740 



Fishery Bulletin 94(4). 1996 



1 2 3 



KDa 1 2 3 



205- 



116- 

 97.4- 



66- 



45- 



29- 



A B 



Figure 5 



SDS-PAGE (A) and corresponding Western blots (B) of the highly 

 purified 66 kDa proteins from 1) L. jocu, 2) L. apodus, and 3) L. 

 griseus. Total protein added to each lane was 2 ug. The protein of L. 

 griseus is slightly heavier compared with the other two species. Com- 

 parative N-terminal amino acid sequences of the three proteins are 

 shown in Table 3. 



Table 3 



The N-terminal amino acid sequence analysis of the 66 kDa glycoproteins purified from L. griseus. L.jocu, and L. apodus.' 



Snappci s 



1 



8 



10 



L. griseus 

 L. jocu 

 L. apodus 



Asp/ Ala 



Asp 



Asp 



Glu Ala 



Glu (Ala; 3 



Glu/Ala His 



N.I. 2 



His 



Ala 



Ala 

 Ala 

 Asp 



Asp 



Asp 

 Ala 



Ala 

 \l.i 

 Glu 



Glu 

 Glu 

 Glu 



Glu 

 Glu 

 Val 



Val 

 Val 



I'm 



Analyzed at the University of Florida. Dep. of Biochemistry and Molecular Biology (ICBR Protein Chemistry Core Facility), Gainesville. FL. 

 Not interpretable. 

 Confidence: I ) = possible/low. 



with the antiserum, we purified to homogeneity simi- 

 lar 66 kDa glycoproteins from L. jocu and L. apodus 

 (Fig. 5). The antiserum reacted weakly in immuno- 

 blots with soluble extracts of seven other species of 

 lutjanid and did not react with extracts of four more 

 lutjanid species and a hybrid of O. chrysurus and L. 

 synagris (Table 2). The results indicated that the 

 protein had both interspecies and species-specific 

 determinants. In additional experiments, adsorption 

 of the IgG fraction of the anti-66 kDa antiserum with 

 glutaraldehyde-insolubilized L. apodus extract re- 

 sulted in an antiserum that remained strongly reac- 

 tive with L. griseus extracts but that was weakly 

 reactive withL. apodus, and negative with L.jocu in 

 Western blots (Fig. 4). Although the N-terminal 



amino acid sequence analyses of the first ten resi- 

 dues of L. griseus and L. jocu were approximately 

 the same, only three of the ten residues were the 

 same from L. apodus. These analyses did not take 

 into account the possibility that the interspecies 66 

 kDa proteins may have different carbohydrate moi- 

 eties that influence the antigenicity of the respec- 

 tive epitopes. For example, the 66 kDa protein of L. 

 griseus was slightly heavier compared with the pro- 

 teins of L.jocu and L. apodus in SDS-PAGE (Fig. 5) 

 and thus could account for, but not prove, differences 

 in glycan-dependent epitopes. 



This approach was used as a model for distinguish- 

 ing the different species of lutjanids. The eventual 

 goal of these studies is to produce species-specific 



