BACKGROUND
glucokinase  is a metabolic enzyme encoded by the gck gene and expressed in glucose-sensitive tissues, principally pancreatic islets cell and hepatocytes.
pancreatic and hepatic gk enzymes are isoforms that differ slightly in the n-terminus amino acid sequence  <cit> .
the isoforms arise from tissue-specific mrnas that are encoded by a single gck gene under control of separate promoters  <cit> .
in pancreas and liver, the gk protein functions as a “glucose sensor” that couples fluctuations in the blood glucose concentration to changes in cellular function.
biochemical analysis has determined that the metabolic control strength of pancreatic gk for glucose metabolism is near unity, a finding supported by results of gene expression studies that shows that the gck gene exerts a dose dependent effect on glucose metabolism  <cit> .
knowledge about feline gk and its metabolic role is incomplete.
the domestic cat  is an obligate carnivore and must have evolved specialized mechanisms suited for consumption of a diet containing large amounts of protein but relatively little carbohydrate .
several early studies reported reduced or absent activity of the gk enzyme and its regulatory protein  in feline liver when compared with other species  <cit> .
recent studies have shown that the feline liver does not express mrna for gk  <cit>  or gkrp  <cit> , which suggests that reduced expression of genes involved in glucose-sensing is responsible for the low gk enzyme activity observed in early studies.
although gk mrna expression has been documented in the feline pancreas  <cit> , expression and activity of the feline pancreatic gk isoform has not been studied in depth.
given the central role of pancreatic gk as glucose-sensor for insulin-producing islet cells and the requirement for gk in normal glucose metabolism and insulin secretion, detailed knowledge about gk expression and function may improve understanding of feline carbohydrate metabolism.
the objective of the current study was to characterize the feline pancreatic gk isoform and compare the feline isoform with gk from non-carnivorous mammals.
study aims were to determine the nucleotide sequence of pancreatic gck cdna, predict the amino acid sequence and structure of the feline gk protein, and perform bioinformatic analysis of the feline cdna and protein.
RESULTS
feline gk cdna coding sequence and alignments
structural features of the complete feline pancreatic gk cdna are shown in figure  <dig>  alignment of dna clones yielded a 2513 bp cdna sequence with  <dig> putative orfs.
an orf beginning at base  <dig> of the cdna sequence spanned 1398 bp and represented the complete feline gk cds.
nucleotide identities between the feline cds and sequences from human, chimpanzee, rat, and mouse were 92%, 92%, 87%, and 88%, respectively.
using the known exonic structure of human gk mrna , it was determined that feline gk contains  <dig> exons.
race cloning yielded sequence information for putative 5’and 3’ utrs contained in the feline gk mrna.
the 5’-utr contained a 139 bp sequence located immediately upstream to the orf that encodes the 1398 bp cds.
the 976 bp immediately following the cds stop codon comprises the 3’-utr.
additional alignments were carried out using the fgk cdna sequence and the feline genomic sequence .
since the linear cdna contains only exon information the alignment was limited to coding regions of the feline gk gene.
the 5’-utr and exon  <dig> of fgk cdna had 100% identity with a short 184 nt genomic sequence located approximately 32 kb upstream from the start of exon  <dig>  overall, nt identity between fgk exons 2– <dig> and the 3’-utr, excluding intervening intron sequences, was  <dig> %.
three a/g nucleotide differences were detected in the 3’-utr sequences.
a t/c change at position  <dig> in exon  <dig> was the only non-identity in a coding region.
predicted feline pancreatic gk protein sequence, alignments and bioinformatic analysis
the feline pancreatic gk protein deduced from the 1398 bp cds found in the cdna is  <dig> amino acids in length, and has a calculated molecular weight of  <dig>  daltons or approximately  <dig> kd .
amino acid sequence identities with human, chimpanzee, rat and mouse sequences ranged from 89-94% .
structural and mutational analyses of human gk have identified residues 78– <dig>   <dig>   <dig>   <dig>  225– <dig>  295– <dig>  331– <dig>   <dig>  and 410– <dig> as important in atp binding  <cit>  and residues 151– <dig>  166– <dig>  204– <dig>  225– <dig>  254– <dig>   <dig>  and  <dig> as important for glucose binding  <cit> .
the amino acids involved in atp- and glucose-binding in human gk are completely conserved in feline gk .
amino acid residues  previously found to mediate gk binding to its regulatory protein  <cit>  are also completely conserved in feline pancreatic gk .
feline pancreatic gk contains a leucine-rich 11-aa sequence  near the c-terminus of the protein that is likely to act as a nuclear export signal  .
fifteen amino acid residues found in feline gk are not found in corresponding positions in human, mouse, rat or chimpanzee gk .
of these,  <dig> are considered moderately or highly conserved substitutions and unlikely to affect protein structure or function.
the remaining  <dig> residues  are non-conserved substitutions.
three non-conserved residues  are located near the n-terminus and away from residues involved in atp or glucose binding and catalysis.
one of the non-conserved residues  occurs at a site of amino acid variability in other species .
the fifth non-conserved residue  is found near the protein c-terminus, where no crucial residues have been identified.
phylogenic analysis of several mammalian gk proteins shows the feline gk protein is closer to primate gks than to rodent gks.
‡alignments performed using human, chimpanzee, rat, and mouse gks.
conserved substitutions are shown in normal text.
non-conserved substitutions are shown in bold text.
r = rodents .
p = primates .
†this residue is ala in rat, human, and chimpanzee; val in mouse.
the  <dig> aa feline gk protein sequence predicted from pancreatic cdna is longer than the  <dig> aa gk protein  coded by a 1353 bp gk cdna  predicted from analysis of feline genomic dna.
the discrepancy in protein size is due to the presence of  <dig> additional amino acids on the n-terminal of the fgk protein.
direct comparison revealed three amino acid differences between the two feline gk sequences.
the fgk protein differs from the ensembl sequence at aa  <dig>  and  <dig> .
in addition, val  <dig> present in fgk corresponds with a gap in the alignment with the ensembl sequence.
the predicted size and sequence of fgk is more compatible with other mammalian gk protein sequences than the ensembl sequence, all of which are  <dig> aa in length and contain lys and val at the  <dig> and  <dig> positions.
however, tyr  <dig> in fgck is not found in other mammalian sequences, which like the ensembl feline protein have a his residue at this location.
gk structural model
homology modeling of the predicted feline gk protein  demonstrated high structural homology with the crystallographic structure of human glucokinase determined by kamata et al.
<cit> .
secondary structure analysis of feline gk reveals the protein has two globular domains separated by a hinge region .
the cleft formed between the globular regions contains the residues involved in glucose and atp binding.
the predicted feline gk structure would permit the globular domains to open and close during substrate binding and is consistent with a currently proposed mechanistic model of gk function  <cit> .
the non-conserved amino acid variants present in feline gk conveyed no appreciable structural consequences.
three of the non-conserved residues lie within a n-terminus 43-aa stretch which forms part of the surface of the gk molecule that is opposite the surface that forms the substrate binding cleft and catalytic region.
the feline gk sequence at positions  <dig> and  <dig> is trp-arg while other species have arg-arg or ser-arg in these same positions.
as a result of the presence of trp- <dig> and ala- <dig>  feline gk lacks a salt bridge that is present in human gk  <cit> .
furthermore, trp- <dig> is fully solvent exposed in the model, which may make this portion of the molecule more lipophilic.
discussion
the results provide the initial description of the feline pancreatic gk mrna and protein, including the complete nucleotide sequence, amino acid sequence, and molecular structure.
the  <dig>  kb feline cdna contains a  <dig> cds that has a high nucleotide identity with pancreatic gk cdna sequences isolated from other mammals.
the feline cdna encodes a  <dig> aa protein that has a high degree of sequence and structural homology with mammalian pancreatic gk proteins.
the coding region of feline cdna has a high overall nucleotide identity with gk mrnas isolated from pancreases of other mammalian species.
percent  nucleotide identity is highest with pancreatic gk cdnas from primates  and slightly less with rodent pancreatic gk cdnas.
the latter finding is consistent with a trend observed in other feline genes involved in carbohydrate metabolism, such as g6pc   <cit>  and khk   <cit> , which have shown greater nucleotide identity with primate and canine sequences than with rodent species.
the ten putative exons identified in feline gk have homology with established exon sequences of human pancreatic gk.
exon-exon borders in the feline sequence are identical to those in human gk and feline exons are similar in length to corresponding human exons.
based on extensive homology with human exon 1a, which is expressed in pancreatic endocrine cells but not in hepatocytes, exon  <dig> of feline pancreatic gk can be designated as exon 1a.
gk mrnas containing alternative exon  <dig> sequences have been isolated from rodent and human tissues  <cit> .
alternatively spliced gk mrna isoforms are typically detected in liver  <cit> .
islet glucokinase mrna is present as a single transcript produced via activation of the neuroendocrine  promoter.
consistent with findings from other mammals, the feline pancreas was found to produce a single gk cdna with homology to mammalian pancreatic gk and no alternatively spliced pancreatic isoforms were detected.
although gk activity has not been demonstrated in the feline pancreas, pancreatic gk activity is essential for normal glucose homeostasis in humans and rodents and a similar role is almost certainly true in cats.
comparative analysis of feline gk at the protein level showed amino acid identities to be slightly higher than at the nucleotide level but the comparisons followed the same general pattern across the species examined .
feline gk has five non-conservative amino acid substitutions that are unique to the feline protein .
non-conserved amino acid substitutions are of interest because the introduction of unique amino acids into gk during feline evolution has the potential to impact global protein function through any of a number of molecular mechanisms, as observed in human gk mutations  <cit> .
among the amino acid residues unique to feline gk, the most interesting for its possible functional consequences is trp <dig>  unlike the cat, rat, mouse, human, and chimpanzee all have an arg residue in this position.
interestingly, an arg→trp mutation at position  <dig>  in gck was detected in a human patient with gck-mody , a monogenetic type of diabetes  <cit> .
in vitro studies that examined the effects of the r36w mutation on the gk enzyme found that the kinetic characteristics and thermal stability of the r36w mutant human gk did not differ from wild type human gk  <cit> .
however, the r36w mutation is located in a region of the gk protein that may be important for interaction with gk regulators and proteins involved in cellular gk localization  <cit> .
the positively-charged arginine residue at position  <dig>  forms a salt bridge with glu  <dig> in human gk but the trp  <dig> substitution in feline gk abolishes the salt bridge with glu  <dig>  in addition, trp  <dig> is fully solvent exposed in the structural model of feline gk, which would suggest this region is more lipophilic than the same region on human gk.
both of these predicted structural changes could potentially impact feline gk protein dynamics, perhaps by influencing protein-protein interactions or protein stability, as postulated for the r36w mutation of human gk.
when gk is expressed in mammalian liver cells its cellular localization is determined by its interactions with gkrp.
gkrp is not expressed in pancreatic endocrine cells but there is evidence that protein interactions are involved in gk localization in beta cells.
the specific gk residues that mediate binding with gkrp are located in the small domain and hinge region in the folded protein and partly overlap with the glucose-binding domain  <cit> .
the amino acids involved in interaction with gkrp are fully conserved in feline gk but since feline liver does not express gkrp  <cit> , retention of these residues in the feline protein likely reflects the high degree of overall amino acid conservation in the gk protein and the dual role of some of these residues in gkrp and glucose binding.
gk interaction with gkrp is needed for entry into the nucleus  <cit>  but gkrp is not required for gk to exit the nucleus.
nuclear export signal  sequences enriched in leucine residues are a characteristic of proteins, like gk, that can traverse the nuclear membrane.
a leucine-rich nes encompassing positions 300– <dig> has been defined in rat gk and similar sequences are found in human, mouse, and chimpanzee gks, including conservation of all lysine residues.
in feline gk, the putative nes sequence is identical to that found in rat gk  <cit> .
direct determination of cdna sequences for transcripts expressed in specific tissues can be difficult and time consuming but the approach has several advantages over methods that employ gene mining software to identify putative genes and predict cdnas from a genomic dna template.
direct cdna sequencing is a particularly apt method for investigation of the gk gene, which is controlled by multiple promoters and exhibits tissue specific mrna expression.
the feline cdna and protein we report here contain structural and sequence features that differ from archived feline cdna and protein sequences in the ensembl data base.
in particular, the fgk cdna and corresponding gk protein sequences we determined are longer than those determined by primary sequencing of feline genomic dna.
comparison of the protein sequences revealed that the emsembl protein lacks  <dig> aa that are present in the  <dig> aa fgk predicted from pancreatic cdna.
these  <dig> aa comprise the exon  specifically encoded by mrna produced by activation of the gk pancreatic promoter.
no sequence corresponding to the pancreatic exon  <dig> is contained in the emsemble cdna or protein sequences.
in human gk, the pancreatic exon  <dig> is located near the pancreatic  promoter, which is far upstream of the remainder of the coding region  common to pancreatic and hepatic gk isoforms.
a similar situation is likely to occur in the feline gk gene since the genomic location of the 184 nt sequence homologous to the feline gk 5’-utr and pancreatic exon  <dig> is approximately 30 kb upstream of exon  <dig>  inability of the predictive software to recognize a short start sequence so distant from the next exon likely accounts for the size discrepancy between our reported cdna and protein and the ensembl sequences.
there are few gk sequences from individual cats available for comparison.
although overall sequence identity between the fgk cdna and the putative gk gene contained in the feline genome is very high, several nucleotide differences were noted.
the majority of the differences occurred in the utr and do not affect protein sequence but one possible non-synonymous snp was identified.
the t/c snp occurs within exon  <dig> and produces either a tyr  or a his .
the tyr at position  <dig> is unique to feline gk as a his residue occurs in that position in most mammalian gk sequences .
this was considered a non-synonymous  difference because tyr is an uncharged, aromatic aa and his is a charged aa.
modeling the fgk protein with tyr <dig> had no appreciable effects on the protein structure but functional studies are required to fully assess the impact of this possible snp on the gk protein.
CONCLUSIONS
in summary, the pancreatic isoform of feline gk is highly conserved at the mrna and protein levels.
all residues crucial for substrate recognition and binding, protein interactions, and localization are completely conserved in the feline protein.
several amino acid substitutions unique to the feline protein were identified but do not significantly impact gk structure in the protein model.
however, the impact of each substitution on gk structure and function was not directly determined.
based on the molecular analysis performed, it is likely that feline pancreatic gk functions in a manner similar to other mammalian pancreatic gk proteins.

 glucokinase is a metabolic enzyme encoded by the gck gene and expressed in glucose-sensitive tissues, principally pancreatic islets cell and hepatocytes. pancreatic and hepatic gk enzymes are isoforms that differ slightly in the n-terminus amino acid sequence. the isoforms arise from tissue-specific mrnas that are encoded by a single gck gene under control of separate promoters. in pancreas and liver, the gk protein functions as a “glucose sensor” that couples fluctuations in the blood glucose concentration to changes in cellular function. biochemical analysis has determined that the metabolic control strength of pancreatic gk for glucose metabolism is near unity, a finding supported by results of gene expression studies that shows that the gck gene exerts a dose dependent effect on glucose metabolism. knowledge about feline gk and its metabolic role is incomplete. the domestic cat is an obligate carnivore and must have evolved specialized mechanisms suited for consumption of a diet containing large amounts of protein but relatively little carbohydrate. several early studies reported reduced or absent activity of the gk enzyme and its regulatory protein in feline liver when compared with other species. recent studies have shown that the feline liver does not express mrna for gk or gkrp, which suggests that reduced expression of genes involved in glucose-sensing is responsible for the low gk enzyme activity observed in early studies. although gk mrna expression has been documented in the feline pancreas, expression and activity of the feline pancreatic gk isoform has not been studied in depth. given the central role of pancreatic gk as glucose-sensor for insulin-producing islet cells and the requirement for gk in normal glucose metabolism and insulin secretion, detailed knowledge about gk expression and function may improve understanding of feline carbohydrate metabolism. the objective of the current study was to characterize the feline pancreatic gk isoform and compare the feline isoform with gk from non-carnivorous mammals. study aims were to determine the nucleotide sequence of pancreatic gck cdna, predict the amino acid sequence and structure of the feline gk protein, and perform bioinformatic analysis of the feline cdna and protein.  feline gk cdna coding sequence and alignments structural features of the complete feline pancreatic gk cdna are shown in figure  alignment of dna clones yielded a 2513 bp cdna sequence with putative orfs. an orf beginning at base of the cdna sequence spanned 1398 bp and represented the complete feline gk cds. nucleotide identities between the feline cds and sequences from human, chimpanzee, rat, and mouse were 92%, 92%, 87%, and 88%, respectively. using the known exonic structure of human gk mrna, it was determined that feline gk contains exons. race cloning yielded sequence information for putative 5’and 3’ utrs contained in the feline gk mrna. the 5’-utr contained a 139 bp sequence located immediately upstream to the orf that encodes the 1398 bp cds. the 976 bp immediately following the cds stop codon comprises the 3’-utr. additional alignments were carried out using the fgk cdna sequence and the feline genomic sequence. since the linear cdna contains only exon information the alignment was limited to coding regions of the feline gk gene. the 5’-utr and exon of fgk cdna had 100% identity with a short 184 nt genomic sequence located approximately 32 kb upstream from the start of exon overall, nt identity between fgk exons 2– and the 3’-utr, excluding intervening intron sequences, was %. three a/g nucleotide differences were detected in the 3’-utr sequences. a t/c change at position in exon was the only non-identity in a coding region. predicted feline pancreatic gk protein sequence, alignments and bioinformatic analysis the feline pancreatic gk protein deduced from the 1398 bp cds found in the cdna is amino acids in length, and has a calculated molecular weight of daltons or approximately kd. amino acid sequence identities with human, chimpanzee, rat and mouse sequences ranged from 89-94%. structural and mutational analyses of human gk have identified residues 78– 225– 295– 331– and 410– as important in atp binding and residues 151– 166– 204– 225– 254– and as important for glucose binding. the amino acids involved in atp- and glucose-binding in human gk are completely conserved in feline gk. amino acid residues previously found to mediate gk binding to its regulatory protein are also completely conserved in feline pancreatic gk. feline pancreatic gk contains a leucine-rich 11-aa sequence near the c-terminus of the protein that is likely to act as a nuclear export signal. fifteen amino acid residues found in feline gk are not found in corresponding positions in human, mouse, rat or chimpanzee gk. of these, are considered moderately or highly conserved substitutions and unlikely to affect protein structure or function. the remaining residues are non-conserved substitutions. three non-conserved residues are located near the n-terminus and away from residues involved in atp or glucose binding and catalysis. one of the non-conserved residues occurs at a site of amino acid variability in other species. the fifth non-conserved residue is found near the protein c-terminus, where no crucial residues have been identified. phylogenic analysis of several mammalian gk proteins shows the feline gk protein is closer to primate gks than to rodent gks. ‡alignments performed using human, chimpanzee, rat, and mouse gks. conserved substitutions are shown in normal text. non-conserved substitutions are shown in bold text. r = rodents. p = primates. †this residue is ala in rat, human, and chimpanzee; val in mouse. the aa feline gk protein sequence predicted from pancreatic cdna is longer than the aa gk protein coded by a 1353 bp gk cdna predicted from analysis of feline genomic dna. the discrepancy in protein size is due to the presence of additional amino acids on the n-terminal of the fgk protein. direct comparison revealed three amino acid differences between the two feline gk sequences. the fgk protein differs from the ensembl sequence at aa and. in addition, val present in fgk corresponds with a gap in the alignment with the ensembl sequence. the predicted size and sequence of fgk is more compatible with other mammalian gk protein sequences than the ensembl sequence, all of which are aa in length and contain lys and val at the and positions. however, tyr in fgck is not found in other mammalian sequences, which like the ensembl feline protein have a his residue at this location. gk structural model homology modeling of the predicted feline gk protein demonstrated high structural homology with the crystallographic structure of human glucokinase determined by kamata et al.. secondary structure analysis of feline gk reveals the protein has two globular domains separated by a hinge region. the cleft formed between the globular regions contains the residues involved in glucose and atp binding. the predicted feline gk structure would permit the globular domains to open and close during substrate binding and is consistent with a currently proposed mechanistic model of gk function. the non-conserved amino acid variants present in feline gk conveyed no appreciable structural consequences. three of the non-conserved residues lie within a n-terminus 43-aa stretch which forms part of the surface of the gk molecule that is opposite the surface that forms the substrate binding cleft and catalytic region. the feline gk sequence at positions and is trp-arg while other species have arg-arg or ser-arg in these same positions. as a result of the presence of trp- and ala- feline gk lacks a salt bridge that is present in human gk. furthermore, trp- is fully solvent exposed in the model, which may make this portion of the molecule more lipophilic. discussion the results provide the initial description of the feline pancreatic gk mrna and protein, including the complete nucleotide sequence, amino acid sequence, and molecular structure. the  kb feline cdna contains a cds that has a high nucleotide identity with pancreatic gk cdna sequences isolated from other mammals. the feline cdna encodes a aa protein that has a high degree of sequence and structural homology with mammalian pancreatic gk proteins. the coding region of feline cdna has a high overall nucleotide identity with gk mrnas isolated from pancreases of other mammalian species. percent nucleotide identity is highest with pancreatic gk cdnas from primates and slightly less with rodent pancreatic gk cdnas. the latter finding is consistent with a trend observed in other feline genes involved in carbohydrate metabolism, such as g6pc and khk, which have shown greater nucleotide identity with primate and canine sequences than with rodent species. the ten putative exons identified in feline gk have homology with established exon sequences of human pancreatic gk. exon-exon borders in the feline sequence are identical to those in human gk and feline exons are similar in length to corresponding human exons. based on extensive homology with human exon 1a, which is expressed in pancreatic endocrine cells but not in hepatocytes, exon of feline pancreatic gk can be designated as exon 1a. gk mrnas containing alternative exon sequences have been isolated from rodent and human tissues. alternatively spliced gk mrna isoforms are typically detected in liver. islet glucokinase mrna is present as a single transcript produced via activation of the neuroendocrine promoter. consistent with findings from other mammals, the feline pancreas was found to produce a single gk cdna with homology to mammalian pancreatic gk and no alternatively spliced pancreatic isoforms were detected. although gk activity has not been demonstrated in the feline pancreas, pancreatic gk activity is essential for normal glucose homeostasis in humans and rodents and a similar role is almost certainly true in cats. comparative analysis of feline gk at the protein level showed amino acid identities to be slightly higher than at the nucleotide level but the comparisons followed the same general pattern across the species examined. feline gk has five non-conservative amino acid substitutions that are unique to the feline protein. non-conserved amino acid substitutions are of interest because the introduction of unique amino acids into gk during feline evolution has the potential to impact global protein function through any of a number of molecular mechanisms, as observed in human gk mutations. among the amino acid residues unique to feline gk, the most interesting for its possible functional consequences is trp unlike the cat, rat, mouse, human, and chimpanzee all have an arg residue in this position. interestingly, an arg→trp mutation at position in gck was detected in a human patient with gck-mody, a monogenetic type of diabetes. in vitro studies that examined the effects of the r36w mutation on the gk enzyme found that the kinetic characteristics and thermal stability of the r36w mutant human gk did not differ from wild type human gk. however, the r36w mutation is located in a region of the gk protein that may be important for interaction with gk regulators and proteins involved in cellular gk localization. the positively-charged arginine residue at position forms a salt bridge with glu in human gk but the trp substitution in feline gk abolishes the salt bridge with glu in addition, trp is fully solvent exposed in the structural model of feline gk, which would suggest this region is more lipophilic than the same region on human gk. both of these predicted structural changes could potentially impact feline gk protein dynamics, perhaps by influencing protein-protein interactions or protein stability, as postulated for the r36w mutation of human gk. when gk is expressed in mammalian liver cells its cellular localization is determined by its interactions with gkrp. gkrp is not expressed in pancreatic endocrine cells but there is evidence that protein interactions are involved in gk localization in beta cells. the specific gk residues that mediate binding with gkrp are located in the small domain and hinge region in the folded protein and partly overlap with the glucose-binding domain. the amino acids involved in interaction with gkrp are fully conserved in feline gk but since feline liver does not express gkrp, retention of these residues in the feline protein likely reflects the high degree of overall amino acid conservation in the gk protein and the dual role of some of these residues in gkrp and glucose binding. gk interaction with gkrp is needed for entry into the nucleus but gkrp is not required for gk to exit the nucleus. nuclear export signal sequences enriched in leucine residues are a characteristic of proteins, like gk, that can traverse the nuclear membrane. a leucine-rich nes encompassing positions 300– has been defined in rat gk and similar sequences are found in human, mouse, and chimpanzee gks, including conservation of all lysine residues. in feline gk, the putative nes sequence is identical to that found in rat gk. direct determination of cdna sequences for transcripts expressed in specific tissues can be difficult and time consuming but the approach has several advantages over methods that employ gene mining software to identify putative genes and predict cdnas from a genomic dna template. direct cdna sequencing is a particularly apt method for investigation of the gk gene, which is controlled by multiple promoters and exhibits tissue specific mrna expression. the feline cdna and protein we report here contain structural and sequence features that differ from archived feline cdna and protein sequences in the ensembl data base. in particular, the fgk cdna and corresponding gk protein sequences we determined are longer than those determined by primary sequencing of feline genomic dna. comparison of the protein sequences revealed that the emsembl protein lacks aa that are present in the aa fgk predicted from pancreatic cdna. these aa comprise the exon specifically encoded by mrna produced by activation of the gk pancreatic promoter. no sequence corresponding to the pancreatic exon is contained in the emsemble cdna or protein sequences. in human gk, the pancreatic exon is located near the pancreatic promoter, which is far upstream of the remainder of the coding region common to pancreatic and hepatic gk isoforms. a similar situation is likely to occur in the feline gk gene since the genomic location of the 184 nt sequence homologous to the feline gk 5’-utr and pancreatic exon is approximately 30 kb upstream of exon inability of the predictive software to recognize a short start sequence so distant from the next exon likely accounts for the size discrepancy between our reported cdna and protein and the ensembl sequences. there are few gk sequences from individual cats available for comparison. although overall sequence identity between the fgk cdna and the putative gk gene contained in the feline genome is very high, several nucleotide differences were noted. the majority of the differences occurred in the utr and do not affect protein sequence but one possible non-synonymous snp was identified. the t/c snp occurs within exon and produces either a tyr or a his. the tyr at position is unique to feline gk as a his residue occurs in that position in most mammalian gk sequences. this was considered a non-synonymous difference because tyr is an uncharged, aromatic aa and his is a charged aa. modeling the fgk protein with tyr had no appreciable effects on the protein structure but functional studies are required to fully assess the impact of this possible snp on the gk protein.  in summary, the pancreatic isoform of feline gk is highly conserved at the mrna and protein levels. all residues crucial for substrate recognition and binding, protein interactions, and localization are completely conserved in the feline protein. several amino acid substitutions unique to the feline protein were identified but do not significantly impact gk structure in the protein model. however, the impact of each substitution on gk structure and function was not directly determined. based on the molecular analysis performed, it is likely that feline pancreatic gk functions in a manner similar to other mammalian pancreatic gk proteins. 
the gk protein acts in pancreatic islets as a “glucose sensor” that couples fluctuations in the blood glucose concentration to changes in cellular function and insulin secretion. glucokinase is a metabolic enzyme encoded by the gck gene and expressed in glucose-sensitive tissues, principally pancreatic islets cell and hepatocytes. the study was undertaken to determine the nucleotide sequence of feline pancreatic gk cdna, predict the amino acid sequence and structure of the feline gk protein, and perform comparative bioinformatic analysis of feline cdna and protein. molecular analysis predicts that feline pancreatic gk functions similarly to other mammalian gk proteins. feline pancreatic gk is highly conserved at nucleotide and protein levels. the deduced amino acid feline protein has amino acid substitutions not found in other mammalian gk proteins but maintains high structural homology with human gk. residues crucial for substrate binding and catalysis are completely conserved in the feline protein. full-length feline pancreatic gk cdna contains a nucleotide coding sequence with high identity to other pancreatic gk cdnas. routine pcr techniques were used with cdna from feline pancreas.
 glucokinase is a metabolic enzyme encoded by the gck gene and expressed in glucose-sensitive tissues, principally pancreatic islets cell and hepatocytes. the gk protein acts in pancreatic islets as a “glucose sensor” that couples fluctuations in the blood glucose concentration to changes in cellular function and insulin secretion. gck and gk have proposed importance in the development and progression of diabetes mellitus and are potential therapeutic targets for diabetes treatment. the study was undertaken to determine the nucleotide sequence of feline pancreatic gk cdna, predict the amino acid sequence and structure of the feline gk protein, and perform comparative bioinformatic analysis of feline cdna and protein. routine pcr techniques were used with cdna from feline pancreas. clones were assembled to obtain the full length cdna. protein prediction and modeling were performed using bioinformatic tools.  full-length feline pancreatic gk cdna contains a nucleotide coding sequence with high identity to other pancreatic gk cdnas. the deduced amino acid feline protein has amino acid substitutions not found in other mammalian gk proteins but maintains high structural homology with human gk. feline pancreatic gk is highly conserved at nucleotide and protein levels. residues crucial for substrate binding and catalysis are completely conserved in the feline protein.  molecular analysis predicts that feline pancreatic gk functions similarly to other mammalian gk proteins. glucose sensorpancreascarbohydrate metabolismhexokinasegene expression 