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Lens iso-precipitin in yellowfin tuna (Thunnus albacares)
Comp. Biochem. Physiol., 1972, Vol. 42B, pp. 497 to 499. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION LENS ISO-PRECIPITIN IN YELLOWFIN TUNA
(THUNNUS ALBACARES) A L B E R T C. S M I T H Division of Natural Science, University of Hawaii at Hilo, Hilo College, P.O. Box 1357, Hilo, Hawaii 96720
(Received 1 November 1971) A b s t r a c t - - 1 . Electrophoresis has been commonly used in the analysis of nuclear eye lens extracts for intraspecific variation. 2. Experiments with extracts from yellowfin tuna indicate the presence of an iso-precipitin which may provide a simpler, but equally precise method.
INTRODUCTION THE ELECTROPHORETICseparation of nuclear eye lens proteins has been a useful method of demonstrating intraspecific variation in a n u m b e r of fish species, e.g. yellowfin tuna (Thunnus albacares) (Smith, 1965, 1970), ocean whitefish (Caulolatilus princeps) (Smith & Goldstein, 1967), Arctic char (Salvelinus alpinus) (Saunders & McKenzie, 1971) and striped mullet (Mugil cephalus) (Peterson & Shehadeh, 1971). However, the electrophoretic procedure has several disadvantages, e.g. the many small steps involved make it tedious, and minor alterations in procedure sometimes distort protein patterns. Therefore, an attempt was made to develop a method of nuclear lens protein analysis that would not have the disadvantages of electrophoresis. MATERIALS AND M E T H O D S The method consisted of testing for substances capable of reacting with other substances varying among individuals of the same species. Lens nuclei from yellowfin tuna were used because of availabilitT and awareness of nuclear lens protein variability in this species (Smith, 1965, 1970). Specifically, eye lenses were collected from three frozen samples of five yellowfin each. The lenses were dissected to obtain entire nuclei or portions of nuclei containing all layers. This tissue was then ground into a powder with a Sorvall Omni-Mixer with the micro-sample holder immersed in ice water. Extracts were prepared by adding a volume of 0"018 g o/ /o saline solution equal to seven times the wet weight of tissue and shaking on a multi-purpose rotator (Scientific Industries, Inc.) for 24 hr at 5°C. After this time, the extracts were cleared by centrifugation at 6000 rev/min for 60 rain at 5°C. Five drops from each extract were mixed at room temperature with five drops from each remaining extract in the same collection. After 5 rain incubation, the extracts were examined against an indirectly illuminated black background for clarity or turbidity. R E S U L T S AND D I S C U S S I O N Table 1 shows that one or two extracts in each group of five produced a m o d e r ate cloudiness when mixed with one to three other extracts in the same group. T h e 497
498
ALBERTC. SMITH TABLE 1--REsuLTS OF MIXINGYELLOWFINNUCLEARLEANSEXTRACTS
Extract No.
1
Collection I
Collection II
Collection III
Extract No.
Extract No.
Extract No.
2
3
4
5
1
1
3 +
2
3 4 5
2
--
+ + +
+ +
4
5
1
2
3
4
5
+
-
+
" -
+ +
+
Key : (+) = moderate cloudiness ; ( - ) = equivocal or no cloudiness. In some cases, reciprocals of precipitate-producing mixtures gave a ( - ) result. The reason for this is under investigation. former extracts, before mixing, developed cloudiness upon refrigeration. This cloudiness, however, disappeared, or was greatly reduced, at room temperature. Thus, within the yellowfin lens nucleus there must be a substance acting like an iso-antibody, since it reacts with another substance acting like an antigen variably present within the same species. Since the former substance is involved in a precipitation reaction, it may be considered an iso-precipitin--the first so reported in eye lens. T h e s e findings are consistent with earlier reports of intraspecific variation in lens proteins of yellowfin and other species, as well as with a report on agglutinogen (blood group antigen) variation in human lenses (Sabnis & Basu, 1963). Iso-precipitation reactions may provide a quick and simple alternative to electrophoresis for demonstrating intraspecific variability, in nuclear lens protein. "Reagent extracts", i.e. those containing the iso-precipitin, need only to be mixed with other extracts to distinguish those with reacting substances from those without. An even simpler determinant appears to be the clouding at low temperature of only those extracts containing the iso-precipitin. Although intraspecific, lowtemperature-dependent variation in clouding was previously noted in yellowfin, it was not related to an iso-precipitin or any other specific substance (Smith, 1965). However, in shark and rat lens extracts, low-temperature clouding of cold precipitable protein (C.P.P.), or gamma crystalline, is observed (Lerman et aL, 1968). T h e cold sensitivity of what appears to be iso-precipitin suggests a method of obtaining it in pure, concentrated solution. T h e method consists of high-speed centrifugation of extract at low temperature, followed by washing of the precipitated iso-precipitin with cold water or weak saline solution. T h e final step is redissolution of the precipitate in one of these fluids at room temperature. T h e volume of this solution must be smaller than that of the original extract. Research currently in progress is directed toward identifying the reacting substances and their combination product; and toward determining whether there
LENS I S O o P R E C I P I T I N I N YELLOV~'FIN T U N A
499
is any variation in concentration of lens iso-precipitin with lens layer, sex, size, reproductive condition, time of year, breeding unit (population), etc. ; whether species other than yellowfin will demonstrate nuclear lens iso-precipitation reactions; and whether lens iso-precipitin from one species will react with substances f r o m lens and other tissues f r o m other species. I n conclusion, this study presents evidence for the existence of an iso-precipitin in vellowfin eye lens. It also suggests the promise of iso-precipitation and differential clouding at low t e m p e r a t u r e of nuclear lens extracts as attractive alternatives to electrophoresis for identifying intraspecific variation. T h e y are simpler and at least as precise. Unlike electrophoresis, they can be conveniently p e r f o r m e d in the field as well as in the laboratory.
Acknowledgements~I wish to thank Mr. Hiro Nishimura, Auctioneer, Suisan's fish auction, Hilo, Hawaii, for the yellowfin lenses; Mr. John Ford and Mr. Myron Yoshioka, students, University of Hawaii, Hilo College, for excellent technical assistance; Professors David R. Miller and Allan J. Kennedy, faculty., Humanities Division, Hilo College, for critical reading of the manuscript; and the Research Council, University of Hawaii, for financial support of this research. REFERENCES LERMAN S., FORBES W. F., Z m M ~ S. & KIRMAN J. (1968) A study on gamma crystallin derived from the rat and dogfish lens. In Biochemistry of the Eye (Edited by DARDENNA M. A. & NORDMANNJ.), pp. 292-300. Symposium, Tutzing Castle. PETERSON G. L. & SHEHADEHZ. H. (1971) Subpopulations of the Hawaiian striped mullet .VIngil cephalus (L.) : analysis of variations of nuclear eye-lens protein electropherograms and nuclear eye-lens weights. 2Viar. Biol. 11, 52-60. SABNIS S. S. & BAS~.'P. K. (1963) A and B antigens in human avascular tissues. Am. J. Ophth. 56, 229-232. SAL'NDERS L. H. & McKENZIE J. A. (I971) Comparative electrophoresis of Arctic char. Comp. Biochem. Physiol. 38B, 487-492. SMITH A. C. (1965) Intraspecific eye lens protein differences in the yellowfin tuna, ThTmmls albacares. Calif. Fish Game 51, 163-167. SMITH A. C. (1970) Electrophoretic, solubility and therrnostability differences in proteins of eye lens nuclei from two closely related fish species, the yellowfin tuna and the bigeye tuna. Comp. Biochem. Physiol. 33, 1-14. SMITH A. C. & GOLDSTEINR. A. (1967) Variation in protein composition of the eye lens nucleus in ocean whitefish, Caulolatilus princeps. Comp. Biochem. Physiol. 23, 533-539.
Key Word Index--Lens protein ; iso-precipitin of proteins ; tuna fish ; Thunnus albacares.
SHORT COMMUNICATION LENS ISO-PRECIPITIN IN YELLOWFIN TUNA
(THUNNUS ALBACARES) A L B E R T C. S M I T H Division of Natural Science, University of Hawaii at Hilo, Hilo College, P.O. Box 1357, Hilo, Hawaii 96720
(Received 1 November 1971) A b s t r a c t - - 1 . Electrophoresis has been commonly used in the analysis of nuclear eye lens extracts for intraspecific variation. 2. Experiments with extracts from yellowfin tuna indicate the presence of an iso-precipitin which may provide a simpler, but equally precise method.
INTRODUCTION THE ELECTROPHORETICseparation of nuclear eye lens proteins has been a useful method of demonstrating intraspecific variation in a n u m b e r of fish species, e.g. yellowfin tuna (Thunnus albacares) (Smith, 1965, 1970), ocean whitefish (Caulolatilus princeps) (Smith & Goldstein, 1967), Arctic char (Salvelinus alpinus) (Saunders & McKenzie, 1971) and striped mullet (Mugil cephalus) (Peterson & Shehadeh, 1971). However, the electrophoretic procedure has several disadvantages, e.g. the many small steps involved make it tedious, and minor alterations in procedure sometimes distort protein patterns. Therefore, an attempt was made to develop a method of nuclear lens protein analysis that would not have the disadvantages of electrophoresis. MATERIALS AND M E T H O D S The method consisted of testing for substances capable of reacting with other substances varying among individuals of the same species. Lens nuclei from yellowfin tuna were used because of availabilitT and awareness of nuclear lens protein variability in this species (Smith, 1965, 1970). Specifically, eye lenses were collected from three frozen samples of five yellowfin each. The lenses were dissected to obtain entire nuclei or portions of nuclei containing all layers. This tissue was then ground into a powder with a Sorvall Omni-Mixer with the micro-sample holder immersed in ice water. Extracts were prepared by adding a volume of 0"018 g o/ /o saline solution equal to seven times the wet weight of tissue and shaking on a multi-purpose rotator (Scientific Industries, Inc.) for 24 hr at 5°C. After this time, the extracts were cleared by centrifugation at 6000 rev/min for 60 rain at 5°C. Five drops from each extract were mixed at room temperature with five drops from each remaining extract in the same collection. After 5 rain incubation, the extracts were examined against an indirectly illuminated black background for clarity or turbidity. R E S U L T S AND D I S C U S S I O N Table 1 shows that one or two extracts in each group of five produced a m o d e r ate cloudiness when mixed with one to three other extracts in the same group. T h e 497
498
ALBERTC. SMITH TABLE 1--REsuLTS OF MIXINGYELLOWFINNUCLEARLEANSEXTRACTS
Extract No.
1
Collection I
Collection II
Collection III
Extract No.
Extract No.
Extract No.
2
3
4
5
1
1
3 +
2
3 4 5
2
--
+ + +
+ +
4
5
1
2
3
4
5
+
-
+
" -
+ +
+
Key : (+) = moderate cloudiness ; ( - ) = equivocal or no cloudiness. In some cases, reciprocals of precipitate-producing mixtures gave a ( - ) result. The reason for this is under investigation. former extracts, before mixing, developed cloudiness upon refrigeration. This cloudiness, however, disappeared, or was greatly reduced, at room temperature. Thus, within the yellowfin lens nucleus there must be a substance acting like an iso-antibody, since it reacts with another substance acting like an antigen variably present within the same species. Since the former substance is involved in a precipitation reaction, it may be considered an iso-precipitin--the first so reported in eye lens. T h e s e findings are consistent with earlier reports of intraspecific variation in lens proteins of yellowfin and other species, as well as with a report on agglutinogen (blood group antigen) variation in human lenses (Sabnis & Basu, 1963). Iso-precipitation reactions may provide a quick and simple alternative to electrophoresis for demonstrating intraspecific variability, in nuclear lens protein. "Reagent extracts", i.e. those containing the iso-precipitin, need only to be mixed with other extracts to distinguish those with reacting substances from those without. An even simpler determinant appears to be the clouding at low temperature of only those extracts containing the iso-precipitin. Although intraspecific, lowtemperature-dependent variation in clouding was previously noted in yellowfin, it was not related to an iso-precipitin or any other specific substance (Smith, 1965). However, in shark and rat lens extracts, low-temperature clouding of cold precipitable protein (C.P.P.), or gamma crystalline, is observed (Lerman et aL, 1968). T h e cold sensitivity of what appears to be iso-precipitin suggests a method of obtaining it in pure, concentrated solution. T h e method consists of high-speed centrifugation of extract at low temperature, followed by washing of the precipitated iso-precipitin with cold water or weak saline solution. T h e final step is redissolution of the precipitate in one of these fluids at room temperature. T h e volume of this solution must be smaller than that of the original extract. Research currently in progress is directed toward identifying the reacting substances and their combination product; and toward determining whether there
LENS I S O o P R E C I P I T I N I N YELLOV~'FIN T U N A
499
is any variation in concentration of lens iso-precipitin with lens layer, sex, size, reproductive condition, time of year, breeding unit (population), etc. ; whether species other than yellowfin will demonstrate nuclear lens iso-precipitation reactions; and whether lens iso-precipitin from one species will react with substances f r o m lens and other tissues f r o m other species. I n conclusion, this study presents evidence for the existence of an iso-precipitin in vellowfin eye lens. It also suggests the promise of iso-precipitation and differential clouding at low t e m p e r a t u r e of nuclear lens extracts as attractive alternatives to electrophoresis for identifying intraspecific variation. T h e y are simpler and at least as precise. Unlike electrophoresis, they can be conveniently p e r f o r m e d in the field as well as in the laboratory.
Acknowledgements~I wish to thank Mr. Hiro Nishimura, Auctioneer, Suisan's fish auction, Hilo, Hawaii, for the yellowfin lenses; Mr. John Ford and Mr. Myron Yoshioka, students, University of Hawaii, Hilo College, for excellent technical assistance; Professors David R. Miller and Allan J. Kennedy, faculty., Humanities Division, Hilo College, for critical reading of the manuscript; and the Research Council, University of Hawaii, for financial support of this research. REFERENCES LERMAN S., FORBES W. F., Z m M ~ S. & KIRMAN J. (1968) A study on gamma crystallin derived from the rat and dogfish lens. In Biochemistry of the Eye (Edited by DARDENNA M. A. & NORDMANNJ.), pp. 292-300. Symposium, Tutzing Castle. PETERSON G. L. & SHEHADEHZ. H. (1971) Subpopulations of the Hawaiian striped mullet .VIngil cephalus (L.) : analysis of variations of nuclear eye-lens protein electropherograms and nuclear eye-lens weights. 2Viar. Biol. 11, 52-60. SABNIS S. S. & BAS~.'P. K. (1963) A and B antigens in human avascular tissues. Am. J. Ophth. 56, 229-232. SAL'NDERS L. H. & McKENZIE J. A. (I971) Comparative electrophoresis of Arctic char. Comp. Biochem. Physiol. 38B, 487-492. SMITH A. C. (1965) Intraspecific eye lens protein differences in the yellowfin tuna, ThTmmls albacares. Calif. Fish Game 51, 163-167. SMITH A. C. (1970) Electrophoretic, solubility and therrnostability differences in proteins of eye lens nuclei from two closely related fish species, the yellowfin tuna and the bigeye tuna. Comp. Biochem. Physiol. 33, 1-14. SMITH A. C. & GOLDSTEINR. A. (1967) Variation in protein composition of the eye lens nucleus in ocean whitefish, Caulolatilus princeps. Comp. Biochem. Physiol. 23, 533-539.
Key Word Index--Lens protein ; iso-precipitin of proteins ; tuna fish ; Thunnus albacares.