. INVERTEBRATES F R O M POSIET BAY, SE~

.~to make any inferences on digesl ical features and related feeding parent with the most thoroughl I ~re, we shall only try to give a gen ant carbohydrases in marine inverl e widely distributed in marine inve viously reported (Molodtsov & Vaflna, 1972), its ri, richest sou ascidium Halocynthia roretzi. The activity of thJ this source lest of the known natural sources, namely that of the th pig epi ,evvy, 1960). Large amounts of N-acetyl-~/-D-glucos.. lucosaminidasq tifferent Mollusca and Arthropoda. Coelenterata, Annelida AI al es, on the other hand, have a low content of this carlbohydras ,lain unambiguously its role in marine invertebrate brates. One t its function in organisms is not exclusively digestiw estlve.

-¢vJ

edrases fferent nollusc of the abiting

~kS w a s

iver of to the :indlay served ermata cult to :esume

N-ac ety l- ~- D-galactosaminidase N-aeetyl-/i-D-galaetosaminidase invariably aceomp~anies N .glucolinidase, and attempts to distinguish their actlvttms aetiviti~ have ] cessful oolen et al., 1961). The relationship of the two activities acl fluctuates in a very [e range from 0-5 to nearly 10. This is an interesting pl~henomenon, to which, howeverr, a suitable explanation has yet to be found. As a ma matter of fact, even within the same le class, wide fluetuations in the relationship of these th, activities are observed. 3. ~-Glucosidase This enzyme splits the /3-1inked glucose residues, particularly in cellobiosis. Apparently, ?arently, it accompanies eellulase. The high activity of fl-glucosidase in the species cies Chaetopterus variopedatus and Urechis umcmetus unicinet of the family Annelida may¢ be readily .y explained by bvy their feedin~ g habits on ve~e egetative food containing large amounts of cellulase; the same ame may be said of Crustacea and certain representatives of Mollusca and Echinodermal lermata, e.g. Ischnochiton hakodadensis and Strongylocentrotus intermedius. At the ae same time, organisms belonging to the classes Anthozoa, Cirripedia (Balanus rostr ~stratus), Brachiopoda and Chordata and attached to the substrate possess a slight enz3 mzyme activity. Mollusca species, as a rule, have a low /~-glucosidase activity. 4. c~-Glucosidase a-Glucosidase activity in the tissues of marine invertebrates is generally quite low or is entirely lacking (Anthozoa and Ascidiaceae). Only a few representatives have this enzyme in significant cant amounts. Sometimes this relates to highly specialized organs, e.g. the gonad in Echinocardiura cordatum. High ~-glucosidase activity was observed in Amphipholis lis kohii, a representative of Ophiuroidea.

T.

Balanus rotratus

CIRRIPEDIA

Hemigrapsus sanguineus Pachycheles stevensff Pagurus sp. Pugettia quadridens Axiopsis princeps

D.tr.

T.

H. T. T. T. T.

M.i.g. M.g. M.i.g. M.i.g. M.i.g. M.i.g. M.i.g. M.i.g.

Cancer amphioetus Hapalogaster dentata

CRUSTACEA

A r t h r o p o d a

Urechis unicinetus

ECHIUROIDEA

Physcosoma japonica

81PUNCULOIDEA

Ul~aetopterus varwpedatus ts Neoamphitrite gray Harmothoe imbricata Arctonoe vittata Serpula vermicularis

330

5450 580 925 6000 2650 3400 1970 3300

63

975

156 72 1250 471 425

160

613 285 333 4850 642 425 281 2250

20~

262

37~ 2~ 334 152 15f

21] 22( 22~

11

1530 3000 270 177 1440

59

- -

15

38 105 445 382 170 640 169 13

2 2 1

~4 ~0 104 -51 80 31

178

--

55

54

66

58

23

14

156

- -

- -

--

--109 -129 177 21

- -

325

35

- -

--

- -

131

--

178 204 216 176 415 83 735

- -

--

--

- -

--

- -

61

7

tum

595 555 583

6

T. T. T.

5

4

2

Organ t II

es* I

1~

THE ORGANS OF MARINE I N E

TABLE 1--GLYcOSIDASES SPECIFIC A(

Coptothyris graya

c

h

i

n

o

d

e

r

m

a

t

a

Scaphechinus mirabilis

Echinocardium cordatum

ECHINOIDEA

Cucumaria f raudatrix

HOLOTHURIOIDEA

E

D.tr. S.gl. D.tr.

D.tr.

T.

Swiftopecten swifti Crassostrea gigas Glycymeris yessoensis Mactra sulcataria Mercenaria stimsoni Modiolus difficilis Mizuhopecten yessoensis Protothaca euglypta Septifer virgatus Spisula sachalinensis Tellina lutea

810 312 534

283

457

114 1710 590 2960 104 71 2990 1150 1240 5400 1280 660 2800 3800

C.st. H. H. H. H. H. H. H. H. H. H. H. H. H.

BRACHIOPODA

2700

H.

5050 11 66

278

D.tr. D.tr. D.tr.

T.

334 151 206

213

164

995 62 2100 181 810 264 89 246 202 251 2340 272 266 228 565

1650 9 159

97

86 ---

25

-158 --

28

[3

~8 ~5

- -

~0 -~7 i6 54 )8 ~7 ~5 ---

- -

!2

~4 ~3 ~6

111 88 60

25

200 476 317 405 142 855 520 127 950 275 151 420 403

6 2

334

535 60 262

278

- -

- -

--

- -

--

--

59 84

- -

77 -81 64 52 108 46 30 99 --

- -

132

192 25 82

- -

39

265 -603 341 143 397 208 86 800 107 137 252 91

- -

415

-15 262

82

2

h

o

r

d

a

t

a

3~ 15, 41! 3,

3(

1 24 1( 4 1~ 4~

7 12 26 3C

:

40 26 140

7 8 21

82

440

6 5

- -

- -

70 325 361 --

- -

6 0

136

- -

128

5 8

84

- -

7 7

95

17 18

32 18 - -

5

4

--/

- -

37 -169

39

128

- -

- -

9 7 32

--

- -

17 9

6

- -

- -

550

- -

--

128

- -

- -

-4 9

--

- -

23 9

7

:lands; H., hepatopancre J" C.st., crystalline style; D.tr., digestive tract; tract M.i.g., M i d - i n t e s t i n e gland sex gland; T., total. ; 3, fl-glucosidase; 4, ~-~ 1, N-acetyl-fi-D-glucosaminidase; 2, N-acetyl-fl-D-galactosaminidase N-acet annosidase; 10, ~-mannosJ sidase; 6, a-galactosidase; 7, fl-xylosidase; 8, ~-xylosidase; 9, fl-mannosid glucosaminidase. All specific activity values x 104.

Possjet Bay of the Sea of Japan, 1971, Nauka, Leningrad. Le

nora oc fauna iauna OI m e r o s s j eett Bay Day of the Sea of Japan. * Classification according to : A list of the flora flor &

Ascidia sp. Halocynthia aurantium Halocynthia roretzi Styella clara

ASCIDIACEA

C

OPttlUROIDEA

620 3660 8775 1210

Amphipholis kohil

T. H. H. T.

543

T.

Lysastrosoma anthosticta Patiria pectinifera

Distolasterias nippon

182 33 860 288 137 261 630

H. S.gl. H. H. S.gl. H. T.

s

65 58 1070 842

D.tr. S.gl. D.tr. S.gl.

medius

15

O r g a n t II

ies* I

r

I N V E R T E B R A T E S F R O M P O S I E T BAY~ SFJ

--rv 7

stribution than/3-glucosidase, the 1 greater number of galactose-cc toplankton. Thus, in Mollusca, titles than fl-glucosidase, at tim~ sm; fl-galactosidase also shows co

~rently ~lysac:lase is e pre:ctivity

a-Galactosidase is a rarer enzyme in marine invertelbrates th~ :asionally, its activity is considerable and may exceed excee that of :ance in Mollusca. This carbohydrase is absent in the Ant [ned; it is virtually absent in Echinodermata (exce except for F • in Annelida possesses considerable activity only in (C. variop,

sidase. Lse, for [es ex-

~-Xylosidase /%Xylosidase was found in considerable amounts in Arthropc ~as also found in C. variopedatus of Annelida and Halocy'n )rdata. The enzyme proved to be most active in Axiopsis Ax~ prit

llusca.

ium of stacea.

x-Xylosidase This is the rarest of the enzymes studied. It showed show considerable activity in

variopedatus and Anadara broughtoni. It is noteworthy that a-xylosidase is present sent in the gonads of such Echinodermata as S. intermedius int and Aphelasterias japonica. mica. However, its functions are still obscure. 9. ~-Mannosidase Due to the significant amounts of mannanes, g~alactomannanes and other pol~ysaccharides containing mannose, one may e x p e c t that j3-mannosidase would play a definite role in the digestion of vegetative food. Indeed, i some invertebrates belonging to Arthropoda, Mollusca and Echinodermata possess /~-mannosidase activity. Maximum activit,ty is not high, but in some invertebrates the enzyme is present predominantly. 10. c~-Mannosidase This enzyme has approximatel tteb the same distribution as fl-mannosidase, some~ximately times surpassing it in activity. In absolute figures, Tellina lutea possesses the highest c~-mannosidase activity. 11. N-acetyl-~-D-glucosaminidase N-acetyl-~-n-glucosaminidase inidase is only slightly distributed in the species studied. It is entirely absent in Anthc hozoa, Echinoidea and Ascidiacea. The highest activity was observed in C. variopedatus.

N . V . MOLODTSOV et al.

tion of the paper from the Russian b

Shapiro

REFERENCES

.,. . . . . . . . . j . . . . . . . .

zn rotatory power and structure in th ounds of D-mannose with calcium ch

~--XX. • Chem•

ion of cellulases and chitinases in n 67-70. E. (1971) Alginases of marine invert~

ebrates.

~p. Bio-

~ .......

Aglucosam: the pig • LEVVY G. A. (1960) Purification of fl-N-acety] .~pididymis. Biochem. J. 77, 170-175. alaktosids u n d fib ~kfilver,FERICH B. (1944) Darstellung des Phenol ~-D-galaktosid 94-197. Mndung dieser Substanz mit Phenol a-D-galaktosid. Bet. dtsch, ehe of their STENSEN J. H. (1972) Carbohydrases of some marine invertebrates. inve tudied. Mt 30-142. ~ood and on the natural occurrence of the carbohydrates stt lrement VRY O. H., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) r ~5-275. with the Folin phenol reagent. J. biol. Chem. 193, 265-2' ainidase LODTSOV N . V . & VAFINA M . G . (1972) The distribution of fl-N-a( in marine invertebrates. Comp. Bioehem. Physiol. 41B, 113-120. 11 Oxf CHON R. D. (1968) The Biology of MoUuska. Pergamon Press, t arinases The distribut A V . V . , ELYAKOVA L. A. & VASKOVSKY V . E. (1970) Th~ in marine invertebrates. Comp. Biochem. Physiol. 32, 459-464. 459dnanten ;TPHAL O. ~x; FEIER H. (1956) Darstellung Kfinstlieher Antigens ¢l-tA-~-glycoslcle yon L-IFucose, ~ucose ¢l-O-~-gb Zuckergruppen--II Mitteil. Synthese derp-aminopheny] L-Rhamnose, D-Galactose und D-Mannose. Bet. dtseh, chem. ch~ Ges. 89, 582-588. on glucosaminidase--III se--III. Stu, W O ODLEN L E N J. W., HEYXVORTHR. & WALKER P. G. (1961) Studies Festicular N-acetyl-fl-glucosaminidase and N-acetyl-fl-galactosaminidase. Bioehem. J.7. Tes 78, 111-116. method for prepara)ara~BJAN S. E., VOLOSYUKT. R. & KHORLINA. JA. (1968) A convenient c ZUP~B khim ttion l o n of p-nitrophenyl-fl-D-glucosaminide. lzvestia Acad. Nauk S S S R , ser. khim. 1612-1614. DLAY J .

Key Word lndex--Glycosidases in marine invertebrates, 2N-acetyl-c~-D-glucosaminidase Ke" alactosaminidase ; a-glucosidase ; /3-glucosi [ucosicetyl-fl-D-glucosaminidase; N-acetyl-fl-D-galactosaminid~ N-acet~ 3-man ,losidase ; c~-mannosidase ; fl-man dase:.~; a-galactosidase; fl-galactosidase : a-xylosidase ; fl-xylosi, nosidase.