Warren H. Evans

Guinea pig heterophil and eosinophil peroxidase

Abstract Guinea pig heterophil and eosinophil peroxidases were purified from bone marrow leukocytes in order to compare certain physical properties. Purified heterophil peroxidase (homogeneous in the ultracentrifuge, by immunologic test, and by gel electrophoresis) had a Soret maximum at 437 nm, an A 437 A 280 ratio of 0.79, and a mol wt of 137,000. The molecule consisted of two subunits of similar size, as assessed by its appearance in the electron microscope and by electrophoresis in SDS polyacrylamide gels. Eosinophil peroxidase required the cationic detergent cetyltrimethylammonium bromide to prevent binding to the materials used in its purification. The purified esoinophil enzyme, homogeneous by immunologic criteria, was resolved by gel filtration into two peroxidase components having mol wts of 75,000 and 150,000, respectively. Each component had the same specific activity, absorption spectrum (Soret maximum at 425 nm), and A 415 A 280 ratio (0.83). Treatment of each with SDS and mercaptoethanol produced a single component with a mol wt of about 65,000. Examination of crude bone marrow extracts resolved only one eosinophil peroxidase component with a mol wt of about 80,000, indicating that aggregation occurred during purification. The leukocyte peroxidases, therefore, have substantially different properties despite their location in analogous organelles of closely related cells.

Factors influencing myeloperoxidase and catalase activities in polymorphonuclear leukocytes.

Abstract 1. 1. Various factors affecting myeloperoxidase (EC 1.11.1.7) and catalase (EC 1.11.1.6) activities in polymorphonuclear leukocytes have been investigated. 2. 2. Under conditions in which the myeloperoxidase-containing granules of leukocytes remain intact, such as in intact cells or in 0.25 M sucrose homogenates, myeloperoxidase is not inhibited by 3-amino-1,2,4-triazole or by endogenously generated H 2 O 2 . Catalase is inhibited by aminotriazole but not by H 2 O 2 under such conditions. When the granules are disrupted, such as in intact phagocytizing cells, in 0.25 M sucrose homogenates that have been frozen and thawed or in sucrose-free homogenates, both enzymes are inhibited by aminotriazole. Exogenous H 2 O 2 , at concentrations employed by us, inhibits myeloperoxidase but not catalase in frozen-thawed sucrose homogenates and in sucrose-free homogenates. Aminotriazole also inhibits purified myeloperoxidase. 3. 3. Myeloperoxidase activity was 18 times greater in lysed versus intact granule preparations. 4. 4. It is postulated that myeloperoxidase, like lysosomal enzymes, is probably inactive in resting leukocytes but might function in phagocytizing cells following lysis of the myeloperoxidase containing granules. A possible role of catalase in the regulation of myeloperoxidase activity in phagocytizing leukocytes is also suggested.

Purification of alkaline phosphatase from guinea pig bone marrow

Abstract A new method of purifying alkaline phosphatase (AP) from specific granules of guinea pig bone marrow granulocytes is described. The method devised resulted in a 500-fold purification of AP with a specific activity of 81,000 μmoles of p -nitrophenyl phosphate hydrolyzed min −1 mg −1 and a 39% yield. The purified enzyme was free of protein contaminants as judged by immunoprecipitation and gel electrophoresis. Its molecular size was estimated by gel filtration to be 4 × 10 5 . The purified enzyme behaved like a lipoprotein on ion-exchange chromatography. Lipid extracted from purified enzyme contained 2–4 μg P/mg of protein. Like other alkaline phosphatases, purified AP appeared to be a zinc metalloenzyme, that is, it was inhibited by various chelating agents, including o -phenanthroline, and contained a stoichiometrically significant amount of zinc, 3–5 g-atoms of zinc/400,000 g of protein. Magnesium activated the purified enzyme 4- to 7-fold.

Enrichment of antibody plaque-forming cells of spleen by sedimentation at unit gravity.

Summary Spleen cells from mice immunized with sheep erythrocytes were sedimented at unit gravity in a sucrose gradient. Cells forming hemolytic antibody plaques sedimented more rapidly than the bulk of the nonplaque-forming cells, resulting in a partial separation with enrichments of up to 18-fold over their starting concentration.

Evidence for a factor in normal human serum that induces human neutrophilic granulocyte end-stage maturation in vitro

The end-stage maturation of neutrophilic granulocyte precursor cells isolated from normal human bone marrow by Ficoll density centrifugation was studied in a liquid culture assay system used previously to study the maturation of guinea pig granulocyte precursors. Dialyzed normal human serum induced end-stage morphological maturation of human granulocyte precursors and this induction was proportional to a serum level of up to 5.0% in the assay medium. At serum concentrations greater than 5.0% a pronounced inhibition of maturation was observed. Passage of serum through a DEAE-Fractogel 650S column equilibrated with 0.01 M phosphate buffer (pH 7.0) resulted in the binding of the end-stage granulocyte maturation factor to the column. The activity eluted from the column in a fraction containing 17% of the starting serum protein that was inhibitor-free and was also capable of inducing the appearance of granulocyte alkaline phosphatase, a specific biochemical marker for granulocyte end-stage maturation. GMF is most likely a protein since it was destroyed by protease digestion. The data also indicate that neither purified human transferrin nor human recombinant granulocyte colony-stimulating factor can substitute for human serum GMF as a granulocyte end-stage maturation factor in this assay system. It was observed, however, that purified human transferrin greatly potentiated the effect of GMF suggesting that transferrin plays a supporting role in the end-stage maturation of human granulocytes in vitro. To our knowledge the evidence presented here indicates for the first time the existence of a neutrophilic granulocyte end-stage maturation factor in normal human serum.

Transferrin induces maturation of neutrophil granulocyte precursors in vitro

Previous studies showed that dialyzed serum from guinea pigs contained a factor which induces guinea pig granulocyte precursors to form mature neutrophil granulocytes in vitro. We show here that this maturation inducing activity of serum is associated primarily with transferrin. This activity is not species specific since both guinea pig and human transferrin serve equally well in this capacity. These findings raise the possibility that transferrin could serve as a physiological regulator of granulocyte maturation.

Differences in protein and glycoprotein patterns between mature and immature neutrophil leukocytes.

The post-nuclear particulate fractions of highly enriched mature and immature neutrophil preparations were dissolved in buffer containing sodium dodecyl sulfate and analyzed for protein and glycoprotein components by polyacrylamide gel electrophoresis. The electrophoretic patterns of mature and immature neutrophils showed pronounced differences in both protein and glycoprotein bands. There were 12 prominent protein bands in the patterns of mature neutrophils which also stained for glycoproteins. With one exception, these bands were either greatly reduced in intensity or undetectable at corresponding positions in the patterns of immature cells. The patterns of immature neutrophils revealed three prominent glycoprotein bands, two of which were absent and the third was greatly reduced in intensity at corresponding position in the mature cell patterns. The results indicate that a number of new glycoproteins appear in the later stages of neutrophil maturation, whereas other glycoproteins, present in immature cells, are either lost or greatly reduced in amount. These changes are presumably related to the development of specific cell functions that also appear in later stages of cell maturation.

Concentration of immature and mature granulocytes from normal human bone marrow.

Summary Highly enriched fractions of mature and immature granulocytes were isolated from human bone marrow cell suspensions by Ficoll density centrifugation. Fractions of immature cells contained myelocytes (45%), promyelocytes plus blasts (31%), and only 8% mature neutrophils. Fractions of mature cells contained 85% cells at the metamyelocyte to polymorphonuclear stages of maturation and only 9% immature neutrophils. The cells were well preserved morphologically and are useful for biochemical studies of cell maturation.

Blast crisis associated with granulocytic leukemia in strain 13 guinea pigs

A transplantable granulocytic leukemia (GL-13) in inbred strain 13 guinea pigs had been shown previously to have many characteristics in common with human CML except that a blast crisis did not occur in the terminal stage of the disease. We report here that, after the 25th transplant generation, leukemic guinea pigs began to develop a blast crisis similar to that seen in human CML. This feature of the disease is still present after 90 transplant generations. The transplantation conditions necessary to produce the leukemia (now referred to as GL-13-BC) with a predictable clinical and hematological course over a short interval are described. Thus, s.c. injection of 3.0 X 10(6) leukemic cells results in a slowly rising leukocyte count, due mainly to increasing numbers of myelocytes and mature granulocytes, at about 3 weeks after injection. At this early stage the spleen weights of the leukemic animals are about seven times greater than those of normal controls. Approximately 4-7 days later the leukocyte count rises sharply due to a blast crisis and the animals die shortly thereafter with a mean survival time of 30 (range 25-32) days. It is proposed that the GL-13-BC leukemia could serve as a useful model for investigating the problem of blast crisis associated with human CML.

Isolation and biochemical characterization of nuclei from immature and mature guinea pig granulocytes.

Abstract Intact nuclei were isolated in high yield from enriched fractions of immature and mature guinea pig granulocytic leukocytes. These nuclei were used to determine whether any changes in synthesis and content of nuclear proteins accompany the striking increase in chromatin condensation and the nuclear lobation which occur during granulocyte maturation. The results indicate that the synthesis of nuclear proteins and the nuclear RNA content decrease markedly during granulocyte maturation. The incorporation of l -[ U - 14 C]leucine into the acid-soluble histone-rich fraction of chromatin from immature cells is about 25 times that of mature cells, and the incorporation into the acid-insoluble, nonhistone proteins of chromatin from immature cells is about 6 times that of mature cells. It appears that there is very little quantitative change with respect to the protein components of nuclei from immature and mature granulocytic leukocytes. No significant differences in the amounts of histone, nonhistone protein, or phosphoprotein between nuclei of immature and mature granulocytes could be detected. No major differences in gel electrophoretic patterns of histones or nonhistone proteins could be detected. The fact that the amount of the chromatin proteins remains relatively constant during cell maturation in spite of the pronounced decrease in the rate of synthesis suggests that the rate of turnover of these proteins decreases significantly as the maturation of granulocytic leukocytes proceeds.