Principal Subunit Research Articles

Dissociation of the extracellular hemoglobin of Nephtys incisa

Although the extracellular hemoglobin of Nephtys incisa possesses the two-tiered hexagonal appearance characteristic of annelid extracellular hemoglobins, it contains an additional subunit in the central cavity of the molecule. 1. 1. Its molecular size as judged by gel filtration on Sepharose C1-6B is similar to that of Lumbricus terrestris hemoglobin. 2. 2. The oxygenation curve of Nephtys hemoglobin is slightly sigmoid with P 50 = 10 torr; the Hill plot is linear with n = 1.7 at half saturation. 3. 3. The pattern of dissociation of the unreduced hemoglobin upon electrophoresis in polyacrylamide gels (PAGE) in the presence of sodium dodecyl sulfate (SDS) was different from that of Lumbricus and other annelid extracellular hemoglobins. It exhibits two dominant subunits with molecular masses of about 66,000 and 50,000 daltons and minor subunits of lower molecular mass; this result was corroborated by gel filtration of the hemoglobin in 0.1% SDS at neutral pH. 4. 4. SDS-PAGE of the reduced hemoglobin showed that it dissociates into five subunits: three “monomeric” subunits with molecular masses in the range 11,000 to 15,000 daltons and two “dimeric” subunits in the range 27,000 to 37,000 daltons. Two-dimensional SDS-PAGE showed that the two principal subunits of unreduced Nephtys hemoglobin produced upon reduction, both “monomeric” and “dimeric” subunits. 5. 5. The iron content of Nephtys hemoglobin was determined to be 0.248 ± 0.013% wt; the corresponding minimum molecular mass of 22,400, together with the results of SDS-PAGE suggests that not all of the polypeptide chains carry heme.

The erythrocruorin of Eisenia fetida: II. Properties of the principal subunit

The erythrocruorin of Eisenia fetida can be dissociated partially into its principal subunits, the putative one-twelfths of the molecule and smaller subunits, by three different methods: freezing and thawing (Af), exposure to alkaline pH (Aa) and aging (Ao). The isolated subunits possess a relative molecular mass of 310000±20000 by gel filtration and their SDS-polyacrylamide gel electrophoretic patterns are identical to (Af) or slightly different from (Aa,Ao) that of the erythrocruorin. The absorption spectra and Hill constant h of the principal subunit Af correspond to the values of the whole erythrocruorin in the state of low cooperativity, which results from freezing and thawing, exposure to alkaline pH, and aging. Electron microscopic studies of the principal subunits and of the ‘treated’ erythrocruorin showed that their dimensions had increased relative to the native erythrocruorin: a diameter of 9 nm vs. 8.5 nm and (26.4±0.4) × (18.3±0.4) nm vs. 25.0×16.5 nm, respectively. Erythrocruorin reconstituted from the Af subunits possessed the dimensions (26.6±0.4)×(18.6±0.4) nm. Based on the subunit model of Eisenia erythrocruorin proposed previously it is suggested that there can exist an association-dissociation equilibrium between the principal subunits, smaller subunits and the erythrocruorin when it is in the state of low cooperativity, but not when it is in its native, high-cooperativity state.

Preliminary X-ray crystallographic study of the principal subunit of the lens structural protein, bovine β-crystallin

A principal subunit of the β-crystallins has been isolated and crystallized from bovine lens. The crystals are orthorhombic with unit cell dimensions a = 154·2 A ̊ , b = 165·2 A ̊ and c = 78·4 A ̊ , corresponding to two dimers of molecular weight 46,400 in the asymmetric unit. The crystals are pseudo-tetragonal, thus indicating that the pair of dimers are related by an approximate crystallographic dyad.

O-phosphoserine content of intermediate filament subunits

Purified subunits of intermediate filaments obtained from a variety of tissues and cell types contain O-phosphoserine and, in some cases, smaller amounts of O-phosphothreonine. The O-phosphoserine content was estimated by reaction of performic acid oxidized subunits with methylamine in NaOH. Decamin of BHK-21 and CHO fibroblasts contained about 1 mol/mol. Avian and mammalian desmin consists of two subunits, an acidic (alpha) subunit which contained 2 mol/mol and a more basic (beta) nonphosphorylated subunit. The principal (Mr approximately 60 000) subunit of squid brain neurofilaments contained 5 mol/mol. Most mouse and bovine keratin subunits contained 3--6 mol/mol, although certain bovine subunits of higher molecular weight contained none. The O-phosphoserine contents of keratin subunits purified from the viable and stratum corneum layers were the same. The O-phosphoserine was located in non-alpha-helical regions of the subunits which presumably project out from the alpha-helical wall of the intermediate filaments. Most subunits could be partially dephosphorylated in vitro with alkaline phosphatase. It was found that the capacity of such partially dephosphorylated subunits for assembly into native-type filaments in vitro was independent of their phosphate content.

Alfalfa Proteins: Isolation and Partial Characterization of the Major Component — Fraction I Protein

ABSTRACTFraction I protein was extracted from fresh alfalfa (Medicago sativa) leaves and purified by ammonium sulfate fractionation, DEAE‐cellulose and Sephadex G‐200 chromatography. The isolate possessed a specific activity for ribulosebisphosphate carboxylase EC 4.1.1.39) of 1.01 units/mg protein. An approximate molecular weight of 573,000 was determined from values of So20,w (18.7) and Do20,w (2.97). The protein contained 26 disulfide and 37 sulfhydryl groups—only one of which was titratable in the undenatured protein complex. It migrated as a single boundry by Tiselius electrophoresis and as a dense zone in polyacrylamide gels (disc). In the presence of SDS, two principal subunits of ∼52,000 and ∼12,500 daltons were revealed. Based on the FAO/WHO (1973) provisional pattern for essential amino acids, the protein was slightly deficient in sulfur‐containing residues.

Chlorophyllase in Phaeodactylum tricornutum Photosynthetic Membranes. Extractability, Small‐Scale Purification and Molecular Weight Determination by SDS‐Gel‐Electrophoresis

AbstractThe extractability of chlorophyllase is used as an indicator for the way in which the enzyme is incorporated in Phaeodactylum tricornutum photosynthetic membranes. Whereas with various aqueous solutions no appreciable amount of chlorophyllase is washed from the membranes even after chloroform‐pretreatment. the enzyme can be extracted with aqueous solutions after practically all the lipids have been removed by means of 80% acetone.The proteins in an aqueous extract of the membranes thus treated were separated by electrophoresis on a 4% polyacrylamide gel; extraction of the active protein from several gels yielded a purified chlorophyllase solution.As with the intramembraneous enzyme, activity of purified, solubilized chlorophyllase depends upon the combined presence of MgCl2 and dithiothreitol. The enzyme is inactivated upon heating at 56°C for 5 min.After SDS‐gel‐electrophoresis of an enriched extract, enzyme activity could be localized in the gels. The molecular weight of SDS‐treated chlorophyllase, or of its principal subunit, was estimated to be about 38 kilodaltons.The results are discussed in terms of the identity of prochlorophyllase.

Biosynthesis of crustacean lipovitellin. III. The incorporation of labeled amino acids into the purified lipovitellin of the crab Pachygrapsus crassipes.

Electrophoretically pure subunits were obtained from the lipovitellin (LV) of the intertidal crab, Pachygrapsus crassipes. The molecular weights of these subunits were determined. In addition, it was demonstrated that the ovaries of Pachygrapsus, incubated in vitro, incorporated 3H-leucine into the two principal subunits of lipovitellin. At the end of a 30 hour incubation the specific activity of each of the principal subunits was approximately 30,000 dpm/mg protein. It is concluded that the ovaries of Pachygrapsus crassipes are capable of synthesizing the proteinaceous yolk found in the mature egg.

Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels

Bacterial polyribosomes, ribosomes and ribosomal subunits were found to possess characteristic electrophoretic mobilities in polyacrylamide-agarose composite gels (containing 2 to 3% acrylamide). The mobilities of these particles were inversely proportional to their size. The smaller (30 s) subunit had the fastest mobility, and migrated at a rate similar to its constituent 16 s RNA. Polyribosomes containing eight ribosomes were the largest that could be resolved. Brief treatment with RNase converted the polyribosomes to faster migrating particles (70 s ribosomes). A unique feature of the electrophoretic analysis was the ability to identify, by a second electrophoresis, the type of RNA contained in ribosomal subunits, ribosomes and polyribosomes. Similarly, the subunit composition of ribosomes and polyribosomes could be established. The principal subunits observed were a faster (30 s) subunit containing 16 s RNA and a slower (50 s) subunit containing 23 s RNA. Polyribosomes resembled each other in their proportional content of these subunits. RNA isolated from polyribosome preparations was composed of three very similar but electrophoretically distinct 23 s RNA's; similarly the 16 s RNA was composed of two distinct RNA's. Another RNA migrating somewhat slower than the 16 s RNA (16 s–17 s) was observed. In addition to these multiple forms of RNA an unanticipated diversity of subunits was observed. One of the smaller subunits contained the slower 16 s–17 s RNA and may represent the 26 s precursor particle of the 30 s subunit. It is likely that some of the variations in subunit mobilities are related to differences in the size of the RNA which they contain. However, provided the protein is not removed, the electrophoretic mobilities of subunits and polyribosomes are not dependent on their containing intact RNA.