Conversion of atriopeptin II to atriopeptin I by atrial dipeptidyl carboxy hydrolase

We are examining the substrate specificity of atrial dipeptidyl carboxyhydrolase, a membrane-bound metallo enzyme that we isolated from bovine atrial tissue homogenates. This enzyme readily removes the dipeptide, Phe-Arg, from Bz-Gly-Ser-Phe-Arg, a stand-in substrate for atriopeptin II, one of several atrial natriuretic factors. We now report that the atrial enzyme cleaves the C-terminal dipeptide, Phe-Arg, from atriopeptin II to form atriopeptin I. The km (pH 7.5) is 25 microM and the ratio of relative Vmax/km as a measure of substrate specificity indicates that atriopeptin II is a 240-fold better substrate than Bz-Gly-His-Leu. Only Phe-Arg was detected as a hydrolysis product, indicating that sequential cleavage of Asn-Ser from atriopeptin II does not occur, and that atriopeptin I is not a substrate. Bz-Gly-Asn-Ser was as good a substrate for the atrial enzyme as Bz-Gly-His-Leu, but Bz-Cys(bzl)-Asn-Ser was not hydrolyzed. This result suggests that the presence of an intact disulfide bond or an S-alkylated residue in the P1 position of a substrate (as in atriopeptin I) prevents hydrolysis by the atrial enzyme. Comparative studies were made with the angiotensin I converting enzyme. Atriopeptin II was not a substrate. The stand-in substrates for atriopeptin I, Bz-Cys(bzl)-Asn-Ser and Bz-Gly-Asn-Ser were barely hydrolyzed, which by itself suggests that atriopeptin I is not a substrate of the angiotensin converting enzyme. Our results strongly suggest that atriopeptin II is converted to atriopeptin I and that hydrolysis is mediated by the atrial enzyme. The angiotensin I converting enzyme plays no role in processing these peptides. We suggest that the atrial enzyme be named atrial peptide convertase.     

Diversity in the immune system: “Preconceived ideas” or ideas preconceived?

Two concepts of the evolution and regulation of expression of the combining site repertoire of the immune system, are compared. One view is based on the Associative Recognition Theory as formulated by the author and the other is based on the Idiotype Network Idea as conceived by Jerne. The two concepts are analyzed from the point of view of their logic, internal consistency and factual support.     

Mesenchymal control of branching pattern in the fetal mouse lung

The effect of mesenchyme on specialization of respiratory epithelium in the fetal mouse was tested in organ cultures. Heterologous combinations were made between respiratory and non-respiratory lung epithelia and the corresponding mesenchymes. Isolated terminal respiratory buds of fetal mouse lungs were recombined with mesenchyme from chick lung parabronchi, mouse trachea or from the avascular, non-respiratory air sacs of chick lungs. Isolated non-branching chick air sacs were combined with mouse terminal bud mesenchyme or mesenchyme from the respiratory branches of chick lungs. Air sac epithelia branched in a pattern characteristic of the chick lung when combined with chick respiratory mesenchyme and in a pattern characteristic of mouse lung when combined with mouse terminal bud mesenchyme. Mouse terminal bud epithelia did not branch with either mouse tracheal mesenchyme or chick air sac mesenchyme but branched in a chick pattern with chick parabronchial mesenchyme. Electron microscopic examination of the cultures showed that all chick air sac epithelial cultures failed to produce surfactant (lamellar bodies) even when they branched. Control cultures of mouse terminal buds contained large numbers of lamellar bodies; mesenchyme which suppressed branching reduced the number of lamellar bodies to only a few in a small proportion of the cells. Culture medium supplemented with growth factors and hormones increased the number of lamellar bodies in heterologous mouse combinations but did not bring the number to control levels. Supplemented medium had no effect on lamellar body production by chick air sac epithelium. The results indicate that branching pattern is determined by the mesenchyme surrounding the epithelial primordium. However, the capacity to synthesize surfactant is determined by the source of the epithelium; mesenchyme may control the degree of expression but not the absolute presence or absence of the differentiated condition.     

Alpha 1- and beta 2-adrenergic receptors co-expressed on cloned MDCK cells are distinct glycoproteins

We have explored the molecular differences between alpha 1- and beta 2-adrenergic receptors that are co-expressed by a clonally-derived cell line, Madin-Darby canine kidney clone D (MDCK-D). MDCK-D membranes were pre-labeled with selective alpha 1- and beta-adrenergic radioligands and were then solubilized with the non-ionic detergent digitonin. Solubilized alpha 1- and beta 2-adrenergic receptors were retained by immobilized wheat germ agglutinin and were eluted following addition of N-acetyl-D-glucosamine or sialic acid. Both receptors were also retained by immobilized Limax flavus lectin, a sialic acid-binding lectin. Lectins that were specific for N-acetyl-D-glucosamine residues did not bind to these receptors. These results indicate that both alpha 1 and beta 2 receptors are sialylated glycoproteins. The solubilized alpha 1- and beta 2-adrenergic receptors migrated with different elution profiles from an Ultragel AcA 34 column. The apparent molecular sizes of the digitonin-receptor complexes were 68A for the alpha 1 receptor and 55A for the beta 2 receptor. These results show that alpha 1- and beta 2-adrenergic receptors can be present on the same cell as distinct sialic acid-containing glycoproteins.     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.     

Antigenic probes locate the myosin subfragment 1 interaction site on the N-terminal part of actin

The interaction of two different anti-actin antibody populations with the myosin subfragment 1-F-actin rigor complex has been studied. In contrast with the 1–7 sequence, the 18–28 sequence appears to be strongly implicated in the contact area of the myosin head on the actin polypeptide chain.     

Analysis of fluorescence transients of DCMU-treated leaves of Triticum species to provide estimates of the densities of photosystem II reaction centres

The fluorescence of the chlorophyll associated with photosystem II was studied in seedling and flag leaves of Triticum species. Seedling leaves of the diploid species T. urartu had higher values of t (the normalised area over the fluorescence induction curve of DCMU treated leaves) than those of the other species studied which included hexaploid T. aestivum. However this difference was not evident when leaves were grown in a low light intensity (40 µmol quanta of photosynthetically active radiation m^–2 s^–1). The smaller total number of chlorophyll molecules per photosystem II reaction centre (chl/RCII) in T. urartu (177) as compared with the other species (mean 234) was deduced from the observed differences in t. As a consequence of its lower chl/RCII, despite slightly lower chlorophyll content (mg m^–2), T. urartu had a greater density of reaction centres than the other species (2880 cf 2230 nmol m^–2 of leaf). Consistent with the lower chl/RCII of T. urartu, it had a higher chlorophyll a/b ratio than the other genotypes. Seedling leaves of T. urartu had higher light saturated rates of photosynthesis than those of the other species, when grown at high light, a difference associated with reaction centre density.In flag leaves, when the complications due to variable development and senescence patterns were eliminated, t of the diploid species including T. urartu was lower than that of T. aestivum. The lower apparent chl/RCII of T. urartu arose partly because the molar extinction coefficient of the chlorophyll in the leaves of T. urartu was greater than in T. aestivum. However, the density of PS II reaction centres was slightly lower for the diploid species studied because their chlorophyll contents were lower than the hexaploids.The validity of the method for estimating chl/RCII from fluorescence transients is discussed. The possibility is considered that the difference in apparent chl/RCII of flag and seedling leaves of R. urartu as compared to the other five genotypes is a consequence of its different adaptive response to the spectral quality of the light.     

Chlorophyll-protein and polypeptide composition of Mn-deficient sugar beet thylakoids

The chlorophyll-protein and polypeptide composition of manganese deficient and control sugar beet thylakoids was examined using three different detergent-electrophoresis systems. On a per chlorophyll basis, manganese deficiency reduced the amounts of CPa complex (separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis), and CP 47 and CP 43 complexes (separated by octylglucoside/SDS-polyacrylamide gel electrophoresis) without decreasing the amounts of light harvesting complexes. Lithium dodecylsulfate/Triton X-100 polyacrylamide gel electrophoresis showed that manganese deficiency decreased several thylakoid polypeptides, including a chlorophyll b containing 30 kilodalton chlorophyll-protein complex, but did not decrease the amounts of 28 and 29 kilodalton light-harvesting chlorophyll b-containing polypeptides.     

Thermodynamics of the charge recombination in photosystem II

The temperature dependence of the electric field-induced chlorophyll luminescence in photosystem II was studied in Tris-washed, osmotically swollen spinach chloroplasts (blebs). The system II reaction centers were brought in the state Z⁺P⁺-Q_A ^-Q_B ^- by preillumination and the charge recombination to the state Z⁺PQ_AQ_B ^- was measured at various temperatures and electrical field strengths. It was found that the activation enthalpy of this back reaction was 0.16 eV in the absence of an electrical field and diminished with increasing field strength. It is argued that this energy is the enthalpy difference between the states IQ_A ^- and I^-Q_A and accounts for about half of the free energy difference between these states. The redox state of Q_B does not influence this free energy difference within 150 s after the photoreduction of Q_A. The consequences for the interpretation of thermodynamic properties of Q_A are discussed.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - I intermediary electron acceptor - Mops 3-(N-morpholino)propanesulphonic acid - P (P₆₈₀) primary electron donor - PS II photosystem II - Q_A and Q_B first and second quinone electron acceptors - Tricine N-tris(hydroxymethyl)methylglycine - Tris tris-(hydroxymethyl)aminomethane - Z secondary electron donor Dedicated to Professor L.N.M. Duysens on the occasion of his retirement     

Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorometer

A newly developed modulation fluorometer is described which operates with 1 sec light pulses from a light-emitting diode (LED) at 100 KHz. Special amplification circuits assure a highly selective recording of pulse fluorescence signals against a vast background of non-modulated light. The system tolerates ratios of up to 1:10⁷ between measuring light and actinic light. Thus it is possible to measure the dark fluorescence yield and record the kinetics of light-induced changes. A high time resolution allows the recording of the rapid relaxation kinetic following a saturating single turnover flash. Examples of system performance are given. It is shown that following a flash the reoxidation kinetics of photosystem II acceptors are slowed down not only by the inhibitor DCMU, but by a number of other treatments as well. From a light intensity dependency of the induction kinetics the existence of two saturated intermediate levels (I₁ and I₂) is apparent, which indicates the removal of three distinct types of fluorescence quenching in the overall fluorescence rise from F₀ to F_max.Abbreviations Q_A and Q_B consecutive electron acceptors of photosystem II - PS II photosystem II - P 680 reaction center chlorophyll of photosystem II - F₀ minimum fluorescence yield following dark adaptation - F_max maximum fluorescence yield - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea - DCCD N,N-dicyclohexylcarbodiimide - PQ plastoquinone - DAD diaminodurene Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.