Trypsin hydrolysis

At this point you should be able to deduce the sequence of five amino acids at the C-terminus of eledoisin.) (4) Trypsin hydrolysis gives two peptides, P and Q, with the indicated compositions ... 

At this point, you can deduce two possible sequences for Q.) (5) Trypsin hydrolysis of L gives a peptide of composition Ala, Asp, Phe which, with 2,4-dinitrofluorobenzene, gives the 2,4-dinitrophenyl derivative of aspartic acid. (6) Partial acid hydrolysis of eledoisin gives several dipeptides, among them Ser-Lys and Pro-Ser. 

Trypsin hydrolysis of SR vesicles cleaves the (Ca2, Mg2+)-ATPase into three fragments, NH2-20 000, 30 000 and 45 OOO-COOH. The phosphorylation site is associated with an aspartic acid residue in the 30 000 fragment and ionophoric activity with the 20 000 fragment. The amino terminus of the enzyme is located on the cytoplasmic side of the membrane.143... 

All five subunits of the E. coli F, ATPase have been purified, and the primary sequence of the a-subunit has been determined. ATPase activity could be reconstituted from the a-, p- and y-subunits. Subunits j3 and y from R. rubrum have been purified and reincorporated into the depleted membrane with full activity.302 The a- and /3-subunits contain sites for nucleotides.303 The sensitivity of the a-subunit of E. coli to trypsin hydrolysis differs when the high affinity site for ATP is saturated, suggesting that binding of ATP produces conformational changes.304... 

Latent forms of a neutral protease and an acid protease (pH optimum 5.3) from cartilage are activated by trypsin hydrolysis to give Ca2+-dependent enzymes that catalyze the hydrolysis of proteoglycan.406 Some Ca2+-dependent proteinases are isolated as proenzymes that can be converted to the active form by high [Ca2+] or by low [Ca2+] in the presence of a digestable substrate.407... 

Trypsin Hydrolysis of proteins Bovine pancreas Wound and ulcer cleansing... 

Trypsin Hydrolysis of polypeptides, amides, and esters at bonds involving the carboxyl groups of L-arginine and l-lysine, yielding peptides of lower molecular weight. 

Proteomics unknown proteins by enzymatic (usually trypsin) hydrolysis. See Brock,... 

Latent forms of a neutral protease and an acid protease (pH optimum 5.3) from cartilage are activated by trypsin hydrolysis to give Ca -dependent enzymes that catalyze the hydrolysis of... 

Combination of two immobilized enzyme columns with HPLC/thermospray MS can be useful for amino acid sequencing and identification. The use of an endopeptidase bioreactor followed by HPLC separation then an exopeptidase column and MS detection can enable sequencing of 3-5 amino acids of each endopeptidase hydrolysis product. The trypsin, hydrolysis/HPLC/ carboxypeptidase A, B, and Y (1 1 1) hydrolysis/ thermospray MS analysis assist in the sequencing of Y-endorphin (Figure 2C,C ). 

Figure 2. A) Trypsin hydrolysis/HPLC/thermospray MS analysis of Y-endorphin. The two tryptic fragments (T1 and T ) were separated by HPLC (10% isopropanol in aqueous ammonium acetate (0.1 M) pH 7.1, 1 mL/min) and detected by thermospray MS. B,B ) Thermospray mass spectra of tryptic fragments and Tg separated by HPLC after hydrolysis. C,Cj) Thermospray spectra from trypsin hydrolysis followed by HPLC then carboxypeptidase A, B, and Y hydrolysis for the partial amino acid sequencing of Y-endorphin.

Figure 3. HPLC/trypsin hydrolysis/thermospray MS analysis of a mixture of two endorphins in a plasma extract. The separation was performed using 25% isopropanol in aqueous ammonium acetate (0.1 H) solution at a pH of 7.5 at a flow of 1.0 mL/min.

The answer is d. (Murray, pp 48-62. Scriver, pp 3 5. Sack, pp 1-3. Wilson, pp 101-120.) Pepsin is secreted in a proenzyme form in the stomach. Unlike the majority of proenzymes, it is not activated by protease hydrolysis. Instead, spontaneous acid hydrolysis at pH 2 or lower converts pepsinogen to pepsin. Hydrochloric acid secreted by the stomach lining creates the acid environment. All the enzymes secreted by the pancreas are activated at the same time upon entrance into the duodenum. This is accomplished by trypsin hydrolysis of the inactive proenzymes trypsino-gen, chymotrypsinogen, procarboxypeptidase, and proelastase. Primer... 

The TCA-alcohol-treated albumin, after removal of acid and solvent by dialysis, shows no change from the untreated material in electrophoretic mobility, sedimentation, solubility, crystallizability (S24), precipitin curves (Kl), or rate of degradation in the rabbit (F9). In the case of bovine albumin, a slight increase in susceptibility to proteolysis by trypsin has been reported (R3). This effect may have been due to removal of traces of calcium, which normally exerts a stabilizing action on albumin toward trypsin hydrolysis (G14). 

As in Problem 20. look lirst lor a piece that ends with an amino acid that. should not be a site ol cleavage by one of the enzymes. All the chymotrypsin fragments end in Phe. Trp. or Tyr. so that s no help. The trypsin results are more u.seful. It only cleaves after Arg or Lys. so the I8-amino acid fragment ending in Phe must be al the end of the intact hormone. Now it s a matter of matching up all the pieces.. Start with this end piece from trypsin hydrolysis and overlap it with chymotrypsin fragments. 

---