John W. Mehl

Purification of aminopeptidase a in human serum and degradation of angiotensin II by the purified enzyme

Abstract 1. 1. Aminopeptidase A, which is activated by Ca 2+ and specifically hydrolyzes N-terminal dicarboxylic amino acids, was purified from human serum approx. 700-fold. α- l -Glutamyl β-naphthylamide was used as substrate throughout the purification. 2. 2. The purified enzyme hydrolyzed α- l -glutamyl β-naphthylamide, α- l -aspartyl β-naphthylamide, and aspartylalanine, which indicated that this enzyme preferentially hydrolyzes N-terminal dicarboxylic amino acids. Leucyl β-naphthylamidase activity was decreased during the purification, but complete removal of leucyl β-naphthylamidase activity from glutamyl β-naphthylamidase activity was not possible. 3. 3. Purified aminopeptidase A removed the N-terminal aspartic acid residue from natural angiotensin II, i.e. α- l -Asp 1 -Val 5 -angiotensin II. Thus aminopeptidase A was proved to be an “angiotensinase”. Leucyl β-naphthylamidase hydrolyzed mainly synthetic asparaginyl angiotensin II.

Mucoproteins of Human Plasma. IV. Electrophoretic Demonstration of Mucoproteins in Serum at pH 4.5.

Summary When serum is subjected to electrophoresis at pH 4.5, two components are seen which have isoelectric points more acid than those of albumin. One of these has been identified with a mucoprotein which has been shown to increase in amount in cancer, pneumonia, and certain other diseases. The second component, with a less acid isoelectric point, may also be a mucoprotein, but this point has not been definitely established.

Studies of the distribution of vitamin A as ester and alcohol and of carotenoids in plasma proteins of several species

Abstract The distribution of vitamin A ester, alcohol, and lutein was studied in fractions of chicken plasma proteins obtained by ammonium sulfate fractionation or dialysis, 6–8 hr. after oral administration of vitamin A and lutein in an oily medium. The losses of vitamin A and carotenoids during fractionation were considerable in some cases, losses of vitamin A being as large as 50% at times. Although this made the interpretation of the distribution between precipitate and supernatant difficult, the following conclusions seem to be possible. Vitamin A ester was found to be associated with the least soluble, and vitamin A alcohol and lutein with the more soluble protein fractions. No mono- or diesters of lutein could be demonstrated in the plasma. In beef plasma, β-carotene and lutein and vitamin A alcohol were found in the more soluble protein fractions. In pig plasma, vitamin A alcohol was likewise associated with the more soluble protein. The hypothesis is put forward that specific plasma or lymph proteins may be responsible for the specificity of absorption of carotenoids and vitamin A.

Mucoproteins of Human Plasma. III. Electrophoretic Studies of Mucoproteins from Perchloric Acid Filtrates of Plasma.

Summary The electrophoretic behavior of mucoproteins isolated from perchloric acid filtrates of human plasma has been studied over the pH range of 2 to 8.4. There are at least 3 electrophoretic components in such preparations, the isoelectric points being approximately 2.3, 3.4 and 4.3. The major component has the lowest isoelectric point and travels with a mobility characteristic of α1-globulin at pH 8.4. The increase in the mucoprotein level isolated from the plasma of a patient with gastric cancer was largely in this acid mucoprotein fraction.

Separation of plasma thromboplastin antecedent (PTA) and Hageman factor (HF) from human plasma.

Summary 1) Hageman factor and plasma thromboplastin antecedent have been separated from human plasma and each other by ion exchange chromatography. 2) The defect in “exhausted plasma” is not corrected by purified PTA, purified HF, or a combination of the two.

Human growth hormone and α2-macroglobulin: A study of binding☆

Abstract The availability of a pure human α 2 -macroglobulin ( α 2 -M) preparation has prompted a re-examination of its reported binding with human growth hormone (HGH). Incubation of labeled HGH preparations with α 2 -M did not show significant binding as measured by paper chromatoelectrophoresis, starch gel electrophoresis, or gel filtration. Although a small proportion of HGH traveled with α 2 -M, an analysis of the results suggests that this is not due to binding by the major component of α 2 -M. These studies indicate that the major component of α 2 -M cannot serve as a carrier protein for HGH in human plasma. An expression for the behavior of a dissociating system in gel filtration, applicable to the particular conditions employed in this study, is developed and discussed.

Lack of identity of hyaluronidase inhibitor and certain mucoproteins in blood serum.

Summary and Conclusions 1. Electrophoretically and chemically separated fractions of human serum containing high concentrations of mucoproteins showed no increase in non-specific hyaluronidase inhibitor when compared to native serum or other mucoprotein-poor fractions of serum. 2. In children suffering from “lipoid nephrosis” the hyaluronidase inhibitor levels of the serum were higher while the muco-protein levels were significantly lower than normal. 3. In spite of the striking statistical correlation existing between the serum levels of mucoprotein and non-specific hyaluronidase inhibitor in a wide variety of human diseases, marked disparity in the serum levels of these substances occasionally occurs. 4. On the basis of these findings the lack of identity of the non-specific hyaluronidase inhibitor and these serum mucoproteins is pointed out.

Separation of Trypsin Inhibitors of Human Plasma on DEAE-Cellulose.

Summary Chromatography of human plasma on DEAE-cellulose permits the separation of 3 fractions which inhibit trypsin. The first of these inhibits trypsin only partially, and is an α2-macroglobulin. The second fraction represents most of the inhibitor in plasma, and must be largely that previously identified with the α1-globulin fraction. The third fraction is very stable to heating at low pH, and is largely made up of material with a mobility very close to that of albumin. A fourth component can be shown to differ from these with respect to its behavior in gel filtration.

Partial purification of bovine intestinal alkaline phosphatase.

Summary A procedure has been presented for obtaining a soluble preparation of intestinal alkaline phosphatase from the microsomes of steer intestine mucosa by extraction of the microsomes with n-butanol. Although the purification achieved is only 15 to 25 times that of the original homogenate of the tissue, the resulting preparation appears to be particularly suitable for further purification by fractional precipitation with acetone.

Zone electrophoresis of intestinal alkaline phosphatase.

Summary A crude preparation of alkaline phosphatase from bovine intestinal mucosa, obtained by treatment with butanol to release enzyme from the microsomes, was subjected to sequential electrophoresis on starch blocks. The results indicate that the phosphatase may have been present initially in a complexed form and that the complex was disrupted during repeated electrophoretic separation. Such behavior, together with the rather small amounts of enzyme protein which appear to be present in the starting material, may account for some of the difficulties encountered in attempts to purify intestinal alkaline phosphatase. An approximately 260-fold purification was achieved, but the amounts of final product obtained were too small for further study.