Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects最新文献

In this work, bovine chymotrypsinogen B has been quantitatively determined and a first attempt has been made towards the identification of a chymotrypsinogen B in porcine pancreas.

The quantitative technique used for bovine chymotrypsinogen B involves acid denaturation of procarboxypeptidase A, separation of anionic from cationic proteins on CM-cellulose at pH 6.0 and determination of the activity of both fractions against acetyl-l-tyrosine ethylester after tryptic activation. By using the specific activity of the pure precursors, the weight ratio chymotrypsinogen A/chymotrypsinogen B is finally calculated. This ratio is shown to be about 4 in bovine pancreas and pancreatic juice.

The anionic proteins of porcine pancreas and pancreatic juice delay, as in the case of bovine pancreas a strong activity against acetyl-l-tyrosine ethylester. This kind of activity is not given by porcine procarboxypeptidase A which, in contrast with its bovine analog, does not hydrolyze acetyl-l-tyrosine ethylester after tryptic treatment, but my several other anionic proteins which can be partly separated on DEAE-cellulose at pH 8.0. It is not yet known whether or not one of these proteins is actually a chymotrypsinogen B.

During the process of activation of chymotrypsinogen B at least 3 stages of activity levels can be distinguished. The first is the least stable, the third the most stable. With the same concentration of enzyme precursors and activating trypsin (EC 3.4.4.4) the initial rate of formation of chymotrypsin B (EC 3.4.4.6) is about 25 times higher than that of α-chymotrypsin (EC 3.4.4.5). With a constant time and varied concentration of trypsin, chymotrypsinogen B reaches a maximum activity level with one-tenth the amount of trypsin required to reach a maximum activity for α-chymotrypsinogen. The first activating cleavage is the same in both enzyme precursors and involves the bond arginine-isoleucine.

Some physico-chemical properties of the crystalline proteinase (peptide peptidohydrolase) from Pseudomonas aeruginosa IFO 3080 were studied. The [α]_D was −30°. The λ_c was 227.6 mμ and b₀ was −30, derived from the optical rotatory dispersion. The helical content was calculated as 5.2% from the value of b₀. The amino acid composition of the proteinase was as follows: aspartic acid 66, threonine 24, serine 42, glutamic acid 35, proline 11, glycine 65, alanine 58, valine 25, isoleucine 17, leucine 37, tyrosine 21, phenylalanine 20, lysine 16, histidine 6, arginine 7, tryptophan 6. Amino acids containing sulfur, such as cystine, cysteine and methionine, were not detected.

Malate dehydrogenase (decarboxylating) (l-malate: NADP oxidoreductase (decarboxylating), EC 1.1.1.40) has been purified 100-fold from Mycobacterium 607, and its properties for oxidative decarboxylation reaction have been studied. The pH optimum was 7.8. Below pH 5.5 the enzyme was unstable. Reaction with NAD was slow. K₃ for NADP was 4·10⁻⁵ M.

Bivalent cations such as Mn²⁺, Mg²⁺, Co²⁺ or Ni²⁺ were essential for activity. The order and extent of effectiveness of these cations depended on the concentration of malate and Ks⁺. K⁺ activated the reaction, but higher substrate concentrations reduced or inhibitory. This inhibition was reversed completely by GSH and partially by increasing the activity bivalent cation concentration. The involvement of a sulfhydryl group in the enzymic reactions is also suggested by other inhibition studies.

The apparent K_s for malate at pH 7.4 was 1☆10⁻³ M and at 8.2 it was 2.5·10⁻³ M. Higher malate concentrations were inhibitory. Raising the pH lowered, and increasing the Mg²⁺ concentration abolished, this effect. The cause of substrate inhibition is concluded to be its chelatioon of bivalent cation.

Anions also affected the activity. Activity with Cl- was more than that with SO₄²⁻ and this effect was more marled at higher pH.

• 1.

  1. Whole cells of Escherichia coli hydrolyze fructose 1,6-diphosphate to fructose 6-phosphate, and when the cells are incubated in an unbuffered medium the product is not isomerized to glucose 6-phosphate. The reason for this lack of isomerization has been investigated.

• 2.

  Ubder specified conditions phosphoglucose isomerase (d-glucose-6-phosphate ketol isomerase, EC 5.3.1.9) is inhibited, probably because of a low pH in the cells which is unfavourable for the phosphoglucose isomerase reaction, but which permits other enzymes in the glycolytic sequence to act.

• 3.

  3. Aerobically incubated cells of E. coli contain more acids than what is found under anaerobic conditions. This may result in a pH inhibition of the phosphoglucose isomerase which is dependent upon the oxygen pressure.

• 4.

  4. In suspensions of E. coli buffered at pH 5.8, hydrolysis in the 1 position is competing favourably woth glycolytic breakdown of fructose 1,6-diphosphate.

• 5.

  5. The specific radioativity of mannitol 1-phosphate observed in experiments with ³²P-labelled orthophosphate, strongly suggests that E. coli contains an active mannitol kinase.

The twice recrystallized proteinase (peptide peptidohydrolase) of Pseudomonas aeruginosa IFO 3080 contained 1–2 gramatoms of Ca per mole (48 400 g) of enzyme protein and insignificant amounts of the other metal ions. Some chelating agents, such as ethylenediaminetetraacetic acid and o-phenanthroline, inhibited the enzymic activity, but the inactivation at 40° or below was easily reversed either by dialysis, dilution or the addition of various metal ions such as Zn²⁺, Co²⁺, Ca²⁺, etc. The Ca content of the enzyme protein was not decreased by the reversible inactivation, showing that the inactivation was produced by masking the Ca²⁺ of the enzyme with the chelating agent. To dissociate the Ca²⁺ from the enzyme protein, the inactivation treatment by chelating agent was made at 50°, but the trial was unsuccessful, that is, reactivation was no longer observed during the autodigestion of the enzyme protein. Thus the proteinase was regarded as a Ca²⁺-metalloenzyme, in which Ca²⁺ was tightly bound to the enzyme protein.