Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography.

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.

Synthesis of angiotensinogen by renin-containing neuroblastomas.

Angiotensinogen in plasma is of hepatic origin, but many organs possess the ability to synthesize this protein because messenger RNA for angiotensinogen is widely distributed in the body. The cell types responsible for the extrahepatic synthesis of angiotensinogen remain to be identified. To examine whether renin-containing cells synthesize angiotensinogen, we have utilized a polyclonal antibody to angiotensinogen and immunoprecipitated metabolically labeled cells of two neuroblastomas known to contain renin. The results indicate that the cell line Neuro 2a synthesizes and releases a protein with a molecular mass of 57 kDa that is specifically recognized by the angiotensinogen antibody, indicating that Neuro 2a synthesizes angiotensinogen. Similarly, the cell line NB41A3 was also found to synthesize a protein specifically recognized by the antibody to angiotensinogen.

An increase in high-molecular weight renin substrate associated with estrogenic hypertension.

We have previously reported that estrogens have the potential to induce new forms of renin substrate in addition to elevating the major circulating form of this protein. One of these estrogen-induced forms had a molecular weight in excess of 150,000. In this study we have compared the plasma concentration of the high-molecular-weight renin substrate in normotensive women receiving estrogen therapy and women with estrogenic hypertension. A statistically significant elevation of this protein was associated with estrogenic hypertension and normotensive pregnant women at term. This form of renin substrate differed from the major form with respect to electrophoretic mobility, isoelectric point, and immunologic cross-reactivity. In addition, kinetic analysis indicated that this high-molecular-weight substrate has a significantly higher affinity for the enzyme renin than the major circulating form (Km = 1800 +/- 290 versus 3520 +/- 260 ng angiotensin I equivalents/ml). These results suggest that in addition to renin substrate concentration, substrate composition may play an important role in blood pressure regulation.

PIP: This study investigates whether qualitative rather than quantitative differences in renin substrate were associated with estrogen induction of hypertension by comparing the plasma concentration of high molecular weight renin substrate (HMS) in 18 healthy normotensive, nonpregnant women aged 35-50 taking no medication; 20 normotensive subjects receiving estrogens as oral contraceptives (OCs) or ethinyl estradiol (EE) 50 mcg; and 5 women on OCs or EE 50 mcg who became hypertensive on estrogen therapy. A significant increase in renin substrate was evident in all women with elevated plasma estrogen levels. The difference in total renin substrate levels of normotensive and hypertensive subjects was not statistically significant. HMS differed from the normal molecular weight substrate (NMS) in electrophoretic mobility, isoelectric point, and immunologic cross-reactivity. Kinetic analysis indicated that it also had a significantly higher affinity for the enzyme renin than the major circulating form (Km=1800 +or- 290 versus 3520 +or- 260 ng angiotensin I equivalents/ml). The results suggest that substrate composition may play an important role in blood pressure regulation.

High molecular weight angiotensinogen levels in hypertensive pregnant women.

An apparent high molecular weight angiotensinogen (H-Aogen) can be separated from the usually predominant low molecular weight angiotensinogen (L-Aogen) by gel filtration of plasma. H-Aogen has been quantitated in plasma from normotensive menstruating women, estrogen treated women, normotensive pregnant women, women with pregnancy-induced hypertension (PIH), and women whose preexisting hypertension was exacerbated during pregnancy. When expressed as a percent of the total angiotensinogen, the H-Aogen levels were: menstruating women 4%, estrogen-treated women 10%, normotensive pregnant women 16%, women with PIH 25%, and pregnant women with exacerbated hypertension 28%. A significant difference (p less than 0.01) was found between H-Aogen concentration in normotensive pregnant women and women with PIH (1.10 +/- 0.12 and 1.73 +/- 0.16 micrograms angiotensin I/ml plasma respectively). In some hypertensive pregnant women, H-Aogen is the predominant form of angiotensinogen. Thus, H-Aogen should be recognized as a component of the renin-angiotensin system.

Purification and characterization of two forms of rat plasma proangiotensin.

Two forms of rat plasma proangiotensin were purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose at pH 6.5, DEAE-Sepharose at pH 8.9, Sephadex G-150, hydroxyapatite and hexyl-agarose. Both forms were finally separated by affinity chromatography on concanavalin-A--Sepharose. Presence or absence of carbohydrate side chains seems to be the only difference between these forms of proangiotensin. Both proteins consist of single polypeptide chains having apparent molecular weights of 52000 and 55000 and isoelectric points around 4.7 and 4.4, respectively. No significant difference between the proteins could be observed with respect to the amino-terminal amino acid sequence which was found to be the same (H2N-Asp-Arg-Val) as for angiotensin I and II. Furthermore, extensive digestion with renin, releasing the decapeptide angiotensin I, did not significantly reduce the molecular weights of both polypeptides. It can therefore be concluded that the angiotensin I peptide is located at the amino terminus of the prohormone. Kinetic constants measured for the release of angiotensin I by renin were found to be Km = 5.0 microM proangiotensin and V = 270 nmol of angiotensin I h-1 unit renin-1 for the concanavalin-A-binding form and Km = 5.6 microM proangiotensin and V = 250 nmol angiotensin I h-1 unit renin-1 for the prohormone which did not bind to concanavalin-A--Sepharose. The form of proangiotensin not bound to concanavalin-A--Sepharose was found to be more thermally labile (tm of 59.0 degrees C) than the form binding to concanavalin A (tm of 61.5 degrees C, where tm = temperature at which 50% reactivity is lost).

Isolation and characterization of human renin substrate.

Human renin substrate (angiotensinogen) was purified from outdated bank plasma. Purification procedures included ammonium sulfate precipitation, DEAE-cellulose column chromatography, concanavalin A-Sepharose column chromatography, Hydroxylapatite column chromatography preparative isoelectric focusing and Ultrogel AcA 44 gel filtration. The final recovery was 10% and the specific angiotensin I content of 10.5 micrograms/mg of protein was obtained. Polyacrylamide gel and SDS-polyacrylamide gel electrophoresis and analytical ultracentrifugal analyses showed the homogeneity of the purified renin substrate. The molecular weight of 60900 was determined by sedimentation equilibrium studies. Human renin substrate was a glycoprotein containing 13% carbohydrate. Cystine could not be detected on amino acid analysis. The purified renin substrate showed the isoelectric point heterogeneity (pI, 4.6 and 4.9).