Jerry L. Brown

Purification, characterization and possible function of α-N-acylamino acid hydrolase from bovine liver

Abstract α-N-Acylamino acid hydrolase from bovine liver has been purified approx. 1700-fold with a yield of 28%. Based on the results of studies using polyacrylamide gel electrophoresis the purified enzyme is homogeneous. This enzyme catalyzes the hydrolysis of α-N-acylated amino acids to yield the acyl group and the corresponding amino acid, α-Acetylmethionine was the most rapidly hydrolyzed substrate tested for this enzyme. Most of the other α-N-acylated amino acids used as substrates were hydrolyzed. Some, however, were cleaved at less than 1% of the rate observed for N-acetylmethionine, α-N-Acylamino acid hydrolase did not catalyze the hydrolysis of N- acetyl- d -alanine , N-acetyl-β-alanine, ϵ-N-acetyllysine, N-acetylglycinamide or α-N-acetylated peptides. When incubated with this enzyme the chloroacetyl derivatives of leucine and valine were hydrolyzed approx. 8- and 3-times, respectively, more rapidly than the corresponding N-acetyl derivatives. On the other hand, N-formylmethionine was hydrolyzed at only 60% the rate of N-acetylmethionine. Other studies on α-N-acylamino acid hydrolase have shown that it is a dimer composed of two 40 000 molecular weight monomers, it is active over a broad pH range with maximal activity at approx. pH 8.5, and requires Zn2+ for enzymatic activity. The substrate specificity and other properties of bovine liver α-N-acylamino acid hydrolase are very similar to those reported for acylase I isolated from porcine kidney [7]. We suggest that this enzyme functions in the catabolism of α-N-acetylamino acids resulting from the turnover of α-N-acetylated proteins.

The intestinal utilization of doubly labeled alpha-monopalmitin.

Doubly labeled alpha -monopalmitin was employed containing H/sup 3/ in the glycol portion of the molecule and C/aup 14/ in the fatty acid. The doubly labeled alpha -monopalmitin was used in studies on the synthesis of glycerides by the intestine. Results offer convincing evidence for the intact incorporation of alpha -monopalmitin into higher glycerides by the intestine and indicate a mechanism for triglyceride synthesis in addition to the phosphatidic acid pathway. (C.H.)

Binding of vitamin e in mammalian tumor cells in culture.

Abstract Radioactive vitamin E (D-α-[5-methyl-3H]tocopherol) bound with the cytosol (100,000g supernatant), pellet (100,000g pellet), crude nuclear, and purified chromatin fractions from mouse neuroblastoma (NBP2) and rat glioma (C-6) cells in culture. The level and type of vitamin E binding proteins in the cytosol depended upon the cell type. When the cytosol proteins containing radioactive vitamin E were separated by gel filtration (Sepharose 4B gel), there were five protein peaks in neuroblastoma, three peaks in glioma, and one peak in mouse B-16 melanoma cells which contained bound radioactivity. The level of binding in the neuroblastoma cells was higher than that in glioma cells or melanoma cells. Vitamin E remained bound to the proteins from the cytosols of neuroblastoma and glioma even after denaturation and separation by electrophoresis. This suggests that vitamin E is tightly bound with the cytosol proteins. There was only one vitamin E binding protein in the pellet and nuclear fractions of NB, glioma, and melanoma cells. The significance of vitamin E binding proteins in the mechanism of the effect of vitamin E on mammalian cells in culture is unknown.

Crossed immunoelectrophoresis of rat serum

A crossed immunoelectrophoresis (X-IEP) reference profile of normal (Sprague-Dawley) rat serum antigens was developed. Twenty-three antigens were detected; 13 were identified. These included inter-alpha-trypsin inhibitor, alpha 1-antichymotrypsin, and fibronectin, which had not previously been identified in rat serum. Many of the antigens were similar electrophoretically to their analogs in human and mouse serum, but some were substantially different. Inter-alpha-trypsin inhibitor, fibronectin, and C3 globulin were more anodic, while transferrin was more cathodic. Rat haptoglobins mobility was between those of human and mouse haptoglobins. As in the mouse, but more so, alpha-macroglobulin in the rat had alpha 1 mobility rather than alpha 2 seen for this protein in human serum. Only rat alpha 1-antichymotrypsin exhibited a double peak. There were variations in haptoglobin and alpha 1-lipoprotein mobilities among different strains of rat. This establishment of a standard X-IEP map of rat serum will simultaneously permit multiple quantitative and qualitative analyses of rat serum antigens for various experimental and clinical purposes.

Selective enrichment for temperature-sensitive secretion mutants of mammalian cells using plant lectin, concanavalin A

The use of conditional mutants as a genetic approach to study protein secretion in mammalian cells requires the isolation of a large number of mutants. Because a procedure for the direct selection of mutants with secretion defencts is not available, their isolation depends upon the selective enrichment of mutant phenotypes in a cell population. We have devised an enrichment strategy in which rat hepatoma cells unable to replace surface membrane receptors of a plant lectin, concanavalin A, are resistant to the cytotoxic effects of this lectin when administered at a nonpermissive temperature. This treatment yields a population highly enriched in cells that demonstrate temperature-sensitive secretion. Therefore, this selection strategy has important application in isolating temperature-sensitive mutants for use in the study of the mammalian cell secretion pathway.