Biomedical subjects

L H Pratt

Publications and source records attributed to L H Pratt.

At least 37 records · Page 2Linked to original sources

PubMed1988

Light-mediated changes in two proteins found associated with plasma membrane fractions from pea stem sections.

Irradiation of etiolated pea (Pisum sativum L.) seedlings with white light affects two proteins, both of monomer molecular mass near 120 kDa. Both proteins have been detected in association with plasma membrane fractions. The first is identifiable in that it becomes heavily phosphorylated when the membranes are incubated with exogenous ATP. The second of these proteins is phytochrome, as determined by electrophoretic transfer (Western) blot analysis. Measurable phosphorylation and phytochrome (the latter detected by antigenicity) decline when the tissue is irradiated with white light prior to membrane isolation and in vitro phosphorylation. The phosphorylated protein is probably not phytochrome for three reasons. (i) It shows a slightly different distribution in sucrose gradients. (ii) Red light causes a gradual decline in the phytochrome that is associated with membrane fractions but has a negligible effect on the phosphorylatable protein; blue light, on the other hand, causes significantly slower loss of phytochrome than does red light but brings about a rapid decline in the phosphorylation signal. (iii) The molecular masses are not identical. The association of both proteins with membrane fractions is probably neither ionic nor, at least for the phosphorylatable protein, the consequence of entrapment of soluble proteins in vesicles formed during tissue extraction. Phytochrome is lost from the membrane fractions during irradiation, as judged by loss of antigenicity. Whether the phosphorylatable protein is lost, a specific kinase is lost, phosphatase activity increases, or phosphorylatable sites are blocked as a consequence of blue light treatment is not known.

Journal Article↗

PubMed1987

A photoreversible circular dichroism spectral change in oat phytochrome is suppressed by a monoclonal antibody that binds near its N-terminus and by chromophore modification.

Accompanying the phototransformation of native 124-kilodalton (kDa) oat phytochrome from red-absorbing form (Pr) to far-red-absorbing form (Pfr), there is a photoreversible change in circular dichroism (CD) in the far-UV region indicative of a 3% increase in alpha-helical folding of apoprotein. To elucidate the conformational change involved in the phytochrome phototransformation, several monoclonal antibodies have been used as epitope-specific probes. Monoclonal antibody oat-25 suppressed the photoreversible CD spectral change using phytochrome with an A666/A280 as Pr of 1.13. Monoclonal antibodies oat-22, oat-13, and oat-31 did not significantly affect the CD spectral change of phytochrome. Oat-25 requires an epitope near the N-terminus of phytochrome. Oat-22, oat-13, and oat-31 recognize epitopes on the N-terminus, chromophore-containing half of phytochrome, albeit further removed from the N-terminus than that recognized by oat-25. Interestingly, oat-13 and oat-31 did, however, induce a time-dependent decrease in the far-UV CD, apparently due to aggregation of phytochrome (both Pr and Pfr forms). Monoclonal antibodies oat-26 and oat-28, which recognize epitopes on the C-terminus half of phytochrome, also did not suppress the photoreversible CD change, although oat-26 and oat-28 slightly inhibited it. The photoreversible CD spectral change can also be inhibited by sodium borohydride, which bleaches the chromophore by reducing it, and by tetranitromethane, which oxidizes the chromophore of phytochrome. Although explanations of these results based on indirect interactions between the chromophore and the N-terminus segment are possible, we propose that an additional alpha-helical folding of the Pfr form of the phytochrome may result from a photoreversible interaction between the Pfr form of the chromophore and the N-terminus segment.

Antibodies, Monoclonal↗

PubMed1986

Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability.

Using monoclonal antibodies to the plant photoreceptor, phytochrome, we have investigated by immunogold electron microscopy the rapid, red light-induced, intracellular redistribution (termed "sequestering") of phytochrome in dark-grown Avena coleoptiles. Pre-embedding immunolabeling of 5-micron-thick cryosections reveals that sequestered phytochrome is associated with numerous, discrete structures of similar morphology. Specific labeling of these structures was also achieved by post-embedding ("on-grid") immunostaining of LR-White-embedded tissue, regardless of whether the tissue had been fixed chemically or by freeze substitution. The phytochrome-associated structures are globular to oval in shape, 200-400 nm in size, and are composed of amorphous, granular material. No morphologically identifiable membranes are present either surrounding or within these structures, which are often present as apparent aggregates that approach several micrometers in size. An immunogold labeling procedure has also been developed to identify the particulate, subcellular component with which phytochrome is associated in vitro as a consequence of irradiation of Avena coleoptiles before their homogenization. Structures with appearance similar to those identified in situ are the only components of the pelletable material that are specifically labeled with gold. We conclude that the association of phytochrome with these structures in Avena represents the underlying molecular event that ultimately is expressed both as red light-induced sequestering in vivo and enhanced pelletability of phytochrome detected in vitro.

Antibodies↗

PubMed1986

Identification of a highly conserved domain on phytochrome from angiosperms to algae.

A monoclonal antibody (Pea-25) directed to phytochrome from etiolated peas (Pisum sativum L., cv Alaska) binds to an antigenic domain that has been highly conserved throughout evolution. Antigenic cross-reactivity was evaluated by immunoblotting sodium dodecyl sulfate sample buffer extracts prepared from lyophilized tissue samples or freshly harvested algae. Pea-25 immunostained an approximately 120-kilodalton polypeptide from a variety of etiolated and green plant tissues, including both monocotyledons and dicotyledons. Moreover, Pea-25 immunostained a similarly sized polypeptide from the moss Physcomitrella, and from the algae Mougeotia, Mesotaenium, and Chlamydomonas. Because Pea-25 is directed to phytochrome, and because it stains a polypeptide about the size of oat phytochrome, it is likely that Pea-25 is detecting phytochrome in each case. The conserved domain that is recognized by Pea-25 is on the nonchromophore bearing, carboxyl half of phytochrome from etiolated oats. Identification of this highly conserved antigenic domain creates the potential to expand investigations of phytochrome at a cellular and molecular level to organisms, such as Chlamydomonas, that offer unique experimental advantages.

Journal Article↗

PubMed1986

Identification with Monoclonal Antibodies of a Second Antigenic Domain on Avena Phytochrome that Changes upon Its Photoconversion.

An enzyme-linked immunosorbent assay that revealed an antigenic difference between the red-absorbing and far-red-absorbing forms of phytochrome (Pr and Pfr, respectively) near its amino terminus (Cordonnier M-M, H Greppin, LH Pratt 1985 Biochemistry 24: 3246-3253) was used to screen eight additional monoclonal antibodies directed to phytochrome from etiolated oats. While six of these antibodies detected Pr and Pfr with equal affinity, two of them, designated Oat-9 and Oat-16, bound to Pfr 1.6 to 2.3 times better than to Pr. Competitive enzyme-linked immunosorbent assays indicate (a) that Oat-9 and Oat-16 probably bind to the same domain on phytochrome and (b) that this domain is at least 3.5 nanometers away from the epitope near its amino terminus that was shown earlier to change upon phototransformation. Neither the absorbance spectra of Pr and Pfr, nor the rate of dark reversion of Pfr to Pr, was influenced by the presence of Oat-9. Immunoblotting of sodium dodecyl sulfate polyacrylamide gels after electrophoretic separation of phytochrome fragments obtained by endogenous proteolytic digestion indicates that Oat-16 binds to an epitope located on the chromophore half of this chromoprotein. The observation that the epitope recognized by Oat-9 and Oat-16 is also present on at least some of the immunochemically distinct phytochrome that is obtained from green oat shoots (Shimazaki Y, LH Pratt 1985 Planta 164: 333-344), together with the evidence that this epitope undergoes a change upon photoransformation, indicates that it may play an important role in phytochrome function.

Journal Article↗

PubMed1984

Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to pisum and Avena phytochrome.

Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome-red-absorbing form and phytochrome-far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action.

Journal Article↗

PubMed1982

Immunopurification and initial characterization of dicotyledonous phytochrome.

Antiserum was prepared against proteolytically undegraded phytochrome obtained from etiolated zucchini squash (Cucurbita pepo L., cv. Black Beauty). The antiserum was prepared by injecting into a rabbit immunoprecipitates between zucchini phytochrome and specific antiserum against undegraded oat (Avena sativa L., cv. Garry) phytochrome. Specific antiphytochrome immunoglobulins were purified from this crude serum by an affinity column consisting of conventionally purified undegraded pea phytochrome covalently linked to cyanogen bromide-activated agarose. These purified immunoglobulins were also linked to cyanogen bromide-activated agarose and were used to immunopurify zucchini, pea (Pisum sativum L., cv. Alaska), and lettuce (Lactuca sativa L., cv. Grand Rapids) phytochrome. All three dicotyledonous phytochromes exhibited a monomer size near 120,000 daltons by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. Absorbance spectra of immunopurified zucchini phytochrome indicated that the ratio of visible to ultraviolet absorbance for purified zucchini phytochrome is lower than that observed for oat phytochrome. The isoelectric point of zucchini phytochrome, which was observed to be heterogeneous by this criterion, was found to be in the range of 6.5 to 7.0, higher than that observed for oat phytochrome. The electrophoretic mobility of zucchini phytochrome was found to be similar to that observed for oat and pea phytochrome under conditions that were nondenaturing and did not involve any molecular sieving effect. The amino acid analysis of zucchini phytochrome is similar to that reported previously for oat and rye (Secale cereale L., cv. Balbo) phytochrome.

Journal Article↗

PubMed1982

Localization of phytochrome in oats by electron microscopy.

Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction.

Journal Article↗

PubMed1982

Comparative Phytochrome Immunochemistry as Assayed by Antisera against Both Monocotyledonous and Dicotyledonous Phytochrome.

Preparation and characterization of antisera against lettuce (Lactuca sativa L., cv. Grand Rapids) and pea (Pisum sativum L., cv. Alaska) phytochrome is described. These antisera, together with previously obtained antisera against zucchini (Cucurbita pepo L., cv. Black Beauty) and oat (Avena sativa L., cv. Garry) phytochrome, were used to compare by Ouchterlony double immunodiffusion phytochrome isolated from etiolated lettuce, pea, bean (Phaseolus vulgaris L., cv. Taylor Horticultural Bush), zucchini, oat and rye (Secale cereale L., cv. Balbo) seedlings. Cross reactivity between monocotyledonous phytochrome and antidicotyledonous-phytochrome serum and between dicotyledonous phytochrome and antimonocotyledonous-phytochrome serum was always weak or not perceptible by this assay. Among the four dicotyledonous phytochromes examined, pea and bean were the most similar immunochemically as anticipated. Pea and lettuce phytochrome somewhat unexpectedly also exhibited similar immunochemical reactivity. Zucchini phytochrome by contrast was immunochemically distinct from pea, bean, and lettuce phytochrome, although it did react with all three antidicotyledonous-phytochrome sera. Initial attempts to identify immunoglobulins that would recognize phytochrome regardless of its source indicated that they may exist. Such immunoglobulins are of interest because they might react with one or more determinants that could be part of an active site of phytochrome. These immunoglobulins, once isolated, could thus serve as a potential probe for the active site of phytochrome.

Journal Article↗

PubMed1982

Localization of phytochrome in oats by electron microscopy.

Journal Article↗

PubMed1981

Physicochemical differences between the red- and the far-red-absorbing forms of phytochrome.

We investigated some of the chemical and physical differences between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of immunoaffinity-purified, undegraded oat (Avena sativa L., ev. Garry) phytochrome. Both Pr and Pfr had identical surface charges as measured by isoelectric focusing and identical secondary structure as judged from their circular dichroic spectra. Modification of specific amino acid residues, however, revealed some chemical differences between Pr and Pfr. Compared to Pr, Pfr had one more His and Cys residue per monomer modified immediately. His residues on Pfr were modified more rapidly than were those on Pr, as opposed to Cys modification which, after the initial burst, occurred more rapidly on Pr than on Pfr. Both His- and Cys-modified phytochromes were fully photoreversible. Both Pr and Pfr had the same number of immediately modified carboxyl functions, but those on Pr reacted slightly more rapidly than those on Pfr. Carboxyl-modified phytochrome was denatured by the acid pH used for modification so that its photoreversibility could not be measured. Modification of Tyr on Pr and Pfr resulted in two residues per monomer being modified very rapidly, with those on Pfr reacting even faster than those on Pr. Subsequent slower modification of remaining Tyr residues, however, occurred more rapidly on Pr than on Pfr. Phytochrome photoreversibility declined as a linear function of the number of Tyr modified and was lost completely when two Tyr per monomer had been modified. These data are consistent with the hypothesis of a small conformational change in phytochrome upon photoconversion and also fit a conformation-stabilization mechanism in which photo-conversion from Pr to Pfr stabilizes phytochrome in one particular conformation.

Chemical Phenomena↗

PubMed1981

Characterization of a molecular modification of phytochrome that is associated with its conversion to the far-red-absorbing form.

Phytochrome that has been photoinduced to pellet by irradiation of intact oat (cv. Garry) shoots and recovered from a pellet obtained by centrifugation of crude extracts exhibits modified behavior when compared to soluble phytochrome isolated from shoots that had never been irradiated. This modified behavior includes retarded mobility during sodium dodecyl sulfate polyacrylamide gel electrophoresis (Boeshore ML, LH Pratt 1980 Plant Physiol 66: 500-504). The electrophoretic mobility of several different kinds of phytochrome preparations were examined to study how this modification might arise.Phytochrome that was extracted in the pelletable condition from red-, far-red-irradiated tissue, but without added divalent cation so that it did not pellet, did not exhibit an altered electrophoretic mobility. Hence, this modification of phytochrome is not required for the expression in vitro of pelletability induced in vivo. Phytochrome that was extracted in the pelletable condition and in the far-red-absorbing form, but without added divalent cation so that it did not pellet, and phytochrome in the far-red-absorbing form that remained in the supernatant after collection of pellets containing pelleted phytochrome both electrophoresed with reduced mobility. Thus, this modification does not arise as a consequence of phytochrome having been pelleted. Differential sensitivity of phytochrome to different handling conditions also is not the cause of this modification since the far-red-absorbing form of phytochrome, which was extracted in the pelletable condition but by the same protocol used to extract soluble phytochrome, also exhibited reduced mobility. Furthermore, the reduced electrophoretic rate is not due to a simple differential lability of the far-red-absorbing form of phytochrome to extraction conditions, since partially purified soluble phytochrome that was exposed in the far-red-absorbing form to the isolation and extraction conditions used for preparation of soluble phytochrome did not exhibit the alteration.The data are instead consistent with the more complex interpretation that phytochrome is modified in vitro if two conditions are met: (a) that phytochrome is extracted in the far-red-absorbing form or is converted to the far-red-absorbing form in the crude extract soon after extraction and (b), that phytochrome remains in the far-red-absorbing form in the crude extract for at least a brief period.The possibility that the phytochrome modification studied here might have arisen because of a change in carbohydrate content was tested by periodic acid Schiff staining of sodium dodecyl sulfate polyacrylamide gels. No carbohydrate was detected in any of the phytochrome preparations that were examined. This inability to detect carbohydrate is in direct contrast to the report of Roux et al. (1975 Physiol Plant 35: 85-90).

Journal Article↗

PubMed1980

Partial characterization of undegraded oat phytochrome.

We characterized immunoaffinity-purified, undegraded oat (Avena sativa L., cv. Garry) phytochrome by several physicochemical techniques. Phytochrome, of greater than 98% purity [Hunt, R. E., & Pratt, L. H. (1979) Plant Physiol. 64, 332-336], existed in solution as a dimer of its 118 000-dalton monomers and had a full complement of the typical amino acids with about 35% nonpolar residues, 115 carboxylic acid groups per monomer, and an average of one phosphate per monomer. Although the dimer was not held together by disulfide bridges, each monomer contained three disulfide bonds and 14 reduced cysteines out of a total of 27 cysteine-half-cystine residues. Phytochrome preparations, although very pure, exhibited heterogeneity by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which revealed three closely spaced bands, and by nondenaturing gel electrophoresis at pH 7.0, which revealed four bands. Amino-terminal analysis indicated two residues, Lys and Ala. Manual Edman degradation yielded Leu and Ala after one round and Val and Leu after a second round. These data indicate a possible amino-terminal sequence of NH3-Lys-Ala-Leu-Val- with some monomers not having Lys.

Amino Acids↗

PubMed1980

Phytochrome Modification and Light-enhanced, In Vivo-induced Phytochrome Pelletability.

Phytochrome that was induced by red irradiation in vivo to pellet with subcellular material and that was released from the pellet by removal of divalent cations exhibited altered characteristics. Compared to phytochrome extracted in a soluble red-absorbing form from etiolated tissue, pelleted and released phytochrome, which was also assayed in the red-absorbing form even though pelleted in the far-red-absorbing form, showed 50% greater micro complement fixation activity, eluted closer to the void volume of a Sephadex G-200 column, and electrophoresed more slowly on sodium dodecyl sulfate-polyacrylamide gels. Data presented here document that phytochrome pelleted in the far-red-absorbing form differs from soluble phytochrome extracted from nonirradiated tissue. These data, however, do not permit the conclusion that there is a causal relationship between pelletability and phytochrome modification.

Journal Article↗

PubMed1980

Phytochrome Pelletability Induced by Irradiation in Vivo: TEST FOR IN VITRO BINDING OF ADDED [S]PHYTOCHROME.

Undegraded, highly purified [(35)S]phytochrome was immunoaffinity-purified either from dark control oat (cv. Garry) shoots or from etiolated oat shoots that were previously irradiated first with red and then with far-red light so that, if proper extraction conditions had been utilized, about 60% of the total phytochrome would have been pelletable. When [(35)S]phytochrome was added to extraction buffer immediately prior to homogenization of etiolated oat shoots, pelletability assays indicated that there was no preferential binding of [(35)S]phytochrome regardless of (a) whether it was purified from dark control or irradiated shoots, (b) whether it was added as phytochrome-red-absorbing form or phytochrome-far-red-absorbing form, or (c) whether it was added to dark control or red-irradiated shoots. Similarly, binding of [(35)S]phytochrome to resuspended pellets obtained from crude oat extracts was not specific for the source of [(35)S]phytochrome, for its form, or for the irradiation treatment given to intact shoots used to prepare the resuspended pellets. No evidence was obtained to support the hypothesis that phytochrome binds with specificity to particulate material in vitro under conditions used to assay for light-enhanced, in vivo-induced phytochrome pelletability.

Journal Article↗

PubMed1979

Characterization of the Destruction of Phytochrome in the Red-absorbing Form.

Both the red-absorbing (Pr) and far red-absorbing (Pfr) forms of phytochrome undergo destruction, defined as the loss of photoreversibly detectable chromoprotein following actinic irradiation of dark-grown tissue, in 4-day-old etiolated oat seedlings. Pr and Pfr destruction follow the same time course, exhibit the same time delay after actinic irradiation when the plants are grown in sealed containers, result in a loss of antigenically detectable phytochrome, as determined by radial immunodiffusion assay, equal to the loss of spectrophotometrically detectable phytochrome, and have the same sensitivity to 2-mercaptoethanol and azide. We suggest that Pr destruction is a consequence of the same mechanism that is responsible for Pfr destruction.

Journal Article↗

PubMed1979

Phytochrome radioimmunoassay.

A phytochrome radioimmunoassay with a detection limit of about 2 nanograms has been developed. The radioimmunoassay does not suffer from the potential drawbacks of the commonly used spectral assay and requires less than 1 microliter of crude extract from dark-grown plants for quantitation of phytochrome. Measurement of phytochrome in crude extracts by radioimmunoassay gives values about 25% greater than those obtained by spectral assay. The amount of phytochrome detected in crude extracts of light-grown oats by radioimmunoassay is approximately 1% of that detected in comparable extracts from dark-grown oats. General interference by crude plant extracts with radioimmunoassays was also observed and corrected for.

Journal Article↗

PubMed1979

Phytochrome immunoaffinity purification.

We have developed a phytochrome immunoaffinity purification procedure that yields undegraded oat (Avena sativa L., cv. Garry) phytochrome of greater than 98% purity within 2 hours when starting with a brushite-purified preparation. Immunoaffinity-purified phytochrome, except for its greater purity, is indistinguishable from conventionally purified phytochrome by gel exclusion chromatography, isoelectric focusing, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have also used the immunoaffinity technique to purify phytochrome from crude oat extracts, and from brushite-purified pea (Pisum sativum L., cv. Alaska) and rye (Secale cereale L., cv. Balbo) preparations.

Journal Article↗