Michael Gagelmann

Urodilatin (CDD/ANP‐95‐126) is not biologically inactivated by a peptidase from dog kidney cortex membranes in contrast to atrial natriuretic peptide/cardiodilatin (α‐hANP/CDD‐99‐126)

Atrial natriuretic peptide (CDD/ANP‐99‐126) is rapidly inactivated by a membrane preparation from dog kidney cortex. Inactivation occurs by cleavage of the ring structure in the position between Cys‐105 and Phe‐106. A unique proteolytic product separated by HPLC on reverse‐phase column appears as a single peak which elutes prior the intact peptide. In contrast, CDD/ANP‐95‐126 (urodilatin) which is released from the kidney is not destroyed by proteolysis using an identical membrane preparation.

Phosphorylation of the myosin light chains and satellite proteins in detergent-skinned arterial smooth muscle

Isoelectric focusing of extracts prepared from detergent-skinned porcine carotid artery showed that contraction was associated with phosphorylation of the regulatory myosin light chains and two additional proteins of the same apparent molecular weight (20,000). These two proteins, previously described as satellites, did not appear to be artifactually derived from the phosphorylated light chains during electrophoresis. That is, each of the phosphorylated proteins migrated as separate and distinct proteins when subjected to a second cycle of isoelectric focusing. Moreover, relaxation of skinned fibers was associated with dephosphorylation of the light chains and both satellites. These findings suggest that the satellites may represent varients of the light chains per se, or another regulatory protein which is reversibly phosphorylated and dephosphorylated during contraction and relaxation of vascular smooth muscle.

Chromatographic separation of two heterogeneous forms of the catalytic subunit of cyclic AMP-dependent protein kinase holoenzyme type I and type II from striated muscle of different mammalian species☆

Electrophoretically homogeneous preparations of catalytic subunit (C) of cAMP-dependent protein kinase isolated according to two different procedures from holoenzyme type I and type II from rabbit and from holoenzyme type II from rat skeletal muscle and from bovine cardiac muscle can be separated on carboxymethyl cellulose or on a Mono S column (Pharmacia) by salt gradient elution into two enzymatically active peaks called A and B, which do not interconvert on rechromatography. Cochromatography of peak A fractions or of peak B fractions derived from both holoenzymes respectively yields single enzyme peaks in each case, thus indicating that both represent different entities, which were named CA and CB. The separate character of both enzyme forms is supported by the fact that CB under all conditions is degraded faster by the C-specific protease (E. Alhanaty et al. (1981) Proc. Natl. Acad. Sci. USA 78, 3492-3495) than CA, a phenomenon which is enhanced in both enzyme forms by substrate (Kemptide). The separation of both subtypes from each other is probably based on differences in isoelectric values (delta pH less than or equal to 0.5 units). The reason for the charge difference is not presently known. CA and CB do not differ significantly in their phosphate content. No differences between CA and CB have been detectable so far with respect to their migration in SDS gels, kinetic behavior regarding both substrates and cosubstrate, pH dependence, inhibition by regulatory subunits of holoenzyme type I (rabbit skeletal muscle) and of type II (bovine cardiac muscle), and inhibition by specific-heat and acid-stable inhibitor-modulator. The peptide pattern of both forms after limited proteolysis exhibits small differences.

Degradation of porcine brain natriuretic peptide (pBNP-26) by endoprotease-24.11 from kidney cortical membranes.

Porcine brain natriuretic peptide of 26 amino acid residues (pBNP-26) is inactivated by endoprotease-24.11 (EC 3.4.24.11) of kidney cortical membranes. In contrast to human alpha atrial natriuretic peptide/cardiodilatin (ANP/CDD) showing a single major cleavage within the disulfide-linked loop between Cys and Phe in position 7 and 8, pBNP-26 is cleaved at several sites. Although both pBNP-26 and ANP/CDD exhibit Cys-Phe peptide bonds at the corresponding positions this bond is not cleaved in BNP-26.

Relaxation of smooth muscle by cardiodilatin/atrial natriuretic peptide is inhibited by cAMP-dependent phosphorylation

Cardiodilatins/atrial natriuretic peptides (CDD/ANP) exhibit a common amino acid sequence: Arg101‐Arg102‐Ser103‐Ser104. Cyclic AMP‐dependent phosphorylation of Ser104 of atrial peptides with [γ‐32P]ATP enables rapid identification of cardiac hormones. The biological activity of in vitro phosphorylated cardiodilatin (CDD‐28/α‐hANP) is dramatically altered compared to the unphosphorylated peptide: the vasorelaxant effect of cardiodilatin 28 is inhibited upon phosphorylation.

Biochemistry of the Differential Release, Processing and Degradation of Cardiac and Related Peptide Hormones

Investigations during the last few years supported the existence of two major forms of cardiac peptides. In the heart, a prohormone of 126 amino acid residues (CDD-1-126) is stored, while CDD-99-126/ANP (= αANP) is the circulating form in the blood plasma. By use of a similar isolation procedure, as used for the isolation of circulating CDD-99-126/ANP, a biological active peptide, strongly related to CDD-99126/ANP, was purified from urine, indicating excretion from the kidneys. Sequence analysis shows that the main form of urinary cardiodilatin is a 32 amino acid residue containing molecule (urodilatin-95-126) similar to CDD-99-126/ANP except N-terminal extension by four amino acid residues. The prolongation first of all indicates that urodilatin is not derived from a plasma form and obtained by filtration and renal clearance. Secondly, it demonstrates that urodilatin undergoes a different posttranslational process during excretion than CDD-99-126/ANP. Both, synthetic human cardiodilatin (CDD-99-126)/atrial natriuretic peptide (ANP) and urodilatin (95–126) are phosphorylated by the catalytic subunit of cAMP-dependent protein kinase at a serine residue in position 104. Phosphorylated peptides exhibit decreased relaxing potency compared to unphosphorylated peptides. CDD-99-126/ANP is rapidly removed from the circulation by clearance and proteolytic degradation. Inactivation of CDD-99-126/ANP in the kidneys occurs by an endoprotease-24.11 present in the cortical membranes. Due to the enzymatic reaction a major cleavage occurs within the ring structure between Cys105 and Phe106. In contrast, N-terminal extension by four amino acid residues (Thr-Ala-Pro-Arg) and cAMP-dependent phosphorylation of CDD-99-126/ANP inhibit proteolysis by kidney cortical membranes.