dpp - iii ( ec 3.4.14.4 ) of the goat brain is a dipeptidylaminopeptidase which removes n - terminal dipeptide from arg - arg-4mna at ph 8.5 .
the enzyme was recently purified and characterized   and found to be affected by thiol compounds and metal ions .
it hydrolysed leu - enkephalin and other bioactive peptides and displayed micromolar affinity for enkephalins thereby suggesting its involvement in regulating enkephalin disposition [ 2 , 4 ] .
all the studied analgesic and antihypertensive drugs inhibited the enzyme . in the present study , it has been demonstrated that the enzyme activity is retained in davis gel electrophoresis at ph 8.3 .
understanding of catalytic mechanisms , structural features of protease , and their inhibitors are very important to explore their applications in medicinal field .
ethylene diamine tetraacetic acid ( edta ) , tris - hcl , dialysis bags , polyacrylamide , n , n-methylene bisacrylamide , ammonium persulphate , and temed were procured from himedia .  
louis , mo , usa . the routine pipetting was done with micropipettes from tarson and very small volumes were pipetted out using hamilton syringes . the digital spectrophotometer from systronics was used to record the absorbance at 520  nm .
dpp was purified and assayed with arg - arg-4mna as substrate at ph 8.5 ( tris - hcl , 50  mm , containing 100  mm nacl and 1  mm -mercaptoethanol ( -me ) .
one unit of enzyme activity was defined as the amount of enzyme that liberated 1  nanomole of 4  mna from the substrate per minute under assay conditions .
the enzyme was incubated with tris - hcl buffer ( 50  mm , ph 8.5 containing 1  mm -me ) with different concentrations of nacl ( 0 to 1000  mm ) at 37c for 10  min .
, the 10% davis gel was prepared . before loading the protein samples , the polymerized gel was pre - electrophoresed for 2  h at a current of 4  ma by using resolving gel buffer .
purified protein ( 25  g ) was loaded on gel and run at a constant current of 4  ma at 4c .
one half was stained   with   coomassie brilliant dye and other half was stained for enzyme activity by thoroughly washing the gel with assay buffer and   incubating the gel with substrate at 37c   and   then   putting   the gel   in   fast   garnet   gbc   ( 1  mg / ml )   for 15  min   for colour development .  
purified dpp - iii was treated with 2.5  mm edta for 10  min and then extensively dialysed against 50  mm tris - hcl buffer , ph 7.0 .
the edta pretreated enzyme was preincubated with appropriate salts in assay buffer at 37c for 10  min .
the reaction was started by adding 150  m of substrate and activity is expressed as percentage of control .
likewise effect of dtnb was studied on edta pretreated enzyme in presence of zncl2 and cocl2 .
the purified enzyme retained its activity during electrophoresis at ph 8.3 , in davis gel as shown in figure 1 . the activity band obtained with arg - arg-4mna as specific substrate corresponded well with the protein band stained with coomassie brilliant blue .
the enzyme was activated by cl ions and maximum activation was achieved at 100  mm final concentration , which accounted for more than 1.5-fold increase in enzyme activity ( figure 2 ) .
this property is similar to that of dpp - iii enzyme of anterior pituitary . beyond 100  mm , enzyme activity decreased with increase in nacl concentration .
incubation of pure dpp - iii with 2.5  mm edta resulted in 60% inhibition of enzyme activity .
the suppressed activity could be partially restored by adding metal ions like zncl2 , cocl2 , nicl2 , feso4 , and mgcl2 but none of the metal ions could completely restore the enzyme activity .
zn at 50  m almost completely restored enzyme activity ( ~97% ) ( figure 3 ) .
these results support our earlier studies with o - phenanthroline thereby confirming this enzyme to be a metalloprotease having zn at the active site .
our results are in agreement with human placental dpp - iii , whereas co was most effective in restoring the activity of edta pretreated dpp - iii from human rbcs .
ca and co were most effective in restoring the activity of edta pretreated guinea pig brain dpp - iii . on the other hand ,
untreated dpp - iii was activated by co and inhibited by zn , ni , and cu .
relationship between binding of metal ions and cysteine residues on dpp - iii was investigated by inactivation of edta - treated enzyme with dtnb in the presence and absence of co and zn .
however zn was more protective than co. it appears that binding of metal ions influence reactivity and accessibility of thiol group(s ) essential for enzyme activity .  
therefore it can be concluded that enzyme is a metalloprotease with the involvement of cysteine residues either located on the catalytic site or involved in regulation .
several other metalloenzymes having sh group at the active site have also been reported earlier [ 12 , 13 ] .
 dipeptidylpeptidase - iii 
                   ( dpp - iii ) from goat brain was purified and 
                   characterized using 
                   arginyl - arginyl-4-methoxy--naphthylamide 
                   ( arg - arg-4mna ) substrate . 
 this enzyme retained 
                   its activity in native 10% polyacrylamide 
                   gel when stained using arg - arg-4mna . 
                   
 the activity was significantly increased by 
                   100  mm chloride . 
 studies for its 
                   inhibition with some peptides and chemical 
                   inhibitors revealed that leu - trp - met - arg - phe - ala 
                   was most potent inhibitor followed by 
                   arg - phe - ala and gly - phe - leu . all the studied 
                   chemical inhibitors caused 4050% 
                   inhibition at 1  mm . 
 metal ions helped to 
                   regain activity of edta pretreated enzyme . 
                   
 zncl2 at 50  m almost completely restored the enzyme activity . 
 further zncl2 and cocl2 exerted protective effects on edta pretreated enzyme for its susceptibility to dtnb inhibition . 
 therefore , dpp - iii is a metalloprotease with the involvement of cysteine residues either located at the catalytic site or involved in regulation . 
1. Introduction
2. Materials
3. Methods
4. Results and Discussion