Chloroplast and Mitochondrial DNA Variation in HORDEUM VULGARE and HORDEUM SPONTANEUM

A survey of restriction fragment polymorphism in Hordeum vulgare and Hordeum spontaneum was made using 17 and 16 hexanucleotide restriction endonucleases on chloroplast (cp) and mitochondrial (mt) DNA, respectively. The plant accessions originated from various places throughout the Fertile Cresent and Mediterranean. The types of changes in cpDNA consisted of nucleotide substitutions and insertions and deletions on the order of 100 base pairs. In contrast, mtDNA has most likely undergone larger insertions and deletions of up to 20 kilobase pairs in addition to rearrangements. Grouping of mtDNA fragment data showed that in some cases geographical affinities existed between the two species, whereas in others there were no clear affinities. Nucleotide diversity estimates derived from the restriction fragment data were used in a number of comparisons of variability. Comparisons of overall mtDNA variability (nucleotide diversity = 9.68 x 10^-4) with cpDNA variability (nucleotide diversity = 6.38 x 10^-4 ) indicated that the former are somewhat more variable. Furthermore, there was no indication that the wild H. spontaneum (cpDNA diversity = 5.57 x 10^-4; mtDNA diversity = 6.04 x 10 ^-4) was more variable than the land races of H. vulgare (cpDNA diversity = 5.88 x 10^-4; mtDNA diversity = 9.79 x 10^-4). In fact, on the basis of mtDNA diversity, H. vulgare was the more variable species. Comparison of organelle nucleotide diversity estimates with an estimate of nuclear nucleotide diversity derived from existing isozyme data provided evidence that both organelle genomes are evolving at a slower rate than the nuclear genome.     

Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Mouse-protective properties of Bordetella pertussis serotypes in passive tests

In a passive protection procedure in which the ed(50) values of Bordetella pertussis antisera were determined, groups of mice were given graded intraperitoneal doses of serum, followed the next day by intracerebral challenge with 100,000 organisms. Antiserum produced with B. pertussis culture 5373, serotype 1.3, protected mice against challenge with culture 18-323, serotype 1.2.3, as effectively as did an antiserum produced with a serotype 1.2.3 culture. When two groups of mice similarly treated with pertussis immune serum were challenged with culture 353Z (serotype 1) and 18-323, respectively, much lower ed(50) values were obtained with the animals challenged with 353Z. Passive protection tests with adsorbed antiserum gave equivocal results, suggesting that some of the adsorbing antigen remained in the serum and interfered with the tests. There was no evidence that serotype is related to protection.     

In Vitro Processing of Aleurain,a Barley Vacuolar Thiol Protease

Aleurain, originally described from its cDNA as a thiol protease [Rogers, J.C., Dean, D., and Heck, G.R. (1985). Proc. Natl. Acad. Sci. USA 82, 6512-6516], is characterized here as a glycoprotein that is targeted to a distinct vacuolar compartment in aleurone cells. Monospecific antibodies to a bacterial trpE-aleurain fusion protein were used to show that aleurain is made as a 42-kilodalton (kD) proenzyme (proaleurain) that is proteolytically processed in a post-Golgi compartment in two steps to form a 32-kD protein. The first processing step is the discrete loss of 9 kD from proaleurain to yield a 33-kD intermediate that is further processed by the gradual loss of 1 kD resulting in mature 32-kD aleurain. Using proaleurain secreted from Xenopus oocytes as a substrate, we established an in vitro system using aleurone cell extracts that correctly processes proaleurain to a stable protein that is indistinguishable from native barley aleurain as judged by partial digestion with staphylococcal V8 protease. Proaleurain is not capable of self-cleavage in the absence of aleurone cell extracts and mature aleurain appears not to participate in processing in vitro.     

Structural changes and lateral redistribution of photosystem II during donor side photoinhibition of thylakoids

The structural and topological stability of thylakoid components under photoinhibitory conditions (4,500 microE.m-2.s-1 white light) was studied on Mn depleted thylakoids isolated from spinach leaves. After various exposures to photoinhibitory light, the chlorophyll-protein complexes of both photosystems I and II were separated by sucrose gradient centrifugation and analysed by Western blotting, using a set of polyclonals raised against various apoproteins of the photosynthetic apparatus. A series of events occurring during donor side photoinhibition are described for photosystem II, including: (a) lowering of the oligomerization state of the photosystem II core; (b) cleavage of 32-kD protein D1 at specific sites; (c) dissociation of chlorophyll-protein CP43 from the photosystem II core; and (d) migration of damaged photosystem II components from the grana to the stroma lamellae. A tentative scheme for the succession of these events is illustrated. Some effects of photoinhibition on photosystem I are also reported involving dissociation of antenna chlorophyll-proteins LHCI from the photosystem I reaction center.     

Breakdown of the photosystem II reaction center D1 protein under photoinhibitory conditions: identification and localization of the C-terminal degradation products

Illumination of a suspension of thylakoids with light at high intensity causes inhibition of the photosystem II electron transport activity and loss from the membrane of the D1 protein of the photosystem II reaction center. Impairment of the electron transport activity and depletion of D1 protein from the thylakoid membrane of pea were investigated with reference to the presence or absence of oxygen in the suspension. The breakdown products of the D1 protein were identified by immunoblotting with anti-D1 polyclonal antibodies which were proven to recognize mainly the C-terminal region of the protein. The results obtained show that (i) the light-induced inactivation of the photosystem II electron transport activity under anaerobic conditions is faster than in the presence of oxygen; (ii) depletion of D1 protein is observed on a longer time scale with respect to loss of electron transport activity and is faster when photoinhibition is performed in the presence of oxygen; (iii) C-terminal fragments of D1 are only observed when photoinhibition is carried out anaerobically and are mainly localized in the stroma-exposed regions; and (iv) the fragments observed after anaerobic photoinhibition are quickly degraded on further illumination of the thylakoid suspension in the presence of oxygen.     

Reactivity of cuprous stellacyanin as a quinone and semiquinone reductase

The reactivity of cuprous stellacyanin as a quinone and semiquinone reductase has been examined. Rate constants (25.0 degrees C) measured for the oxidation of stellacyanin by 1,4-benzoquinone and benzosemiquinone are 2.3 X 10(4) M-1 s-1 (delta H not equal to = 4.4 kcal/mol, delta S not equal to = -24 eu) and 5.1 X 10(6) M-1 s-1, respectively [pH 7.0, I = 0.1 M (phosphate)]. The agreement of these rate constants with those calculated on the basis of relative Marcus theory is discussed. Stellacyanin is more effective than laccase in quenching benzosemiquinone, suggesting that the physiological role of this metalloprotein is to regulate the concentration of free radicals generated through the laccase-catalyzed oxidation of phenols.     

Kinetics of the oxidation of Rhus vernicifera stellacyanin by the Co(EDTA)-- ion.

A kinetic study of the oxidation of the copper(I) form of the blue copper protein stellacyanin (St(I) by Co(EDTA)-- has been performed. Observed rate constants approach a saturation limit with increasing [Co(EDTA)--] at pH 7, consistent with a mechanism involving rapid pre-equilibrium oxidant-protein complex formation followed by rate-limiting intramolecular Cu(I) to Co(III) electron transfer: Co(EDTA)-- + St(i Qp in equilibrium Co(EDTA)-- ---St(I) Co(EDTA)-- ---St(I) k2 leads to Co(EDTA)2-- ---St(II) (Qp = 149 M--1, k2 = 0.169 sec--1; 25.1 degrees, pH 7.0 mu 0.5 M (phosphate)). Activation parameters based on k2 (deltaH not equal to = 1.8 kcal/mol, deltaS not equal to = --56 cal/mol-deg) indicate that the electron transfer process is substantially nondiabatic, in marked contrast with results obtained for Co(phen) 3 3+ as the oxidant. Linear kobsd VS. [Co(EDTA)--] plots are reported for the Co(EDTA)-- oxidation of cuprous stellacyanin at pH 10 (k = 8.9 M--1 sec--1; 25.0, pH 10, mu 0.5 M (carbonate); DELTaH not equal to 11.3 kcal/mol, deltaS not equal to = -16 cal/mol-deg) and at pH 7 in the presence of excess EDTA (k = 21.2 M--1 sec--1; 25.1 degree, pH 7.0, mu 0.5 M (phosphate), [EDTA] tot = 5 X 10(--4) M; deltaH not equal to = 5.9 kcal/mol, delta S not equal to = --33 cal/mol-deg). It is concluded that Co(EDTA)-- adopts an electron transfer mechanism similar to that preferred by Co(phen)33+ under conditions where the oxidant is prevented from binding strongly to reduced stellacyanin.