Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Ovarian steroid production in vitro during gonadal regression in the turkey. II. Changes induced by forced molting.

In the turkey, the onset of incubation behavior is associated with altered ovarian steroidogenesis, ovarian regression, decreased, LH secretion, and increased serum prolactin (Prl) levels. To clarify the relative contribution of circulating LH and Prl to the initiation of ovarian regression, laying hens were exposed for 0, 3, 7, or 14 days to a forced molting procedure (exposure to reduced day length of 6L:18D and removal of feed and water for the initial 3 days) that induces ovarian regression and decreased LH levels but does not increase Prl levels. On each of these days, hens were killed and granulosa and theca interna cells from the largest (F1) and fifth largest (F5) preovulatory follicles and total cells from the small white follicles (SWF) were incubated for 5 h in the presence or absence of ovine LH (oLH; 0-1,000 ng/ml). Force-molted hens exhibited diminished levels of circulating LH, Prl, progesterone (P), androgen (A), and estradiol (E) by Day 3 of treatment. Ovarian atresia began in F1 by the third day of treatment, and included F1 and F5 by the seventh day. No preovulatory follicles were present on the fourteenth day. With both F1 and F5 granulosa cells, production of P in the presence of oLH was initially enhanced (Day 3) and later absent (Day 7). In contrast, production of A by F5 theca interna cells in the presence of oLH was initially suppressed (Day 3) and then returned to pretreatment levels (Day 7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ion exchange and affinity chromatography in the scaleup of the purification of alpha-galactosidase from soybean seeds

Soybeans (Glycine max) contain an alpha-galactosidase that makes up a small fraction of the total protein of the seed. The properties of this enzyme are of interest because of its potential to convert the galactooligosaccharides, stachyose and raffinose, in soybean meal to sugars digestible in the human gastro intestinal tract and thereby increase potential uses of this vegetable protein source in human and animal foods. Study of this enzyme required the isolation of milligram quantities of electrophoretically pure protein from ground soybeans and therefore, scaleup of laboratory procedures by a factor of 300 times. Large scale acid precipitation, ammonium sulfate precipitation, and centrifugal recovery of the precipitated protein allowed alpha-galactosidase to be isolated from 45.5 kg soybean meal containing 17.1 kg protein, to obtain an enzyme extract with a specific activity of 90 to 100. A novel combination of strong anion exchange and cation exchange chromatography followed by Concanavalin-A affinity chromatography with a methyl alpha-D mannoside gradient gave alpha-galactosidase with an average specific activity of 56,000. Ion exchange chromatography preceding Concanavalin-A affinity chromatography allowed elimination of a relatively costly melibiose affinity chromatography step (which followed the Concanavalin-A column In the laboratory procedure) thereby making scaleup practical.     

Effects of cold on vascular permeability and edema formation in the isolated cat limb.

We investigated the effects of cold temperatures on microvascular protein permeability in the isolated constant-flow perfused cat hindlimb. The perfusates were 20% cat plasma-80% albumin-electrolyte solution (low-viscosity perfusate, approximately 1 cP) or whole blood (high-viscosity perfusate, approximately 4 cP). The time at low temperature (less than 10 degrees C) was less than 3 h (short term) or greater than 5 h (long term). Decreases in the solvent drag reflection coefficient (sigma f) indicated increases in permeability. The sigma f's were determined with the integral-mass balance method from measurement of changes in protein concentration and hematocrit induced by fluid filtration into the tissues. Short-term cold exposure did not increase permeability with either a low- or a high-viscosity perfusate, whereas long-term exposure with limb temperatures of approximately 5 degrees C significantly increased permeability when the perfusate was whole blood. In addition, we verified our previous prediction that flow had to be reduced to 6-8 ml.min-1.100 g-1 to avoid the hydrostatic edema caused by short-term perfusion with whole blood at approximately 5 degrees C. Also, we found that at approximately 3 degrees C histamine's permeability-increasing effect was totally abolished, whereas at approximately 20 degrees C this effect was partially inhibited. Hence, constant-flow perfusion at low temperature with whole blood can cause edema by a pressure-dependent mechanism, whereas long-term perfusion with this perfusate at low temperatures can cause a permeability increase that further compounds edema formation. Histamine is not responsible for this permeability increase.     

Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum.

Chromosomal transformation of Agmenellum quadruplicatum PR-6 (= Synechococcus sp. strain 7002) was characterized for phenotypic expression, for exposure time to DNA, and for dependence on DNA concentration with regard to Rifr donor DNA. Exponentially growing cells of PR-6 were competent for chromosomal transformation. Competence decreased in cells in the stationary phase of growth or in cells deprived of a nitrogen source. Dark incubation of cells before exposure to donor DNA also decreased competence. Homologous Rifr and Strr DNA and heterologous Escherichia coli W3110 DNA were used in DNA-DNA competition studies, which clearly showed that DNA binding by PR-6 was nonspecific. DNA binding and uptake by PR-6 exhibited single-hit kinetics. Single-stranded DNA failed to transform competent cells of PR-6, and DNA eclipse was not observed, suggesting that double-stranded DNA was the substrate for the binding and uptake reactions during the transformation of PR-6. A significant improvement in transformation frequency was achieved by increasing the nitrate content of the culture medium and by lowering the temperature at which cells were exposed to donor DNA from 39 degrees C (the optimal temperature for growth) to 30 degrees C.     

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.     

NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein

cDNA clones to rat liver NADPH-cytochrome P-450 oxidoreductase were used to isolate genomic clones from a Wistar-Furth inbred rat genomic DNA library. Fifteen exons containing the coding region and 3'-nontranslated segment of the P-450 reductase gene were identified, spanning 20 kilobases of DNA contained in 3 lambda-Charon 35 clones. The organization of this single copy gene reveals a general correspondence between exons and structural domains of the protein, with the segment responsible for anchoring the reductase to the microsomal membrane and several segments involved in FMN, FAD, and NADPH binding encoded by discrete exons.     

Photolysis and deacylation of inhibited chymotrypsin

Inhibited chymotrypsin was reactivated through the photolysis of the covalently bound light-reversible cinnamates described in our previous paper [Stoddard, B.L., Bruhnke, J., Porter, N.A., Ringe, D., & Petsko, G. (1990) Biochemistry 29, 4871-4879]. The light-induced deacylation was accomplished both in solution and in protein crystals, with the release of inhibitor from the crystal monitored and confirmed by X-ray diffraction. The product of photolysis has been characterized as a 3-methylcoumarin, leading to a mechanism for light-driven deacylation of an internal lactonization that is dependent on the presence of an internal hydroxyl nucleophile. The acyl enzyme formed from cinnamate A is not suitable for photochemical studies, as the complex has a short half-life in solution and does not have a chromophore that is well separated from protein absorbance. Cinnamate B, with a p-diethylamino substituent, shows an enzyme deacylation rate enhancement of 10(9) for the cis photoisomer relative to the trans starting material. The half-life and deacylation rate of this compound in the E-I complex after photon absorption have been directly measured by subsecond UV absorption studies. X-ray diffraction studies of photoactivation using a flow cell show that the cinnamate B acyl enzyme complex is fully capable of light-induced isomerization and regeneration of native enzyme in the crystalline state. The E-I complex formed upon binding of cinnamate A, however, shows little if any effect from irradiation due to competitive absorbance by the highly concentrated protein at the shorter UV wavelengths. Photolysis of cinnamate B appears to occur on a time scale fast enough for applications in crystallographic studies of enzymatic intermediate-state structures.     

Integral kinetics of alpha-galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose

alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.     

Purification of human C3b inactivator by monoclonal-antibody affinity chromatography

Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.