Demonstration that the BchH protein of Rhodobacter capsulatus activates S-adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase.

The bchH gene of Rhodobacter capsulatus has been cloned into an expression strain of Escherichia coli. Following induction of expression of the BchH protein, it was found that the E. coli strain also accumulated porphyrins with the fluorescence properties of protoporphyrin and zinc protoporphyrin. It was also found that the soluble BchH protein increased the activity of S-adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase, when mixed with membranes of an expression strain of E. coli into which the bchM gene (which encodes the methyltransferase) had been cloned, as well as membranes of a bchH mutant of R. capsulatus.

Effect of the PufQ protein on early steps in the pathway of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus.

The addition in trans of the pufQ gene to a strain of Rhodobacter capsulatus from which the entire puf operon had been deleted, increased its ability to synthesize coproporphyrinogen from both delta-aminolevulinic acid and porphobilinogen. Studies at the enzyme level indicated that the conversion of porphobilinogen to uroporphyrinogen III had about a 2-fold higher level of activity in the anaerobically-grown pufQ-containing strain. This increase in activity over the puf-deletion strain appeared to occur during transitions from aerobic to semiaerobic growth conditions. These results indicated that the PufQ protein may exert a stimulatory effect quite early in the pathway of bacteriochlorophyll biosynthesis.

Identification of the PufQ protein in membranes of Rhodobacter capsulatus.

The PufQ protein has been detected in vivo for the first time by Western blot (immunoblot) analyses of the chromatophore membranes of Rhodobacter capsulatus. The PufQ protein was not visible in Western blots of membranes of a mutant (delta RC6) lacking the puf operon but appeared in membranes of the same mutant to which the pufQ gene had been added in trans. It was also detected in elevated amounts in a mutant (CB1200) defective in two bch genes and unable, therefore, to make bacteriochlorophyll. The extremely hydrophobic nature of the PufQ protein was also apparent in these studies since it was not extracted from chromatophores by 3% (wt/vol) n-octyl-beta-D-glucopyranoside, a procedure which solubilized the reaction center and light-harvesting complexes. During adaptation of R. capsulatus from aerobic to semiaerobic growth conditions (during which time the synthesis of bacteriochlorophyll was induced), the PufQ protein was observed to increase to the level of detection in the developing chromatophore fraction approximately 3 h after the start of the adaptation. The enzyme, S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase, also increased in amount in the developing chromatophore fraction but was present in a cell membrane fraction at the start of the adaptation as well.

Association of protochlorophyllide with the PufQ protein of Rhodobacter capsulatus.

The PufQ protein, prepared by the recombinant expression of the pufQ gene of Rhodobacter capsulatus, has been reconstituted into liposomes in the presence of the bacteriochlorophyll precursor, protochlorophyllide. The liposomes were separated from liposomes free of the PufQ protein by sucrose density gradient ultracentrifugation and analyzed for their content of protochlorophyllide, protein, and phospholipid. The results indicated a 3.5 times higher level of association of protochlorophyllide with the PufQ protein-containing liposomes than with liposomes containing either the hydrophobic protein, apolipoprotein A-I, or the water-soluble maltose-binding protein. Only the association of the protochlorophyllide with the PufQ protein corresponded to a small but reproducible shift in its fluorescence emission maximum to a lower wavelength, consistent with a change in its environment.

Recombinant expression of the pufQ gene of Rhodobacter capsulatus.

Genetic studies have shown that the expression of the pufQ gene is required for normal levels of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus. Yet, the exact function of the pufQ gene is unknown, and a pufQ gene product has never been isolated. We describe the recombinant overexpression of pufQ in Escherichia coli, as well as the purification and characterization of its gene product, the 74-amino-acid PufQ protein. Site-directed mutagenesis was used to facilitate the cloning of the pufQ gene into various expression vector systems of E. coli, including pKK223-3, pLcII-FX, and pMal-c. Although high levels of pufQ transcription were evident from constructs of all three vectors, high levels of protein expression were apparent only in the pMal-c system. In vector pMal-c, the recombinant PufQ protein is expressed as a fusion with an amino-terminal maltose-binding domain. After affinity purification on an amylose column, full-length PufQ protein was released from the fusion protein by limited proteolysis with the enzyme factor Xa. The PufQ protein demonstrated a strong tendency to associate with phospholipid vesicles, consistent with the view that it is an integral membrane protein. The PufQ protein was subsequently purified by high-performance liquid chromatography and identified by amino-terminal sequence analysis. A possible role for the PufQ protein in the transport of bacteriochlorophyll biosynthetic intermediates is discussed.

Genograms: a psychosocial assessment tool for hospice.

Genograms are a valuable and non-threatening evaluation tool for hospice patients and families. The genogram provides basic information about the family including the role of each member and the family dynamics. As the diagram is drawn, family life cycle issues and relationships between family members become evident. The genogram may go beyond the household to include supportive neighbors, friends, and community resources. Religious and spiritual support is also noted. The information is used to assess family needs and to provide interventions both before the death and during bereavement.

Use of the membrane-impermeable guanidinating reagent 2-S-[14C]thiuroniumethanesulfonate to demonstrate the orientation of light-harvesting proteins in Rhodobacter sphaeroides.

The radiolabeled guanidinating reagent 2-S-[14C]thiuroniumethanesulfonate reacts with the epsilon-amino groups of accessible lysyl residues of membrane proteins under relatively mild labeling conditions, yielding labeled homoarginyl residues. Model studies have shown that the resulting homoarginyl residues do act as new cleavage sites for trypsin, but only at a very slow rate of hydrolysis. The reagent has been shown to be impermeable to the intracytoplasmic membranes of Rhodobacter sphaeroides: when cytoplasmic-side-out chromatophores were treated with the reagent, it reacted with all four of the light-harvesting proteins, all of which have one or more lysyl residues on the N-terminal sides of their hydrophobic regions. However, when periplasmic-side-out vesicles, prepared by cytochrome c affinity chromatography, were treated with the guanidinating reagent, three of the light-harvesting proteins (B850 alpha, B850 beta, and B870 beta) were not labeled. The only light-harvesting protein to be labeled (B870 alpha) was the only one of the four to have a lysyl residue on the C-terminal side of its hydrophobic region. Guanidinated B870 alpha polypeptides from both the cytoplasmic-side-out chromatophores and the periplasmic-side-out membrane vesicles were purified and digested with trypsin. The resulting peptide fragments were then separated by high-performance liquid chromatography and analyzed for radioactivity. The results have confirmed the asymmetric orientation of the light-harvesting proteins of R. sphaeroides, with their N-termini on the cytoplasmic side of the intracytoplasmic membrane. In the case of the B870 alpha subunit, the protein has been shown to be transmembrane with its C-terminus on the periplasmic side of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Confirmation of a ping-pong mechanism for S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase of etiolated wheat by an exchange reaction.

An exchange reaction between unlabeled S-adenosyl-L-methionine and radiolabeled S-adenosyl-L-homocysteine has been used to confirm the occurrence of a ping-pong mechanism in S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase of etiolated wheat. The enzyme, S-adenosyl-L-homocysteine hydrolase, has been used to prepare radiolabeled S-adenosyl-L-homocysteine from labeled adenosine and DL-homocysteine. The exchange reaction was accomplished with a methyltransferase preparation purified by affinity chromatography on hemin-linked Sepharose 4B, and radioactivity was exchanged into unlabeled S-adenosyl-L-methionine to an extent of 70% of the theoretical maximum value.

Synthesis of 4R- and 4S-tritium labeled NADPH for the determination of the coenzyme stereospecificity of NADPH: protochlorophyllide oxidoreductase.

A rapid and easy method for the production of both the 4R and 4S tritium labeled isomers of either NADH or NADPH has been developed. The method requires the use of only a single labeled compound (D-[1(-3)H] glucose), and two enzymes (glucose dehydrogenase from Bacillus sp. and alcohol dehydrogenase from Thermoanaerobium brockii) which are specific for the pro S and pro R hydrogens, respectively, of either NADH or NADPH. The 4R and 4S tritium labeled isomers of NADPH have been used to determine that NADPH:protochlorophyllide oxidoreductase from etiolated wheat was specific for the pro S hydrogen of NADPH.

Localization of photosynthetic membrane components in Rhodopseudomonas sphaeroides by a radioactive labeling procedure.

Reduction with [3H]KBH4 of Schiff's bases generated by reaction with pyridoxal 5'-phosphate (which cannot penetrate the intact cytoplasmic membrane) yields tritium-labeled derivatives of both proteins and lipids accessible on the periplasmic side of the cytoplasmic membrane. Application of this technique to phototrophically grown Rhodopseudomonas sphaeroides labeled both the cell envelope and chromatophore fractions. The technique was also applied to R. sphaeroides harvested at various times during an adaptation from heterotrophic to phototrophic growth conditions. The specific activity of the chromatophore fraction after 20 h of adaptation was 76% of that found at the beginning, indicating that the intracytoplasmic membranes and cytoplasmic membrane form a continuous membrane system, with the majority of the intracytoplasmic membranes accessible to the external medium throughout the adaptation. The identity of the proteins labeled by this technique was investigated in two fractions labeled after cell disruption: normal "inside-out" chromatophores and "right-side-out" membrane vesicles isolated by lysozyme--osmotic shock treatment of cells grown in high light intensity (15000 lx). The results after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and fluorography indicated that the 28000-dalton subunit (and to a lesser extent the 21000-dalton subunit) of the reaction center complex and two polypeptides in the light-harvesting region of the gel were heavily labeled in the chromatophores and were thus accessible on the cytoplasmic side of the membrane. At least one of the latter two polypeptides was also labeled in the membrane vesicles and was thus also accessible on the periplasmic side of the membrane. None of the reaction center subunits was significantly labeled in a reaction center complex prepared from the membrane vesicle sample.

The nature of a pgiment-protein complex excreted from mutants of Rhodopseudomonas sphaeroides.

Pigment-protein complexes excreted from three bacteriochlorophyll-less mutants (mutants 8, 8-29, and 8-47) of Rhodopseudomonas sphaeroides have been isolated and purified. In the absence of detergents the complexes remained in an aggregated state, but were disaggregated by 0.2% Triron X-100. Sepharose 6B gel filtration indicated that the disaggregated complex from each of the mutants had a particle weight of about 165000, and contained 30 +/- 3% protein. This complex was further dissociated by 1% sodium dodecyl sulfate. Sephadex G-100 gel filtration now indicated that the majority of the protein was present as a small polypeptide with a molecular weight of about 9000. The pigment-protein complex from one of the mutants was treated with a bacteriochlorophyll extract. The bacteriochlorophyll was converted to bacteriopheophytin and became bound to the protein, replacing the endogenous tetrapyrrole (a bacteriocholorophyll precursor). The red absorption maximum of the bacteriopheophytin was shifted during this process to 840-865 nm. These properties are consistent with the possibility that the pigment-protein complexes contain a protein normally associated with light-harvesting bacteriochlorophyll in the wide-type strain.

A possible alternate pathway of bacteriochlorophyll biosynthesis in a mutant of Rhodopseudomonas sphaeroides.

A previously uncharacterized bacteriochlorophyll-less mutant (mutant 8) of Rhodopseudomonas sphaeroides has been found to excrete a tetrapyrrole-protein complex into the incubation medium. The structure of the major pigment of the complex was characterized as 2-desacetyl-2-vinylbacteriopheophorbide. The corresponding magnesium derivative does not fit into the currently proposed biosynthetic pathway for bacteriochlorophyll, and thus may indicate the existence of an alternate pathway of bacteriochlorophyll synthesis in this organism. Such an alternate pathway would be possible if reduction from the chlorin to the tetrahydroporphyrin stage can occur either before or after hydration of the 2-vinyl substituent of chlorophyllide a to an alpha-hydroxyethyl group.