N-terminal sequence of swine pepsinogen and pepsin. The site of pepsinogen activation

The sequence of 119 amino acids of swine pepsinogen comprising the fragment released during the zymogen activation as well as the N-terminal part of pepsin is established. The activation of swine pepsinogen is shown to be accompanied by specific cleavage of Leu-Ile bond in the sequence:

$\text{Ala}\text{41}\text{Ala Ala Leu Ile}\text{Gly}\text{46}$

where Ile-45 represents the N-terminal residue of pepsin. This sequence is attacked in the course of pepsinogen activation by external enzymes — neutral proteinases and elastase.     

Enzymic dephosphorylation of pepsin and pepsinogen

It has been shown by the work presented in this paper that it is possible to dephosphorylate enzymically pepsin and pepsinogen with a variety of phosphatases. With the aid of a phosphodiesterase and the prostate phosphatase it has been established that the phosphorus in the two proteins is present as a diester and connects two sites of the peptide chain in a cyclic configuration. Removal of the phosphorus does not affect the proteolytic activity against hemoglobin or the synthetic substrate acetyl-L-phenylalanyl diiodotryosine, nor the pepsinogen pepsin transformation. However, an increase of the autodigestion of pepsin is observed.     

The purification and properties of a single chicken pepsinogen fraction and the pepsin derived from it

1. Evidence is given for the presence of at least five pepsinogens in a crude extract of mixed chicken stomachs. One of these was purified and could be activated to yield a single pepsin. 2. The molecular weights of the pepsinogen and pepsin were 36000 and 34000 respectively. The pepsin associated at low pH values and low ionic strength. 3. The amino acid analyses of both proteins are given. The pepsin was devoid of phosphate but contained carbohydrate. 4. The N-terminal amino acids of pepsinogen and pepsin were serine and threonine respectively. Five amino acids were released by carboxypeptidase A and it was deduced that serine may be the C-terminal one. 5. Each protein contained one thiol group per molecule as determined by titration with p-chloromercuribenzoate. The rate of the reaction was very rapid with pepsin, but much slower with pepsinogen, although the same group appeared to react in both instances. The enzymic activity of pepsin was unaffected by the modification. 6. The isoionic point of the pepsin was close to pH4.0 and the enzyme was stable for long periods at pH values up to 7.0. 7. The enzyme hydrolysed bisphenyl sulphite almost as rapidly as did pig pepsin A.     

A new fluorescent microassay method for pepsin using succinyl-albumin.

A method was developed for fluorescent microassay of pepsin with a fluorescent reagent, fluorescamine, and a nonquenching substrate, succinyl-albumin. In this method hydrolysis of succinyl-albumin by pepsin at pH 2,0 was stopped by adding phosphate buffer, pH 6.1, and newly liberated amino groups in the reaction mixture were determined quantitatively by fluorescence after adding fluorescamine. Fluorescence increased linearly with 1.0 to 18 ng of hog pepsin. The assay was 200 times more sensitive than the modified micromethod of Anson [(1939) J. Gen. Phys.22, 79–89].     

Modification of swine pepsin and pepsinogen by p-nitrophenyldiazonium chloride

It was found that at pH 5.2 and 40-fold excess of p-nitrophenyldiazonium chloride the inhibitor incorporation into the porcine pepsin molecule involves 1.9 residues, one residue being bound to tyrosine 189. Besides, tyrosines 44, 113, 154 and 174 enter the reaction. Modified pepsin retains 25% of the native enzyme activity. In the pepsinogen molecule the degree of tyrosine 189 modification diminishes 5 times; of 1.5 inhibitor molecules incorporated into the protein 0.78 residues are bound to tyrosine 113. The potential proteolytic activity of modified pepsinogen towards haemoglobin cleavage makes up to 60% of the original one. It is concluded that the activation peptide in the pepsinogen molecule masks the substrate binding site bearing tyrosine 189, thus preventing its modification with p-nitrophenyldiazonium chloride. The activation peptide in the pepsinogen molecule is presumably located in the vicinity of the wide loop bend carrying tyrosine residue 113, which may be the reason for the decreased pKa value of this residue and of its increased reactivity in the azocoupling reaction.     

A new method for purification of mature elastin.

Stable low-noise dual-beam spectrophotometric detection systems have been built to measure protein in the effluent from chromatographic columns. Measurements are carried out at the magnesium 285.2 nm atomic resonance line isolated either by the technique of selective modulation or by the use of a narrow bandpass interference filter. The noise level is about 0.0005-0.001 absorbance units and the drift rate is ±0.001 absorbance units in 24 hr. While slightly better noise performance could be obtained using the interference filter, selective modulation gives better linearity at higher absorbance values (up to 2.0).     

On the apparent inhibition of intramolecular activation of pepsinogen by pepsin substrates.

Marciniszyn et al. (Marciniszyn, J., Huang, J. S. Hartsuch, J. A., Tang, J. (1976) J. Biol. Chem. 251, 7095-7102) have recently suggested an intermediate in the intramolecular activation of pepsinogen. As evidence, they showed apparent competitive inhibition of activation by globin, indication a pepsinogen-globin complex. Previous work had shown pepsinogen activation to occur very rapidly in the presence of high concentrations of hemoglobin, a very similar pepsin substrate (McPhie, P. (1974) Biochem. Biophys. Res. Commun. 56, 789-792). This contradiction has been resolved by a re-evaluation of the techniques used in the two investigations. The experimental conditions of Marciniszyn et al. Were inadequately defined to ensure denaturation of pepsin, a prerequisite of their method. A small decrease in pH, caused by the presence of extraneous protein, prevents this denaturation and leads to consistent underestimates of the rate of zymogen activation.     

Conversion of pepsinogen to pepsin. Further evidence for intramolecular and pepsin-catalyzed activation.

Exposure of pepsinogen to acid for less than 2 min yields a product with proteolytic activity. This activity is due to intramolecular and intermolecular formation of pepsin from pepsinogen. We find no evidence for intermolecular proteolytic activity in the zymogen. These conclusions are based upon two sets of experiments. First, chemical cleavage of pepsinogen during short activation is demonstrated by quantitative analysis of the NH2-terminal 2 residues of the pepsin and pepsinogen in an activation mixture. In addition, quantitative NH2-terminal analyses after activation under different conditions confirm our previous inference that the product of unimolecular pepsinogen activation is homogeneous whereas bimolecular activation produces a pepsin product with a variety of NH2 termini. Second, spectral changes which occur upon acidification of a pepsinogen solution and are reversed by neutralization are shown to be consistent with the chemical cleavage of pepsinogen during acidification. The first order rate constant for pepsinogen activation, calculated from these spectral experiments, agrees well with the value we had determined previously.     

Molecular characteristics of pepsinogen and pepsin from duck glandular stomach

1. Two procedures were developed for the preparation of duck pepsinogen, an enzyme from the family of aspartic proteases (EC 3.4.23.1) and its zymogen. 2. The amino acid composition, sugar content and the partial N- and C-terminal sequences of both the enzyme and the zymogen were determined. These sequences are highly homologous with the terminal sequences of chicken pepsin(ogen). 3. Duck pepsinogen and pepsin are unlike other pepsin(ogen)s in being relatively stable in alkaline media: pepsinogen is inactivated at pH 12.1, pepsin at pH 9.6. 4. Duck pepsin is inhibited by diazoacetyl-D,L-norleucine methyl ester (DAN), 1,2-epoxy-3(p-nitrophe-noxy)propane (EPNP), pepstatin and a synthetic pepsin inhibitor Val-D-Leu-Pro-Phe-Phe-Val-D- Leu. The pH-optimum of duck pepsin determined in the presence of synthetic substrate is pH 4. 5. Duck pepsin has a marked milk-clotting activity whereas its proteolytic activity is lower than that of chicken pepsin. 6. The activation of duck pepsinogen is paralleled by two conformational changes. The activation half-life determined in the presence of a synthetic substrate at pH 2 and 14 degrees C is 20 sec.     

Pig kidney diamine oxidase. A new method of purification.

Diamine oxidase has been purified from pig kidney by a new method to rapidly obtain larger amounts of pure enzyme with a good yield. The enzyme obtained gives only one band in SDS gel electrophoresis. The specific activity and the absorption spectra were identical to those of already preparations homogeneous reported by different methods of purification.