Separation of a blue fluorescence protein from bacterial luciferase

Luciferase preparations from two species of marine bioluminescent bacteria, $\text{Photobacterium phosphoreum}$ and $\text{Photobacterium fischeri}$, are shown to contain a low molecular weight protein, containing a blue fluorescence chromophore having an emission maximum in the 470 nm region. A procedure for separating the luciferase and purifying this protein is described. On disc gel electrophoresis the bulk of the protein is observed to migrate along with the blue fluorescence.     

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO : Relationship to Large Subunit-Binding Protein Complex Dissociation

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.     

Structural Aspects of Photosystem I from Dunaliella salina

A native PSI complex and a PSI core complex have been isolated from the halophilic green alga, Dunaliella salina. The composition and properties of these complexes are similar to previously described PSI complexes from spinach membranes. By growth on ¹⁴C-NaHCO₃, it has been possible to isolate uniformly labeled ¹⁴C-PSI complexes in order to determine PSI subunit stoichiometry. This analysis has shown a ratio of one copy of three low molecular weight subunits (22,000; 15,000; 8,000) per two copies of high molecular weight subunits (84,000). Using a ¹⁴C-labeled cytochrome b₆-f complex as an internal protein standard, it has been possible to estimate the molecular weight of a PSI core complex as about 330,000. This complex contains one P700, two 84,000 subunits, and one subunit of 22,000, 15,000, and 8,000.     

New artificial electron donors for in vitro assay of nitrate reductase isolated from cultured tobacco cells and other organisms

The capacity of bromphenol blue and its analogs to act as electron donors for measurement of in vitro nitrate reductase activity from tobacco cells (Nicotiana tabacum var Techné SP 25 strain) was determined. Competitive inhibition was demonstrated to occur between NADH, the natural electron donor, and bromphenol blue, the artificial electron donor, suggesting that both donors bind to a similar active site on the enzyme. NADH-dependent or bromphenol blue-dependent nitrate reductase activity was carried out by a similar molecular weight protein exhibiting similar antigenic sites. Following ammonium sulfate precipitation, sucrose density gradient and two chromatographic steps, nitrate reductase activity from tobacco cells was purified near homogeneity using bromphenol blue as an electron donor in the absence of measurable NADH-dependent activity. The enzyme is composed of two identical subunits of 83 kilodaltons < ω < 94 kilodaltons.     

Evolution of glutathione S-transferase subunits in culicidae and related nematocera: Electrophoretic and immunological evidence for conserved enzyme structure and expression

The antigenic relatedness and molecular weights of glutathione S-transferase (GST) protein subunits expressed by representative members of the Dipteran suborder Nematocera were analyzed by immunoblotting and affinity chromatography. Ten species within the genus Aedes expressed two subunits which cross-reacted with an antiserum developed against the GST-1b isozyme purified from Aedes aegypti. One of these two subunits showed no discernible molecular weight variability within the ten Aedes species. Its molecular weight (25,900 Da) was identical to the GST-1a subunit found in A. aegypti. The molecular weight of the other subunits varied among the ten species, but in all cases was larger than 25,900 Da. In ten species within the genus Anopheles (considered to be among the more primitive genera in the Culicidae), only one immunologically related GST subunit was expressed. Its molecular weight was also identical to the GST-1a subunit found in the Aedes species. Members of genera considered phylogenetically intermediate between Anopheles and Aedes had either one or two cross-reacting subunits. Representative species of families closely related to the Culicidae (the Chaoboridae, Chironomidae, Simuliidae, and Cecidomyiidae) also expressed immunologically related GST subunits. GST isozymes were partially purified from Anopheles freeborni and A aegypti adults, larvae, and ovaries using S-hexylglutathione affinity chromatography. Adult and larval Anopheles freeborni expressed not only the immunologically related subunit, but an additional, immunologically unrelated subunit. In fully developed ovaries of both species, however, only subunits immunologically related to GST-1b were expressed. These results suggest that there is an immunologically conserved domain shared among specific GST isozymes within the Nemotocea. The conserved nature of these GST subunits, their correlation with oocyte development, and their localization within ovary tissue, suggests that they share a conserved in vivo function during oocyte maturation. In addition, the conserved antibody binding domain within these subunits apparently has been duplicated in several species of Culicidae.     

Purification and characterization of a blue-colored red-fluorescent protein from the silkworm (Bombyx mori L.) larvae

• 1.1. A red-fluorescent blue protein (P600) was purified from the digestive juice of the silkworm (Bombyx mori L.) larvae raised on mulberry leaves.
• 2.2. The purified protein was electrophoretically homogeneous and showed the absorption maxima at 601.5 nm and 278 nm, and the fluorescence maximum at 621 nm.
• 3.3. The molecular weight was estimated to be 540,000 by gel filtration on Sepharose CL-6B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggests that the protein consists of two heterogeneous polypeptide subunits with a mol. wt of 15,000 and 18,000.
• 4.4. The P600 contains excess acidic amino acid residues over basic groups. The polarity and pI were 45.5% and 4.6, respectively.
• 5.5. The production of H₂O₂ was observed in the presence of P600 upon illumination.

     

Tissue distribution and characterization of predominant hemolymph carrier proteins from Dermacentor variabilis and Ornithodoros parkeri

The tissue distribution of the predominant hemolymph protein found throughout tick development was examined in the hard tick, Dermacentor variabilis, and in the soft tick, Ornithodoros parkeri. In D. variabilis, the predominant (purified) hemolymph protein was a lipoglycoheme-carrier protein (DvCP) with a molecular weight of 200 K. A protein with a similar mobility on native-PAGE was found in fat body, salivary gland, muscle and ovary from partially fed females which was most abundant in the plasma and salivary gland. DvCP from plasma, salivary gland and fat body of partially fed females consisted of two subunits on SDS-PAGE (98 and 92 K). In replete females, only salivary gland exhibited protein subunits equivalent to hemolymph CP. CP in salivary gland and fat body stained positive for lipids. The concentration of CP in tissues varied between partially fed and replete females, indicating a difference in the expression and/or sequestration of CP during adult development. The predominant hemolymph carrier protein from O. parkeri (OpCP) was purified to homogeneity for the first time and is presumed to have similar functions to CP from D. variabilis. Purified OpCP exhibited a molecular weight of 668 K by native-PAGE. Unlike CP from D. variabilis, OpCP was not detected in fat body or salivary gland tissues but occurred abundantly in coxal fluid. By SDS-PAGE, purified hemolymph OpCP consisted of two major subunits (114 and 93 K) and a less abundant protein with an apparent molecular weight of 48 K. Purified native OpCP was a lipoprotein like DvCP. A spectral analysis of purified OpCP failed to demonstrate the presence of heme like that found for CP from D. variabilis, purified by the same methods. However, plasma from O. parkeri contained heme with a λ_max of 410 nm.     

Myosin : Subunits and their interactions

There is fairly general agreement that myosin isolated from rabbit skeletal muscle has a molecular weight of about 500,000. The higher values that have been reported apparently reflect protein aggregation related to the method of preparation. On the basis of present evidence, the myosin molecule has an elongate helical core of two f subunits (average weight about 215,000) that extend into a globular head region containing three g subunits (average weight about 20,000). Myosin may be dissociated into subunits by a number of methods. In 5 M guanidine, the myosin molecule is dissociated into f and g subunits, while at pH above 10, the g subunits are dissociated from the intact fibrous core of myosin. The dissociation of g subunits at pH 10 is accompanied by the loss of both ATPase activity and actin-binding capacity; however, the exact biological significance of the g subunits is presently uncertain. In preliminary studies, the f subunits appear to contain the sulfhydryl residues currently implicated in myosin ATPase, and there is some indication of allosteric regulation of enzymic activity.     

Molecular and spectroscopic characterisation of a low molecular weight seed storage protein from yellow mustard (Sinapis alba L.)

• 1.1. A 1.7S protein has been purified from mustard seeds (Sinapis alba L.). This protein, soluble in water and dilute salt solutions, is considered as an albumin and constitutes about 10% of the total soluble protein in mustard seeds.
• 2.2. Its molecular weight is approximately 15,000 and is composed of two polypeptide chains (M_r = 9500 and 5000), linked by two disulfide bridges.
• 3.3. The amino acid compositions of both subunits as well as of the native protein are reported, showing a strong homology with napins from Brassica napus L.
• 4.4. The ultraviolet absorption, fluorescence emission and circular dichroism spectra of the purified protein have been obtained. The mustard protein exhibits about 50% α-helix with a very low β-structure content. Based on its structural characteristics, a zein-like packing is proposed for this protein from mustard seeds.

     

Active center and subunit structure of transaldolase from Candida utilis.

Crystalline transaldolase (type III) isolated from Candida utilis is composed of two identical subunits, as shown by the following lines of evidence. 1. Tryptic digestion of the performic acid oxidized enzyme yields the number of ninhydrin- and arginine-positive peptides expected for identical subunits. 2. All attempts to separate both subunits by molecular weight or charge differences have failed. 3. Cyanogen bromide cleavage and sodium dodecyl sulfate gel electrophoresis of S-carboxymethylated transaldolase revealed four distinct peptides designated C₂ to C₅ according to their decreasing molecular weight and one additional peak, C₁, in low yield, presumably an aggregate or partially degraded peptide.By chromatography on Sephadex G-100 the maleylated cyanogen bromide digest from ¹⁴C-labeled β-giyceryl-transaldolase could be separated into four peptide peaks which have been analyzed for their amino acid composition. The largest peptide C₂ with a molecular weight of 16,800 was identified as the active site containing fragment. The four fragments together account for all amino acid residues in the entire protein.From transaldolase (type I) containing four methionine residues three cyanogen bromide peptides could be identified. By addition of the individual peptides a molecular weight of 37,100 ± 3500 could be calculated, which is half the molecular weight of the native enzyme. From experimental data presented so far both isoenzymes of transaldolase can be regarded as “half-of-the-sites” enzymes.     

Ribulose Diphosphate Carboxylase from Autotrophic Euglena gracilis

Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s_{20, w} at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.     

Isolation and characterization of pigment-protein particles from the light-harvesting complex of Phaeodactylum tricornutum

The light-harvesting complex of the marine diatom Phaeodactylum tricornutum was fractionated into two large pigment-protein particles. One pigment-protein particle, which was contained in a yellow fraction, has a molecular weight, determined by gel filtration, of approx. 230 000 and can be dissociated in sodium dodecyl sulfate/mercaptoethanol solution to apopolypeptides of approx. 15 000. Characterization of particles with regard to molecular weights, subunits, protein and pigments suggests approx. 12 subunits per particle. The other pigment-protein particle, which was found in a green fraction, of approx. 95 000 molecular weight also reduces to apopolypeptide subunits of approx. 15 kDa. The relative molar proportions of chlorophyll a, chlorophyll c, fucoxanthin and total other accessory pigments in the former fraction are 3:1.3:6:2, whereas the proportions in the latter fraction are 5:1:3:1.     

Equal Expression of the Maternal and Paternal Alleles for the Polypeptide Subunits of the Major Storage Protein of the Bean Phaseolus vulgaris L

Discontinuous sodium dodecyl sulfate slab gel electrophoresis of G1 globulin from several strains of Phaseolus vulgaris L. seed permitted clear resolution of the constituent polypeptides. Three strains (Tendergreen, Canadian Wonder, and BBL 240) had subunits of molecular weight 53,000, 47,000 and 43,000 while two strains (Seafarer and PI 229,815) had 50,500, 47,000 and 43,000 molecular weight subunits. F₁ seed from the cross BBL 240 × PI 229,815 showed four polypeptides on dissociation of the G1 protein; however, the amount of each of the 53,000 and 50,500 subunits was half that of the 47,000 subunit. This is interpreted as evidence that both the maternal and paternal loci for these polypeptides are transcribed and translated with similar efficiency. All of the polypeptides were found to have associated sugar residues.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Purification and properties of ribulosebisphosphate carboxylase large subunit binding protein

The large subunit binding protein, an abundant plastid protein implicated in the assembly of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), has been highly purified from leaves of Pisum sativum. The 720 kilodaltons purified binding protein is composed of two types of subunits of 60 and 61 kilodaltons. Highly specific polyclonal antibodies have been raised against the binding protein. The antibodies do not cross-react with the large subunit nor do anti-RubisCO antibodies cross-react with the binding protein. A higher molecular weight form of the binding protein is immunoprecipitated from products of P. sativum polysomes translated in a wheat-germ system, indicating that the binding protein is synthesized by cytoplasmic ribosomes. Immunoblotting reveals the presence of binding protein in extracts of tobacco, wheat and barley leaves and castor bean endosperm.

The previously reported dissociation of the binding protein-large subunit complex upon addition of ATP in vitro has been confirmed and the fates of the dissociated subunits further investigated. The dissociated binding protein subunits are not phosphorylated or adenylated in vitro by added ATP.

     

Deoxyribose-5-P aldolase: subunit structure and composition of active site lysine region

Deoxyribose-5-P aldolase, a Class I aldolase, from Salmonella typhimurium has a molecular weight of 57,000 and is composed of two subunits of 28,500 molecular weight. Evidence from fingerprint analysis of tryptic digests and carboxypeptidase digestion suggests that the two subunits are identical. The COOH-terminal tyrosine residue which is removed by carboxypeptidase digestion appears to be necessary for catalytic activity but not for substrate binding. A tryptic peptide containing the “active site” lysyl residue modified by acetaldehyde has been purified and the following amino acid composition determined: (Ala₃, Gly₃, Thr₃, Asx₂, Ser, Ile, Phe, 1N-Lys)-Lys.     

Further characterization of the sialic acid-binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda

A sialic acid-binding lectin, named carcinoscorpin, has been isolated from the horseshoe crab Carcinoscorpius rotunda cauda. It is a glycoprotein of molecular-weight 420,000, having two subunits of molecular weight 27,000 and 28,000, both subunits responding to glycoprotein stain. Leucine was detected as the only NH₂-terminal amino acid. The sedimentation constant of the native lectin was found to be 12.7 s. On digestion with trypsin, the lectin gave 18 soluble tryptic peptides. This lectin was found to be antigenically unrelated to another sialic acid-binding lectin, limulin, isolated from the horseshoe crab Limulus polyphemus. A lectin-specific disaccharide alcohol namely O-(N-acetylneuraminyl) (2 → 6)2-acetamido-2-deoxy-d-galactitol was found to quench the typical tryptophan fluorescence of the native lectin at 332 nm. The association constant for this interaction was determined spectrofluorimetrically and found to be 1.82 × 10³m^?1.     

Purification and Properties of Catechol 1,2-Dioxygenase from Rhizobium leguminosarum biovar viceae USDA 2370

The catechol 1,2-dioxygenase of Rhizobium leguminosarum biovar viceae USDA 2370 was purified 296-fold, yielding a homogeneous preparation with a specific activity of 51.1 U mg of protein^-1. The molecular weight of the native protein was 70,000, with two identical subunits of 34,500 and 1 g-atom of iron per mol. The optimum pH for catalytic activity was 9.0 to 9.5.     

Role of endoplasmic reticulum in biosynthesis of oat globulin precursors

Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt-soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin α-β dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced α-β dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the α + β subunits, were labeled in protein bodies.

The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller α and β subunits.

     

Protein and electroretinogram changes in the alleles of the norp Ap12Drosophila phototransduction mutant

The protein differences found in the norp A^P12 phototransduction mutant were studied in six other norp A alleles. The two proteins which had altered concentrations in the norp A^P12 mutant were found to have altered concentrations in each of the other alleles. The amount of concentration change varied with the particular allele and appeared related to the reduced visual receptor potentials observed in the mutant. The alleles with the more altered protein concentration exhibited the smaller receptor potentials while alleles with less altered protein concentrations exhibited the larger receptor potentials.Each of the observable protein subunits in the Drosophila melanogaster eye were characterized as to mobility and molecular weight. Fourteen protein subunits were characterized. No mobility changes were detected in any of the eye proteins nor were there any clear concentration changes observed, except for the two proteins altered in all norp A alleles.