Ralph A. Bradshaw

Automatic systems for detecting cystine and cystinyl peptides during column chromatography.

Abstract Two automated methods are described for the determination of cystine and the detection of cystinyl peptides in solutions containing high levels of pyridine and acetic acid. While both methods are effective with free cystine, only one of these methods, an adaptation of the DTE/arsenite/DTNB method of Zahler and Cleland, is sufficiently sensitive to be useful as a technique for continuous detection of cystinyl peptides in the effluent of ion-exchange chromatograms of peptide mixtures. The other, utilizing cyanide and nitroprusside, is not sufficiently sensitive for general applicability in systems of this type. With the DTE/arsenite/DTNB method, the effluent pattern during the chromatography of a peptic digest of a protein has been examined on both an analytical and a preparative scale. The specific application of this detection method to the isolation and identification of cystinyl peptides in carboxypeptidase A is detailed in a separate communication (12).

The solution conformation of bovine carboxypeptidase a: Reaction with 2-hydroxy-5-nitrobenzyl bromide and N-methylnicotinamide chloride.

The recent completion of the amino acid sequence [l] and of the high resolution electron density map [2] of crystalline bovine carboxypeptidase A lead to a study of the position of various amino acid side chains of the enzyme in an aqueous environment. For this reason, the availability of the tryptophanyl residues of this enzyme have been examined by two methods: chemical modification by 2-hydroxy-%nitrobenzyl bromide (I-INBB) [3,4] and complex formation with N-methylnicotinamide chloride [ 151. These experiments revealed that the seven tryptophanyl residues present in the enzyme [ 1 ] are unavailable. However, in contrast to earlier reports, [3,4] modification of the protein by 2-hydroxy-S-nitrobenzyl bromide at pH 7.0 occurs rapidly and apparently specifically at the o-amino group, suggesting that under certain conditions, the specificity of 2-hydroxy-5nitrobenzyl bromide is wider than previously thought.

Structural and functional relationships of human antithrombin III and alpha-antitrypsin.

One of the principal mechanisms for the control of blood coagulation in man is the interaction of serum inhibitors with proteolytically active clotting factors, with concomitant losses in thrombogenic activity. One of the inhibitors, antithrombin III, which migrates as an α2-globulin on gel electrophoresis (1, 9, 13, 20), has received considerable attention. It appears to be identical with heparin cofactor and factor xa inhibitor (21) and provides the major site of action of heparin in inhibiting thrombin formation. However, it should be noted that the inhibition of thrombin is not limited to antithrombin III. Two other plasma proteins, an α2- macroglobulin (1, 3, 4, 11, 17), and the a α1,-proteolytic inhibitor (2, 14, 17) also show such activity. The latter inhibitor is probably identical with α1-antitrypsin (14), the protein responsible for most of the inhibition of trypsin activity in human serum.

Homology and Phylogeny of Proteolytic Enzymes

Publisher Summary This chapter discusses the homology and phylogeny of proteolytic enzymes. The process of evolution has been in the direction of greater diversity both in chemical structure and biological specificity, but as the proteins of the current era contain vestiges of their evolutionary precursors they bear discernible relations to one another, and such relationships provide additional means for the elucidation of the structure and function of a given protein. The concepts of chemical and conformational homology serve to define the structural relationships among members of a given class. Chemical homology refers to the similarity in the amino acid sequence of proteins that is greater than would be predicted by chance alone. To the extent that the linear assembly of the amino acid residues determines the conformation of proteins, it may be expected that a pair of chemically homologous proteins should reveal similar three-dimensional features and, thus, will exhibit conformational homology. The functional relationships among members of a class of homologous proteins are documented by the findings that, enzymes belonging to the same class operate by the same mechanism.

Increased output and accelerated analysis time with the automatic amino acid analyzer.

Abstract Modifications of the automatic amino acid analyzer (Spinco model 120) are described that allow for doubling the output of amino acid analyses by incorporating a second or “satellite” analytical system. The “satellite” system can simultaneously perform an independent analysis (lacking only proline) without impairment of the conventional operation of the analyzer. In addition, an accelerated buffer system has been described that allows for a complete amino acid analysis in a period of 3 hours.

Neuronal Cell Adhesion

Investigations in a variety of systems have shown that dissociated single cells will, under appropriate conditions, aggregate and, if maintained in culture, will morphologically differentiate to resemble the original organ or tissue (23, 33, 34). It can further be shown that in mixed aggregates of cells prepared from different organs (for example, neural retina and liver) there occurs cell segregation such that cells originally derived from the same organ attached to each other and separate from the cells derived from a different organ. The simplest explanation for this observation is that cells have a preferential or higher affinity for homologous cells, and if free to move within the aggregate, will rearrange so as to be adjacent to homologous rather than hetero-logous cells. This simple view is probably an oversimplification, because not only do cells migrate to be adjacent to homologous cells, but they also take up characteristic positions within the aggregate which cannot be explained simply by differential affinities (33).

Specific Interaction of Nerve Growth Factor with Receptors in the Central and Peripheral Nervous Systems

Nerve growth factor (NGF), a protein originally discovered as a diffusable substance produced by two mouse sarcomas (180 and 37)(28), stimulates marked neurite outgrowth by embryonic sympathetic and sensory ganglia both in vivo and in vitro. The latter phenomenon provided the basis for the bioassay (27). Antiserum to NGF blocks this effect, and if given to neonatal animals, produces a profound immunosympathectomy (26). Subsequent workers have noted wide-spread effects of NGF and its antisera on degeneration and regeneration of adrenergic fibers in both the central and peripheral nervous systems in embryonic and adult animals (3, 4). Its biological effects in the sensory nervous system, however, have been demonstrated only during a brief time span in chick embryonic development(27). To date no biological effects on the motor nervous system have been demonstrated. However, effects of the hormone on the development of neuroblastoma and pheochromocytoma cells in culture have been reported (22, 24, 31, 32, 39). In addition to the pronounced morphological changes, i.e. neurite production, several anabolic effects have been noted (7, 25, 27, 34). The sum of these findings suggests a hormone-like mode of action, pleiotypic in in character, that is important in regulating the development of peripheral neurons during embryonic development, and in some cases, in the mature animal.

Structural studies of 2.5 S mouse submaxillary gland nerve growth factor.

During the more than twenty years since its discovery (11), the nerve growth factor (NGF) has been indeed demonstrated to be an essential protagonist in the growth and development of the sympathetic nervous system (12). The NGF-stimulated growth response has been amply described from both metabolic and ultrastructural viewpoints.