Multiple Focusing Research Articles

Human angiotensinogen. Purification partial characterization, and a comparison with animal prohormones.

The renin-angiotensin system appears to play a major role in the regulation of sodium excretion and fluid intake in a wide variety of animal species from mammals to teleosts. In mammals the system has evolved further importance in terms of blood pressure homeostasis. This hormonal system in all species appears to involve a serum protein prohormone, angiotensinogen, a proteolytic enzyme, renin, and angiotensin I, the decapeptide product of the reaction between renin and angiotensinogen. The importance of this system to the organism appears to correlate directly with the necessity to conserve sodium while an abnormality of this process may underlie the development of hypertension in man. As the starting point of the system, angiotensinogen assumes special importance as a possible index of evolutionary development. In addition, it has been known for many years that human (viz. primate) angiotensinogen differs from that found in other mammals in its inability to be a substrate for animal renins while animal angiotensinogens readily react with human renin. Thus, the enzymatic specificity appears to reside with the prohormone. The biochemical basis for this difference is unresolved due primarily to the lack of purified human angiotensinogen. In this paper we describe methods for the purification of human angiotensinogen which have direct applicability to animal angiotensinogens. Our approach utilizes ammonium sulfate precipitation, Sephadex G-150 chromatography, multiple isoelectric focusing, and concanavalin A-Sepharose affinity chromatography. With the availability of highly purified human angiotensinogen we compared the molecular weights, heterogeneity, isoelectric points, and thermal lability of hog, rabbit, and human angiotensinogen in order to define the biochemical basis of the species variation in renin reactivity...

The isoelectric focusing pattern of LDH isozymes

The rabbit liver, heart, and muscle extracts showed isoelectric focusing peak patterns of LDH (Ampholine pH 3.5-10), which considerably differed from the LDH zymograms characteristic to those tissues. Ampholine did not give any significant influence on enzymic activity and electrophoretic mobility (agar gel plate) of the five fractions of purified LDH isozymes. Isoelectric focusing of LDH band 1 (H4) produced an acidic peak but with a very poor recovery of activity (1-2%), while that of LDH band 5 (M4) gave an alkaline peak with a good recovery of activity (88%). LDH bands 2 (H3M), 3 (H2M2), and 4 (HM3) exhibited multiple isoelectric focusing peaks, of which an alkaline peak was dominant. Isoelectric focusing of the DANSylated LDH isozyme, band 3, revealed 3 to 4 fluorescent protein peaks not corresponding to the activity peaks. Dissociaton and partial reactivation of LDH (tetramer), accompanying aggregation-denaturation (inactivation), especially in acidic pH, are responsible for producing the modified activity peak pattern of LDH isozymes in the isoelectric focusing experiments.

Multiple—focusing time of flight mass spectrometers Part I. Tofms with equal momentum acceleration

New time of flight mass spectrometers (TOFMS) are described, which combine stigmatic imaging with vanishing velocity dispersion in time of flight and imaging. In such analysers the time of flight of an ion is a function of the mass to charge ratio only. Velocity focusing is achieved by combining linear drift spaces with sector fields. Mass spectrometers based on this principle will be capable of a mass resolution of several hundred and will be insensitive to initial ion energy spread. In this paper TOFMS with equal momentum acceleration are treated. Extension of the principle to TOFMS with equal energy acceleration will be subject of a subsequent paper.