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Protein and oxalate in mineral granules from the kidney of Pecten maximus (L.)
J. exp. mar. Biol. Ecol., 1981, Vol. 52, pp. 173-183 Elsevier/North-Holland Biomedical Press
PROTEIN
173
AND OXALATE IN MINERAL THE KIDNEY OF PECTEN
J. N.E.R.C.
Institute
of Marine
Biochemistry,
GRANULES
MAXZMUS
FROM
(L.)
OVERNELL St. FittickS
Road, Aberdeen
ABI
3RA,
Scotland
Abstract: The mineral-rich granules present in the kidney of the scallop, Pecten maximus (L.), have been found to contain 11%by weight of carbon and to lose at least 25% of their dry weight on ashing. Protein corresponding to 69% by weight and oxalate corresponding to 7% by weight have been identified. The sodium dodecylsulphate-soluble fraction of the protein (71%) consists of a large number of components having molecular weights of 30000 or higher, but the whole of the protein has an aminoacid analysis similar to erythrocyte membrane protein. Carbonate is absent from the granules.
INTRODUCTION
Dakin (1909) seems to have been the first to report the presence of large intracellular particles of refractile “excrete” material in scallop renal organs. Using
Fig. 1. Light micrograph of Pecten maximus kidney: tissue fixed in 2.5% glutaraldehyde in sea water; unstained section viewed under phase contrast; mc, mantle cavity; g, granule; I, lumen. 0022-0981/81/0000-0000/$2.50
0 Elsevier/North-Holland
Biomedical
Press
J. OVERNELl
174
normal histological procedures large numbers of extremely refractile bodies are obvious using phase contrast light microscopy (Fig. 1). Recently there have been reports on the mineral content of Pet&w granules (George et al., 1980) and Argopecten granules (Doyle cutal., 1978; Carmichael et al.. 1979). which have established that the granules are mostly composed of amorphous metal-phosphates. Thus, an average composition calculated from the data of George PI al. is: Zn 10.7p;. Mn 8.40/i, Ca 7.6Y.b.Mg 1.5’?;,.PO, 48.2”,,. Pcrren granules are probably destined to be excretion products (as suggested by Dakin. 1909) but an alternative role as a buffer for metal storage has not been ruled out (Carmichael et ~1.. 1979). Simkiss (1976) has reviewed evidence for these two functions of metal granules in various or~nisms. While the chemistry of the formation of such mineral concretions is still obscure, it seemed likely that metal-binding proteins might have been involved in the initial stages of the accumulation of the metals and might still be present in the matrix of the granules. This paper reports studies on the characterization of the organic components of Pecren ma~~imus(L.) kidney granules. MATERIAL
AND METHODS
P. maximus adults were purchased at different times of the year from the University Marine Biological Station, Millport, Isle of Cumbrae, Scotland, and held in tanks of circulating sea water until used. They were not fed. The isolation of kidney granules was carried out essentially according to the method described by George et al., (1980), except that the final sedimentation through 2.2 M sucrose was carried out at 1 x g overnight at 4 “C. AMINO-ACID
ANALYSES
These were carried out using a Jeof JLC-6AH liquid phase analyser after hydrolysis in 6 M HCl at 110 “C for 24 h, no correction being ma& for losses of serine or threonine. c‘. H AND N ANALYSES
AND ASH WEIGHTS
These were carried out by Dr. F. B. Strauss, Microanalytical Laboratory, Oxford, and by Elemental Micro-Analysis Ltd., Kingston-upon-Tharnes. Combustion for micro-analysis yielded a residue. This ashed residue was also determined after heating the dry sample at 500 “C in a phtinum crucible for 2 days. Dry weights were obtained after drying in a vacuum desiccator over PzO,.
PECTEN KIDNEY GRANULES DETERMINATION
175
OF OXALATE
Potassium permanganate titration Potassium permanganate titrations were carried out in lo-ml conical flasks using the evaporated residue obtained from extraction of * 6 mg of granules with 1 M HCl. The acid-soluble extract was dissolved in 3 ml of 0.5 M H,SO, and heated to 60-70°C. This was titrated with 0.02 M KMnO, solution. The potassium permanganate solution was standardized by titration against oxalic acid (AR). ~e~~rnpo~iti~n with ~lphur~~ acid Concentrated sulphuric acid is known to decompose oxalate according to the following equation. (COOH), %?$ZO + CO, + H,O.
This was used as the principle for an estimation of oxalate in the granules using manometric measurement of the carbon monoxide liberated after trapping the carbon dioxide with potassium hydroxide pellets. The author is not aware of any previous study on the use of this method for quantitative determination of oxalate. The reaction was carried out in borosilicate glass manometer flasks of = 20 ml having a single side arm. The otherwise conventional manometer apparatus was modified by insertion of a narrow bore tap between the flask and the manometer. Rrodie’s solution was used in the manometer. A standardized suspension of granules (containing w 6 mg) was evaporated in the side arm and covered with 50 ,ul of boiled sulphuric acid (AR). Five pellets of KOH (w 0.5 g) were put in the bottom of the flask. The apparatus was assembled and allowed to equilibrate in the water bath with the connecting stop-cock open. When ~uilibration had been achieved both the connecting stop-cock and the manometer air-vent were closed, the apparatus was removed from the water bath and the side arm heated with a flame until the sulphuric acid just started to condense in the upper parts of the side arm. The flask was allowed to cool and to re-equilibrate in the water bath, at which time the connecting stop-cock was opened and readings taken until the difference in column heights was constant. The weight of oxalate was calculated using the above equation and the usual manometer formulae. DETERMiNATrON
OF SOLUBILITY OF HCI-rNSOLUBLE FRACTION
Granules were transferred to four Eppendorf reaction vessels, dried in vacua over P205 and extracted with 1 ml of 1 M HCl (AR) for 30 min. After centrifugation for 1 min on an Eppendorf centrifuge the supernatant was discarded, the pellets washed twice with water, the residues dried in vacua over P,Os and weighed. The pellets were then suspended in one of the following solutions and left overnight at
.I. OVbRNtLI
176
room temperature: (1) 0.1 M EDTA pH 7.0; (2) 0.1’:” SDS, 0.01 M morpholinopropanesulphonic acid pH 7.0; (3) I .O’!, lubrol, 0.01 M morpholinopropanesulphonic acid pH 7.0; and (4) 0. l”,, cetyltrimethylammonium bromide. 0.01 M morpholinopropanesulphonic acid pH 7.0. The tubes were centrifuged for 1 min in an Eppendorf
centrifuge,
the pellet
washed
four times with distilled
water,
dried
in
vacua over P,O, and weighed. DODECYLSULPHATE
POLYACRYLAMIDE
GEL ELECTROPHORESIS
This was carried out using the method of Weber & Osborn (1969) using as molecular weight standards: bovine serum albumin, ovalbumin, bovine erythrocyte carbonic anhydrase, bovine myoglobin, and equine cytochrome C. The granule sample (100 ~1 of the SDS-soluble, HCl- and water-insoluble. fraction) was treated overnight with 5 ~1 of mercaptoethanol.
REX LTS ELEMENTAL
ANALYSIS
Table I gives a series of analyses of successive batches of kidney granules. The reason for the differences of ash residue found by Strauss on the one hand and Elemental Micro-Analysis and the author on the other hand, is not known. It could be due to batch variation, but more probably is due to different methods of analyses because animals taken 6 months apart gave the same results from one analyser, and animals taken at the same time of year gave different values from different analysers. The most important finding is that the carbon content is x 11”;).
Elemental
analyses
ol’kidney “,, Ash 67.X) 65.55 73.84 72.44 7-l
granules Analyser Strauss straus:, Elementd Micro-Analysis Elemental Micro-Analysis ‘The atrthor
No attempt has been made to calculate an elemental composition for the presumed organic component assuming that the balance is due to oxygen. because of (a) the uncertainty of the ash residue and (b) the possibility that a portion of the hydrogen and nitrogen values may be due to water of hydration and ammonium salts. To illustrate point (b) an acid-soluble mixture of metal phosphates was precipitated by alkali from a solution which also contained 0.7cj; ammonium. After
PECTEN
KIDNEY
GRANULES
177
drying for 4 days under high vacuum over P,Os the precipitate was found to contain 27: H and 0.8% N. Phosphate determined by the method of Gomori (1942) and metals determined by atomic absorption spectrometry after nitric acid digestion were substantially similar to those published by George et al. (1980). SOLUBILITY
STUDIES
The inorganic component of the granules can be dissolved in hydrochloric acid which also extracts a considerable amount of colour, and so presumably some organic components. Thus, when 39.25 mg of a mixture of batches of granules was incubated with 1 ml of 1 M hydrochloric acid for 30 min, the residue after washing with water was 4.37 mg indicating that 88.9% was soluble in the acid. (The head gases smelt of skunk; presumably indicating the presence of liberated sulphides and/or mercaptans.) The hydrochloric acid-insoluble, water-insoluble fraction showed varying solubilities in protein-solubilizing media as shown below:
Medium
Solubility (%)
0.1 M ethylenediaminetetraacetic acid (EDTA) 0.1% sodium dodecylsulphate (SDS) 1% lubrol 0.1% cetyltrimethylammonium bromide (CTB)
19 71 40 22
In addition the 0.1 M EDTA (pH 7.0) was capable of dissolving the inorganic component as well when whole granules were treated with this reagent. ULTRA-VIOLET
ABSORPTION
The portion of the granules soluble in EDTA (pH 7.0) was diluted ten-fold with water and the UV spectrum measured v. 0.01 M EDTA. There was an absorption maximum at 258 nm with E:& = 17 and an absorption tail into the visible (giving the solution a brown colour). If this absorption were due to a purine nucleotide it would correspond to z 3% of the original weight. THE PROTEIN
COMPONENT
Amino-acid analyses of the 6 M HCl hydrolysate of two batches of granules showed very similar amino-acid compositions. The average composition expressed as the mole “/oof the total is shown in Table II and for comparison the compositions of erythrocyte membrane protein, calcium-binding protein and cadmium-binding
178
J. OVERNELL
protein are also given. The total amount of protein by amino-acid analysis was found to be 69% (in different batches) of which = SO”,;,was found to be extractable by 0.1 M EDTA from the whole granules. Tiztne II Amino-acid Guidotti.
analysis of scallop kidney granules: comparison with data i‘or human erythrocyte membrane 1972). calcium-binding protein (~~sser~n er ai., 1968). and cadmium-binding protein (Ragi ef uf.. 1974): t1.m.. not measured. Mole percentages
Amino
acid
Lysine Histidine Arginine Aspartic acid + asparagine Threonine Serine Glutamic acid t gfutamine Prohne <;lycine Alanine Cysteine + f (cystine) Valine Methionine lsoleucine Leucine Tyrosine Pheny~alanine Tryptophan
Sdhp kidney granules ._. _.
Human erythrocyte membrane
Calciumbinding protein
Cadmiumbinding protein
7.2 2.6 5.0
5.2 -.?4 4.5
9.7 1.h 7.5
11.1 0.0 2.3
10,s 5.1 h.?
x.5
5.2 ?.I
h7
13.Y 4, I 3.1
I I.0
10.0 4.3 17.7 1.4
12.’ 4.3 6.1 x.t
15.6 I.5 7.1 7.1
4.3 4.3 IO.0 9.7
I .ir
6. I 1.7 s.1 7.4 2 .‘6 3.6 II 113
5.Y
33.6 ?,I 1.7 0.2 0.6 0.0 0.0 0.0
Separation of the protein component of the EDTA-solubihzed granules from the mineral component was attempted using Sephadex G25 PD 16 columns equilibrated with 0.01 M EDTA pH 7.0. The cohtmn effluent was monitored by alkaline hydrolysis of each fraction followed by ninhydrin treatment (Hirs, 1967). Protein determination on EDTA extracts by the method of Lowry (Miller, 1959) or by dye-binding methods (Schaffner & Weissmann, 1973; Bradford. 1976) did not prove satisfactory. Ninhydrin-positive components were detected throughout the chromatogram from the void volume to beyond the bed volume. It was concluded that there was binding to the column.
PECTEN ELEMENTAL
ANALYSES
KIDNEY
OF HCI-INSOLUBLE,
GRANULES
179
WATER-INSOLUBLE
FRACTION
The average of three analyses was as follows: C 51.55x, ash 4.1% (of dry wt).
H 7.10x,
N 9.75x,
GEL ELECTROPHORESIS
Fig. 2 is a reproduction of densitometer traces of the SDS gel electrophoregram obtained by the electrophoresis of the SDS-soluble extract of the HCl-insoluble, water-insoluble fraction and for comparison a trace of the electrophoregram of a
b
rnw
BSA
A
cb anh ll
ova
0
1
Migration
2
distance
I
3
4
5
6
(cm)
Fig. 2. Gel electrophoresis of granule proteins and of standard: A, dodecylsulphate-soluble fraction of hydrochloric acid-insoluble residue from extraction of Pecten kidney granules; wt 210 pg per tube stained with coomassie blue; reproduced from densitometer scan; B, mixture of proteins electrophoresed, stained, and scanned under identical conditions to A; approximate molecular weights as follows in parentheses; BSA, bovine serum albumin (70000); ova, ovalbumin (40000); cb anh, bovine erythrocyte carbonic anhydrase (30000); myo, bovine myoglobin (17000); cyt c, equine cytochrome C (12000); wt. 6 pg each protein per tube.
J. OVERNELL
I MI
mixture
of standards.
blue positive
Without
components
the mercaptoethanol
of the granules
entered
pre-treatment
no coomassie
the gel. As may be seen there are
a large number of components, all having a molecular weight of 30000 or more, with a significant amount of material of very high molecular weight which has only just entered SUGARS
the gel.
1N WHOLE
GRANULES
Acid hydrolysis for amino-acid analysis produced considerable charring, suggesting the presence of carbohydrate. The following tests, however, proved negative : total carbohydrate using anthrone-H,SO, (Yemm & Willis, 1954). neutral sugars by hydrolysis in 1 M HCl followed by gas chromatography of the alditol acetates (Met2 et al.. 1971), hexosamines by acid hydrolysis and detection on amino-acid analyser (Allen & Neuberger, 1975), and uranic acids by use of carbozole-H,SO, (Bitter & Ewins, 1961). LIPIDS
AND
PHENOLS
IN 1 M EDTA
No significant amount methanol, and no phenol CARBONATE
IN WHOLE
No gas was liberated CITRATE
IN WHOLE
EXTRACT
OF GRANULES
of lipid was detected after extraction with chloroform was detected using diazotized sulphanilic acid.
GRANULES
with 1 M HCl in a calibrated
manometer
GRANULES
No positive reaction was observed of Spencer & Lowenstein, 1967.
using the acetic anhydride-pyridine
method
OXALATE
Titration of a hot acid solution of the granules with potassium permanganate proceeded rapidly at a speed consistent with the oxidation of oxalate. The end-point was, however, difficult to determine with accuracy because the rapid decolourization was followed by a slow phase presumably due to oxidation of protein and/or other organic components. Finally, addition of potassium permanganate produced a precipitate of manganese dioxide. The volume of potassium permanganate decolourized in the rapid phase was equivalent to ox&late accounting for w 7”); of the original dry weight of granules. The decomposition of standard amounts of oxalate with H,SO, in the manometer gave somewhat variable results with calculated efficiencies of determination of between 70 and 90:;. It is suspected that the stoichiometry of the reaction is not accurately as implied by the above equation. It is thought that lower amounts of CO may be obtained because of some reduction of the sulphuric acid. The
PECTEN
KIDNEYGRANULES
181
manometric determination gave a value of between 6 and 7.5% of oxalate in the original granules. This compares well with the value for the oxalate determined by potassium permanganate titration.
DISCUSSION AND CONCLUSION The analyses of four batches of scallop kidney granules show that carbon is present to the extent of = 11% on a dry wt basis. This is in contrast to the results of George et al. (1980) who found 1.2% carbon. As carbonate is absent, organic material must represent a significant proportion of the total weight. The organic components positively identified in the granules are protein 69% 3% nucleotide has been suggested. The protein and oxalate NN 7%. The presence of NN together with the oxalate, therefore, corresponds to 56.5% by weight of carbon in the original granules. Thus, identified components only account for about half of the carbon found by analysis, but we can conclude that much of the remainder of the carbon is probably present in an acid-soluble form which probably has a high oxygen content to account for the observed weight loss on ashing. Now 11% by weight of the granules is insoluble in 1 M HCI but the protein content is only 6-9x. One explanation for this disparity could be the presence of carbon in insoluble lipid-protein complexes such as lipfuchsin or age pigment. Elemental analysis of the HCI-insoluble portion is, however, similar to that of protein although the nitrogen is low. Thus a carbohydrate-protein complex would tit the analysis better. The amino-acid composition of the granules is quite similar to that of human erythrocyte membrane protein, less similar to calcium-binding protein and totally different from cadmium-binding protein. The only significant difference between the granule protein composition and that of membrane lies in the glycine values: glycine being higher in the scallop granules. This tends to support the proton pumping mechanism for granule secretion rather than a binding-protein mechanism (Simkiss, 1977). In granular mineral concretions, the anions present are usually phosphate and carbonate, but m-ate and oxalate have also been found (Simkiss, 1976; Hignette, 1980). The lack of carbonate found here is in agreement with the qualitative observation of George et al. (1980) for Pecten kidney granules, but they were unable to detect the oxalate by an (unspecified) qualitative procedure. Oxalate has recently been reported to be present in granules from the kidney of the giant clam and a mussel, detected by methods quite different from those used here (Hignette, 1980). George et al. (1980) found a ratio of metals to phosphate of x 1. Data from Sillen & Martel (1964, 1971) give the log of the solubility products of M2+ + HPqas follows: Ca -6.5, Mn - 12.9, and Mg - 5.8 to -6.7 but for zinc no value is given (but it is presumably low). The same authors also gave the log of the solubility product for Ca’+ + oxalatti- as -7.9. The other metal-oxalate solubility products
J. OVERNELI
182
are not given but are presumably also low. Thus, on the basis of these data it is quite possible for the oxalate to be deposited as the calcium salt along with the ~nso~ub~~phosphates. From the results of George et cd. (1980). and the oxalate determined here, we have average values as follows : Species ._ .._-_.-_-.__-._._.
Concentrations ~rno~es~kg~ _.__-~_~___-...-__.~_~_.~ _ _ ._“.._. _ _ __~ _~______
Divalent metals Phosphate Oxalate Chloride
6.2 6.0 0.8 0.8
In order to balance charge one-third of the phosp~te has to exist as H,PO; and two-thirds as HP@-. This implies that the granules would exert a buffering effect and that they may have been deposited from a solution of %pH 7.0-7.4. This would argue against the proton pumpirrg mechanism for granule secretion because intracellular pH is generally MpH 7.4.
ACKNOWLEDGEMENT
The author thanks Dr. T. L. Coombs for suggesting the project and for helpfui discussions. REFERENCES ALLEN.A.K. & A. NfUmRmR.
1975.The qu~ntitation of gIuc{~sami~e and gala~tosamine in glycoproteins after hydrolysis in p-toluenesulphonic acid. FEBS Lett., Vol. 60. pp. 76.-80. BII,>ER,T. & R. RWINS,1961. A modified carhazole reaction for uranic acids. Biochenl. J.. Vol. 81, p. 43Ponly. BHADFORD. M. M., 1976. A rapid and sensitive method for the quantitation of microgram quantities ot protein utilizing the principle of protein-dye binding. Anafyt. Bfochem., Vol. 72, pp. 248-254. CARMICHAEL, N. G., K. S. SQUIBB&B. A.FOWLER, 1979. Metalsin the molfuscan kidney: a comparison of two closely related bivalve species (Argopecten), using X-ray microanalysis and atomic absorption spectroscopy. J. Fish Res. Bd Can., Vol. 36, pp. 1149-1155. DAKIN,W. J.. 1909. Pecten. L.M.B.C. Mew. typ. Bv. mar. PI. Aniw.. Vol. 17 DOYLE, L. J.. N. J. BLAKE.C, C. WOO & P. YEVI~N, 1978. Recent biogenic phosphorite: concretions in moliusc kidneys. Science, Vol. 199, pp. 1431.--1433. GEORGE, S.G.. B. J. S. %.IE & T. L. COOMRS.1980. Isolation and elemental amatysis of metal-rich granules from the kidney of the SCaJfOpPecten malrimus (L.) J. exp. mar. Viol. Ew/.. Vol. 42, pp. 143-156. GOMORI, G., 1942. A modification of the calorimetric phosphorus determination for use with the photoelectric calorimeter. J. Lab. c&n. Med.. 27, pp. 955960. GUIDOTTI.G., 1972. Membrane proteins. Ann. Rw. 3i~i~~~z.. Vol. 41, pp. 731 752. HICINETTE. M., 1980. Accumulation de metaux dans les concretions minerales drs reins des mollusques
PECTEN
KIDNEY GRANULES
183
lamellibranches. In, Les mPtaux en milieu marin phosphore et &rids phosphor&s, fiditions du CNRS, Paris, pp. 195-206. HIRS, C. H. W., 1967. Detection of peptides by chemical methods. In, Methodr in enzymology, Vol. II, edited by S. P. Colowick & N. 0. Kaplan, Academic Press, London, pp. 325-329. K.Xcr, J.H.R., S.R. HIMMELHOCH, P.D. WHANGER, J.L. BETHUNE& B.L. VALLEE, 1974. Equine hepatic and renal metallothioneins. Purification, molecular weight, amino acid composition and metal content. J. biol. Chem., Vol. 249, pp. 3537-3542. METZ, J., W. EBERT& H. WEICKER,1971. Quantitative Analyse von Neutral- und Aminozuckern mit der Gas-Liquid-Chromatogaphie. Chromatographia, Vol. 4, pp. 345-350. MILLER, G. L., 1959. Protein determination for large numbers of samples. Analyt. Chem., Vol. 31, p. 964 only. SCHAFFNER,W. & C. WEISSMANN,1973. A rapid, sensitive and specific method for the determination of protein in dilute solution. Analyt. Biochem., Vol. 56, pp. 502-514. SILL~N,L. G. & A. E. MARTELL,1964. Stability constants of metal-ion complexes. Spec. Publ. No. 17, The Chemical Society, London. SILL&N,L. G. & A. E. MARTELL,1971. Stability constants of metal-ion complexes. Suppl. No. I to Spec. Publ. No. 17, The Chemical Society, London. SIMKISS,K., 1976. Intracellular and extracellular routes in biomineralization. Proc. Sot. exp. Biol., Vol. 50, pp. 423-444. SIMKISS,K., 1977. Biomineralization and detoxification. Cal&$ Tissue Res., Vol. 24, pp. 199-200. SPENCER, A.F. & J. M. LOWENSTEIN,1967. Citrate content of liver and kidney of rat in various metabolic states and in fluoroacetate poisoning. Biochem. J., Vol. 103, pp. 342-348. WASSERMAN,R. H., R.A. C~RRADINO& A. N. TAYLOR,1968. Vitamin D-dependent calcium-binding protein. Purification and some properties. J. biol. Chem., Vol. 243, pp. 3978-3986. WEBER, K. & M. OSBORN, 1969. The reliability of molecular wei& determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. biol. Chem., Vol. 244, pp. 4406-4412. YEMM, E. W. & A. J. WILLIS, 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem. J., Vol. 57, pp. 508-514.
PROTEIN
173
AND OXALATE IN MINERAL THE KIDNEY OF PECTEN
J. N.E.R.C.
Institute
of Marine
Biochemistry,
GRANULES
MAXZMUS
FROM
(L.)
OVERNELL St. FittickS
Road, Aberdeen
ABI
3RA,
Scotland
Abstract: The mineral-rich granules present in the kidney of the scallop, Pecten maximus (L.), have been found to contain 11%by weight of carbon and to lose at least 25% of their dry weight on ashing. Protein corresponding to 69% by weight and oxalate corresponding to 7% by weight have been identified. The sodium dodecylsulphate-soluble fraction of the protein (71%) consists of a large number of components having molecular weights of 30000 or higher, but the whole of the protein has an aminoacid analysis similar to erythrocyte membrane protein. Carbonate is absent from the granules.
INTRODUCTION
Dakin (1909) seems to have been the first to report the presence of large intracellular particles of refractile “excrete” material in scallop renal organs. Using
Fig. 1. Light micrograph of Pecten maximus kidney: tissue fixed in 2.5% glutaraldehyde in sea water; unstained section viewed under phase contrast; mc, mantle cavity; g, granule; I, lumen. 0022-0981/81/0000-0000/$2.50
0 Elsevier/North-Holland
Biomedical
Press
J. OVERNELl
174
normal histological procedures large numbers of extremely refractile bodies are obvious using phase contrast light microscopy (Fig. 1). Recently there have been reports on the mineral content of Pet&w granules (George et al., 1980) and Argopecten granules (Doyle cutal., 1978; Carmichael et al.. 1979). which have established that the granules are mostly composed of amorphous metal-phosphates. Thus, an average composition calculated from the data of George PI al. is: Zn 10.7p;. Mn 8.40/i, Ca 7.6Y.b.Mg 1.5’?;,.PO, 48.2”,,. Pcrren granules are probably destined to be excretion products (as suggested by Dakin. 1909) but an alternative role as a buffer for metal storage has not been ruled out (Carmichael et ~1.. 1979). Simkiss (1976) has reviewed evidence for these two functions of metal granules in various or~nisms. While the chemistry of the formation of such mineral concretions is still obscure, it seemed likely that metal-binding proteins might have been involved in the initial stages of the accumulation of the metals and might still be present in the matrix of the granules. This paper reports studies on the characterization of the organic components of Pecren ma~~imus(L.) kidney granules. MATERIAL
AND METHODS
P. maximus adults were purchased at different times of the year from the University Marine Biological Station, Millport, Isle of Cumbrae, Scotland, and held in tanks of circulating sea water until used. They were not fed. The isolation of kidney granules was carried out essentially according to the method described by George et al., (1980), except that the final sedimentation through 2.2 M sucrose was carried out at 1 x g overnight at 4 “C. AMINO-ACID
ANALYSES
These were carried out using a Jeof JLC-6AH liquid phase analyser after hydrolysis in 6 M HCl at 110 “C for 24 h, no correction being ma& for losses of serine or threonine. c‘. H AND N ANALYSES
AND ASH WEIGHTS
These were carried out by Dr. F. B. Strauss, Microanalytical Laboratory, Oxford, and by Elemental Micro-Analysis Ltd., Kingston-upon-Tharnes. Combustion for micro-analysis yielded a residue. This ashed residue was also determined after heating the dry sample at 500 “C in a phtinum crucible for 2 days. Dry weights were obtained after drying in a vacuum desiccator over PzO,.
PECTEN KIDNEY GRANULES DETERMINATION
175
OF OXALATE
Potassium permanganate titration Potassium permanganate titrations were carried out in lo-ml conical flasks using the evaporated residue obtained from extraction of * 6 mg of granules with 1 M HCl. The acid-soluble extract was dissolved in 3 ml of 0.5 M H,SO, and heated to 60-70°C. This was titrated with 0.02 M KMnO, solution. The potassium permanganate solution was standardized by titration against oxalic acid (AR). ~e~~rnpo~iti~n with ~lphur~~ acid Concentrated sulphuric acid is known to decompose oxalate according to the following equation. (COOH), %?$ZO + CO, + H,O.
This was used as the principle for an estimation of oxalate in the granules using manometric measurement of the carbon monoxide liberated after trapping the carbon dioxide with potassium hydroxide pellets. The author is not aware of any previous study on the use of this method for quantitative determination of oxalate. The reaction was carried out in borosilicate glass manometer flasks of = 20 ml having a single side arm. The otherwise conventional manometer apparatus was modified by insertion of a narrow bore tap between the flask and the manometer. Rrodie’s solution was used in the manometer. A standardized suspension of granules (containing w 6 mg) was evaporated in the side arm and covered with 50 ,ul of boiled sulphuric acid (AR). Five pellets of KOH (w 0.5 g) were put in the bottom of the flask. The apparatus was assembled and allowed to equilibrate in the water bath with the connecting stop-cock open. When ~uilibration had been achieved both the connecting stop-cock and the manometer air-vent were closed, the apparatus was removed from the water bath and the side arm heated with a flame until the sulphuric acid just started to condense in the upper parts of the side arm. The flask was allowed to cool and to re-equilibrate in the water bath, at which time the connecting stop-cock was opened and readings taken until the difference in column heights was constant. The weight of oxalate was calculated using the above equation and the usual manometer formulae. DETERMiNATrON
OF SOLUBILITY OF HCI-rNSOLUBLE FRACTION
Granules were transferred to four Eppendorf reaction vessels, dried in vacua over P205 and extracted with 1 ml of 1 M HCl (AR) for 30 min. After centrifugation for 1 min on an Eppendorf centrifuge the supernatant was discarded, the pellets washed twice with water, the residues dried in vacua over P,Os and weighed. The pellets were then suspended in one of the following solutions and left overnight at
.I. OVbRNtLI
176
room temperature: (1) 0.1 M EDTA pH 7.0; (2) 0.1’:” SDS, 0.01 M morpholinopropanesulphonic acid pH 7.0; (3) I .O’!, lubrol, 0.01 M morpholinopropanesulphonic acid pH 7.0; and (4) 0. l”,, cetyltrimethylammonium bromide. 0.01 M morpholinopropanesulphonic acid pH 7.0. The tubes were centrifuged for 1 min in an Eppendorf
centrifuge,
the pellet
washed
four times with distilled
water,
dried
in
vacua over P,O, and weighed. DODECYLSULPHATE
POLYACRYLAMIDE
GEL ELECTROPHORESIS
This was carried out using the method of Weber & Osborn (1969) using as molecular weight standards: bovine serum albumin, ovalbumin, bovine erythrocyte carbonic anhydrase, bovine myoglobin, and equine cytochrome C. The granule sample (100 ~1 of the SDS-soluble, HCl- and water-insoluble. fraction) was treated overnight with 5 ~1 of mercaptoethanol.
REX LTS ELEMENTAL
ANALYSIS
Table I gives a series of analyses of successive batches of kidney granules. The reason for the differences of ash residue found by Strauss on the one hand and Elemental Micro-Analysis and the author on the other hand, is not known. It could be due to batch variation, but more probably is due to different methods of analyses because animals taken 6 months apart gave the same results from one analyser, and animals taken at the same time of year gave different values from different analysers. The most important finding is that the carbon content is x 11”;).
Elemental
analyses
ol’kidney “,, Ash 67.X) 65.55 73.84 72.44 7-l
granules Analyser Strauss straus:, Elementd Micro-Analysis Elemental Micro-Analysis ‘The atrthor
No attempt has been made to calculate an elemental composition for the presumed organic component assuming that the balance is due to oxygen. because of (a) the uncertainty of the ash residue and (b) the possibility that a portion of the hydrogen and nitrogen values may be due to water of hydration and ammonium salts. To illustrate point (b) an acid-soluble mixture of metal phosphates was precipitated by alkali from a solution which also contained 0.7cj; ammonium. After
PECTEN
KIDNEY
GRANULES
177
drying for 4 days under high vacuum over P,Os the precipitate was found to contain 27: H and 0.8% N. Phosphate determined by the method of Gomori (1942) and metals determined by atomic absorption spectrometry after nitric acid digestion were substantially similar to those published by George et al. (1980). SOLUBILITY
STUDIES
The inorganic component of the granules can be dissolved in hydrochloric acid which also extracts a considerable amount of colour, and so presumably some organic components. Thus, when 39.25 mg of a mixture of batches of granules was incubated with 1 ml of 1 M hydrochloric acid for 30 min, the residue after washing with water was 4.37 mg indicating that 88.9% was soluble in the acid. (The head gases smelt of skunk; presumably indicating the presence of liberated sulphides and/or mercaptans.) The hydrochloric acid-insoluble, water-insoluble fraction showed varying solubilities in protein-solubilizing media as shown below:
Medium
Solubility (%)
0.1 M ethylenediaminetetraacetic acid (EDTA) 0.1% sodium dodecylsulphate (SDS) 1% lubrol 0.1% cetyltrimethylammonium bromide (CTB)
19 71 40 22
In addition the 0.1 M EDTA (pH 7.0) was capable of dissolving the inorganic component as well when whole granules were treated with this reagent. ULTRA-VIOLET
ABSORPTION
The portion of the granules soluble in EDTA (pH 7.0) was diluted ten-fold with water and the UV spectrum measured v. 0.01 M EDTA. There was an absorption maximum at 258 nm with E:& = 17 and an absorption tail into the visible (giving the solution a brown colour). If this absorption were due to a purine nucleotide it would correspond to z 3% of the original weight. THE PROTEIN
COMPONENT
Amino-acid analyses of the 6 M HCl hydrolysate of two batches of granules showed very similar amino-acid compositions. The average composition expressed as the mole “/oof the total is shown in Table II and for comparison the compositions of erythrocyte membrane protein, calcium-binding protein and cadmium-binding
178
J. OVERNELL
protein are also given. The total amount of protein by amino-acid analysis was found to be 69% (in different batches) of which = SO”,;,was found to be extractable by 0.1 M EDTA from the whole granules. Tiztne II Amino-acid Guidotti.
analysis of scallop kidney granules: comparison with data i‘or human erythrocyte membrane 1972). calcium-binding protein (~~sser~n er ai., 1968). and cadmium-binding protein (Ragi ef uf.. 1974): t1.m.. not measured. Mole percentages
Amino
acid
Lysine Histidine Arginine Aspartic acid + asparagine Threonine Serine Glutamic acid t gfutamine Prohne <;lycine Alanine Cysteine + f (cystine) Valine Methionine lsoleucine Leucine Tyrosine Pheny~alanine Tryptophan
Sdhp kidney granules ._. _.
Human erythrocyte membrane
Calciumbinding protein
Cadmiumbinding protein
7.2 2.6 5.0
5.2 -.?4 4.5
9.7 1.h 7.5
11.1 0.0 2.3
10,s 5.1 h.?
x.5
5.2 ?.I
h7
13.Y 4, I 3.1
I I.0
10.0 4.3 17.7 1.4
12.’ 4.3 6.1 x.t
15.6 I.5 7.1 7.1
4.3 4.3 IO.0 9.7
I .ir
6. I 1.7 s.1 7.4 2 .‘6 3.6 II 113
5.Y
33.6 ?,I 1.7 0.2 0.6 0.0 0.0 0.0
Separation of the protein component of the EDTA-solubihzed granules from the mineral component was attempted using Sephadex G25 PD 16 columns equilibrated with 0.01 M EDTA pH 7.0. The cohtmn effluent was monitored by alkaline hydrolysis of each fraction followed by ninhydrin treatment (Hirs, 1967). Protein determination on EDTA extracts by the method of Lowry (Miller, 1959) or by dye-binding methods (Schaffner & Weissmann, 1973; Bradford. 1976) did not prove satisfactory. Ninhydrin-positive components were detected throughout the chromatogram from the void volume to beyond the bed volume. It was concluded that there was binding to the column.
PECTEN ELEMENTAL
ANALYSES
KIDNEY
OF HCI-INSOLUBLE,
GRANULES
179
WATER-INSOLUBLE
FRACTION
The average of three analyses was as follows: C 51.55x, ash 4.1% (of dry wt).
H 7.10x,
N 9.75x,
GEL ELECTROPHORESIS
Fig. 2 is a reproduction of densitometer traces of the SDS gel electrophoregram obtained by the electrophoresis of the SDS-soluble extract of the HCl-insoluble, water-insoluble fraction and for comparison a trace of the electrophoregram of a
b
rnw
BSA
A
cb anh ll
ova
0
1
Migration
2
distance
I
3
4
5
6
(cm)
Fig. 2. Gel electrophoresis of granule proteins and of standard: A, dodecylsulphate-soluble fraction of hydrochloric acid-insoluble residue from extraction of Pecten kidney granules; wt 210 pg per tube stained with coomassie blue; reproduced from densitometer scan; B, mixture of proteins electrophoresed, stained, and scanned under identical conditions to A; approximate molecular weights as follows in parentheses; BSA, bovine serum albumin (70000); ova, ovalbumin (40000); cb anh, bovine erythrocyte carbonic anhydrase (30000); myo, bovine myoglobin (17000); cyt c, equine cytochrome C (12000); wt. 6 pg each protein per tube.
J. OVERNELL
I MI
mixture
of standards.
blue positive
Without
components
the mercaptoethanol
of the granules
entered
pre-treatment
no coomassie
the gel. As may be seen there are
a large number of components, all having a molecular weight of 30000 or more, with a significant amount of material of very high molecular weight which has only just entered SUGARS
the gel.
1N WHOLE
GRANULES
Acid hydrolysis for amino-acid analysis produced considerable charring, suggesting the presence of carbohydrate. The following tests, however, proved negative : total carbohydrate using anthrone-H,SO, (Yemm & Willis, 1954). neutral sugars by hydrolysis in 1 M HCl followed by gas chromatography of the alditol acetates (Met2 et al.. 1971), hexosamines by acid hydrolysis and detection on amino-acid analyser (Allen & Neuberger, 1975), and uranic acids by use of carbozole-H,SO, (Bitter & Ewins, 1961). LIPIDS
AND
PHENOLS
IN 1 M EDTA
No significant amount methanol, and no phenol CARBONATE
IN WHOLE
No gas was liberated CITRATE
IN WHOLE
EXTRACT
OF GRANULES
of lipid was detected after extraction with chloroform was detected using diazotized sulphanilic acid.
GRANULES
with 1 M HCl in a calibrated
manometer
GRANULES
No positive reaction was observed of Spencer & Lowenstein, 1967.
using the acetic anhydride-pyridine
method
OXALATE
Titration of a hot acid solution of the granules with potassium permanganate proceeded rapidly at a speed consistent with the oxidation of oxalate. The end-point was, however, difficult to determine with accuracy because the rapid decolourization was followed by a slow phase presumably due to oxidation of protein and/or other organic components. Finally, addition of potassium permanganate produced a precipitate of manganese dioxide. The volume of potassium permanganate decolourized in the rapid phase was equivalent to ox&late accounting for w 7”); of the original dry weight of granules. The decomposition of standard amounts of oxalate with H,SO, in the manometer gave somewhat variable results with calculated efficiencies of determination of between 70 and 90:;. It is suspected that the stoichiometry of the reaction is not accurately as implied by the above equation. It is thought that lower amounts of CO may be obtained because of some reduction of the sulphuric acid. The
PECTEN
KIDNEYGRANULES
181
manometric determination gave a value of between 6 and 7.5% of oxalate in the original granules. This compares well with the value for the oxalate determined by potassium permanganate titration.
DISCUSSION AND CONCLUSION The analyses of four batches of scallop kidney granules show that carbon is present to the extent of = 11% on a dry wt basis. This is in contrast to the results of George et al. (1980) who found 1.2% carbon. As carbonate is absent, organic material must represent a significant proportion of the total weight. The organic components positively identified in the granules are protein 69% 3% nucleotide has been suggested. The protein and oxalate NN 7%. The presence of NN together with the oxalate, therefore, corresponds to 56.5% by weight of carbon in the original granules. Thus, identified components only account for about half of the carbon found by analysis, but we can conclude that much of the remainder of the carbon is probably present in an acid-soluble form which probably has a high oxygen content to account for the observed weight loss on ashing. Now 11% by weight of the granules is insoluble in 1 M HCI but the protein content is only 6-9x. One explanation for this disparity could be the presence of carbon in insoluble lipid-protein complexes such as lipfuchsin or age pigment. Elemental analysis of the HCI-insoluble portion is, however, similar to that of protein although the nitrogen is low. Thus a carbohydrate-protein complex would tit the analysis better. The amino-acid composition of the granules is quite similar to that of human erythrocyte membrane protein, less similar to calcium-binding protein and totally different from cadmium-binding protein. The only significant difference between the granule protein composition and that of membrane lies in the glycine values: glycine being higher in the scallop granules. This tends to support the proton pumping mechanism for granule secretion rather than a binding-protein mechanism (Simkiss, 1977). In granular mineral concretions, the anions present are usually phosphate and carbonate, but m-ate and oxalate have also been found (Simkiss, 1976; Hignette, 1980). The lack of carbonate found here is in agreement with the qualitative observation of George et al. (1980) for Pecten kidney granules, but they were unable to detect the oxalate by an (unspecified) qualitative procedure. Oxalate has recently been reported to be present in granules from the kidney of the giant clam and a mussel, detected by methods quite different from those used here (Hignette, 1980). George et al. (1980) found a ratio of metals to phosphate of x 1. Data from Sillen & Martel (1964, 1971) give the log of the solubility products of M2+ + HPqas follows: Ca -6.5, Mn - 12.9, and Mg - 5.8 to -6.7 but for zinc no value is given (but it is presumably low). The same authors also gave the log of the solubility product for Ca’+ + oxalatti- as -7.9. The other metal-oxalate solubility products
J. OVERNELI
182
are not given but are presumably also low. Thus, on the basis of these data it is quite possible for the oxalate to be deposited as the calcium salt along with the ~nso~ub~~phosphates. From the results of George et cd. (1980). and the oxalate determined here, we have average values as follows : Species ._ .._-_.-_-.__-._._.
Concentrations ~rno~es~kg~ _.__-~_~___-...-__.~_~_.~ _ _ ._“.._. _ _ __~ _~______
Divalent metals Phosphate Oxalate Chloride
6.2 6.0 0.8 0.8
In order to balance charge one-third of the phosp~te has to exist as H,PO; and two-thirds as HP@-. This implies that the granules would exert a buffering effect and that they may have been deposited from a solution of %pH 7.0-7.4. This would argue against the proton pumpirrg mechanism for granule secretion because intracellular pH is generally MpH 7.4.
ACKNOWLEDGEMENT
The author thanks Dr. T. L. Coombs for suggesting the project and for helpfui discussions. REFERENCES ALLEN.A.K. & A. NfUmRmR.
1975.The qu~ntitation of gIuc{~sami~e and gala~tosamine in glycoproteins after hydrolysis in p-toluenesulphonic acid. FEBS Lett., Vol. 60. pp. 76.-80. BII,>ER,T. & R. RWINS,1961. A modified carhazole reaction for uranic acids. Biochenl. J.. Vol. 81, p. 43Ponly. BHADFORD. M. M., 1976. A rapid and sensitive method for the quantitation of microgram quantities ot protein utilizing the principle of protein-dye binding. Anafyt. Bfochem., Vol. 72, pp. 248-254. CARMICHAEL, N. G., K. S. SQUIBB&B. A.FOWLER, 1979. Metalsin the molfuscan kidney: a comparison of two closely related bivalve species (Argopecten), using X-ray microanalysis and atomic absorption spectroscopy. J. Fish Res. Bd Can., Vol. 36, pp. 1149-1155. DAKIN,W. J.. 1909. Pecten. L.M.B.C. Mew. typ. Bv. mar. PI. Aniw.. Vol. 17 DOYLE, L. J.. N. J. BLAKE.C, C. WOO & P. YEVI~N, 1978. Recent biogenic phosphorite: concretions in moliusc kidneys. Science, Vol. 199, pp. 1431.--1433. GEORGE, S.G.. B. J. S. %.IE & T. L. COOMRS.1980. Isolation and elemental amatysis of metal-rich granules from the kidney of the SCaJfOpPecten malrimus (L.) J. exp. mar. Viol. Ew/.. Vol. 42, pp. 143-156. GOMORI, G., 1942. A modification of the calorimetric phosphorus determination for use with the photoelectric calorimeter. J. Lab. c&n. Med.. 27, pp. 955960. GUIDOTTI.G., 1972. Membrane proteins. Ann. Rw. 3i~i~~~z.. Vol. 41, pp. 731 752. HICINETTE. M., 1980. Accumulation de metaux dans les concretions minerales drs reins des mollusques
PECTEN
KIDNEY GRANULES
183
lamellibranches. In, Les mPtaux en milieu marin phosphore et &rids phosphor&s, fiditions du CNRS, Paris, pp. 195-206. HIRS, C. H. W., 1967. Detection of peptides by chemical methods. In, Methodr in enzymology, Vol. II, edited by S. P. Colowick & N. 0. Kaplan, Academic Press, London, pp. 325-329. K.Xcr, J.H.R., S.R. HIMMELHOCH, P.D. WHANGER, J.L. BETHUNE& B.L. VALLEE, 1974. Equine hepatic and renal metallothioneins. Purification, molecular weight, amino acid composition and metal content. J. biol. Chem., Vol. 249, pp. 3537-3542. METZ, J., W. EBERT& H. WEICKER,1971. Quantitative Analyse von Neutral- und Aminozuckern mit der Gas-Liquid-Chromatogaphie. Chromatographia, Vol. 4, pp. 345-350. MILLER, G. L., 1959. Protein determination for large numbers of samples. Analyt. Chem., Vol. 31, p. 964 only. SCHAFFNER,W. & C. WEISSMANN,1973. A rapid, sensitive and specific method for the determination of protein in dilute solution. Analyt. Biochem., Vol. 56, pp. 502-514. SILL~N,L. G. & A. E. MARTELL,1964. Stability constants of metal-ion complexes. Spec. Publ. No. 17, The Chemical Society, London. SILL&N,L. G. & A. E. MARTELL,1971. Stability constants of metal-ion complexes. Suppl. No. I to Spec. Publ. No. 17, The Chemical Society, London. SIMKISS,K., 1976. Intracellular and extracellular routes in biomineralization. Proc. Sot. exp. Biol., Vol. 50, pp. 423-444. SIMKISS,K., 1977. Biomineralization and detoxification. Cal&$ Tissue Res., Vol. 24, pp. 199-200. SPENCER, A.F. & J. M. LOWENSTEIN,1967. Citrate content of liver and kidney of rat in various metabolic states and in fluoroacetate poisoning. Biochem. J., Vol. 103, pp. 342-348. WASSERMAN,R. H., R.A. C~RRADINO& A. N. TAYLOR,1968. Vitamin D-dependent calcium-binding protein. Purification and some properties. J. biol. Chem., Vol. 243, pp. 3978-3986. WEBER, K. & M. OSBORN, 1969. The reliability of molecular wei& determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. biol. Chem., Vol. 244, pp. 4406-4412. YEMM, E. W. & A. J. WILLIS, 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem. J., Vol. 57, pp. 508-514.