kD Subunit Research Articles

Variation and inheritance of the arachin polypeptides of groundnut (Arachis hypogaea L.)

Variation in the arachin polypeptides of groundnut genotypes was observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Three regions could be observed on the electropherogram. Region 1, corresponding to conarachin, did not show any variation; region 2, consisting of arachin acidic subunits, showed variation; region 3, containing the arachin basic subunits, did not show any variation. There are four varietal classes of arachin polypeptide patterns: class A comprised three acidic subunits of arachin of molecular weights 47.5, 45.1 and 42.6 kd and a basic subunit of 21.4 kd; class B, with three acidic subunits of molecular weights 47.5, 45.1 and 41.2 kd and a basic subunit of 21.4 kd; class C of an additive pattern of class A and class B; class D, of two acidic polypeptides of 47.5, 45.1 kd and the basic 21.4 kd subunit. Of the 90 genotypes studied, 73% belong to class A, 15% to class B and 6% each to class C and D. Analysis of F2 seeds from a cross between class A and class B genotypes showed that the two polypeptides (42.6 kd and 41.2 kd) are coded by nonallelic genes and also revealed that class C and class D patterns arose as a result of hybridisation between class A and class B. A. monticola, the progenitor of A. hypogaea, showed a pattern similar to the additive pattern of class A and class B while some diploid Arachis species had the 41.2 kd polypeptide. Based on arachin polypeptide patterns the probable origin of A. hypogaea has been suggested.

Nonhematopoietic cells selected for resistance to tumor necrosis factor produce tumor necrosis factor.

TNF-resistant lines of L cells can be derived from TNF-sensitive populations by repeated exposure to TNF, and these resistant L cells, in contrast to sensitive L cells and other types of cells, lack demonstrable cell surface receptors for TNF. We have now found that TNF-resistant L cells produce a factor that is cytotoxic for L cells and has the following distinguishing characteristics of mouse TNF: it is a protein of 43 kD, composed of 16 kD subunits, that competes with TNF for receptor binding, induces hemorrhagic necrosis of the TNF-sensitive mouse sarcoma Meth A, has synergistic cytotoxic action with interferon, and its activity is neutralized by antibody to TNF. The two conclusions of this study are that cells selected for TNF resistance spontaneously produce a molecule resembling macrophage TNF, and that cells of nonhematopoietic origin are capable of producing TNF.

Biosynthesis and processing of the α subunit of the voltage-sensitive sodium channel in rat brain neurons

The sodium channel from rat brain is a complex of α (260 kd), β1 (36 kd), and β2 (33 kd) subunits. The α and β2 subunits are linked by disulfide bonds. The earliest biosynthetic precursor of the α subunit is a 203 kd core polypeptide with sufficient high-mannose carbohydrate chains to increase its apparent size to 224 kd. It is processed to 224 kd and 249 kd precursor forms containing complex carbohydrate chains before it achieves the mature size of 260 kd. Most newly synthesized α subunits are not disulfide-linked to β2 subunits, but remain as a metabolically stable pool of intracellular subunits. α subunits disulfide-linked to β2 are found preferentially at the cell surface. A possible role for this intracellular pool as a rate-limiting step in the regulation of the cell surface density and localization of sodium channels in developing neurons is proposed.

Mediation of platelet adhesion to fibrillar collagen in flowing blood by a proteolytic fragment of human von Willebrand factor

The effect of purified von Willebrand Factor (vWF) fragments, SpII (dimer of two 110 kd subunits) and SpIII (dimer of two 170 kd subunits) obtained with S aureus V-8 protease was tested upon platelet adhesion to collagen. Purified fibrillar human collagen coated onto cover slips was incubated with SpII, SpIII, or undigested vWF and exposed to reconstituted human blood in a parallel-plate perfusion chamber at a high shear rate. Platelet-collagen interactions were estimated using 51Cr-platelets and quantitative morphometry. When blood was reconstituted with citrated autologous plasma, SpIII and vWF strikingly enhanced platelet adhesion to collagen whereas SpII had no effect. When blood was reconstituted with human albumin and divalent cations, SpIII and vWF again promoted platelet adhesion to collagen. In conclusion, our data suggest that (1) SpIII, the N-terminal portion of vWF which binds to platelet membrane glycoprotein Ib, functionally substitutes for vWF in supporting platelet adhesion to collagen; (2) SpII, the C- terminal portion which binds to glycoprotein IIb/IIIa, has no such effect; (3) in addition to its platelet binding domain, SpIII contains another site for binding to collagen; and (4) the multimeric structure of vWF is not required for platelet adhesion to collagen.

Mediation of Platelet Adhesion to Fibrillar Collagen in Flowing Blood by a Proteolytic Fragment of Human von Willebrand Factor

The effect of purified von Willebrand Factor (vWF) fragments, SpII (dimer of two 110 kd subunits) and SpIII (dimer of two 170 kd subunits) obtained with S aureus V-8 protease was tested upon platelet adhesion to collagen. Purified fibrillar human collagen coated onto cover slips was incubated with SpII, SpIII, or undigested vWF and exposed to reconstituted human blood in a parallel-plate perfusion chamber at a high shear rate. Platelet-collagen interactions were estimated using 51Cr-platelets and quantitative morphometry. When blood was reconstituted with citrated autologous plasma, SpIII and vWF strikingly enhanced platelet adhesion to collagen whereas SpII had no effect. When blood was reconstituted with human albumin and divalent cations, SpIII and vWF again promoted platelet adhesion to collagen. In conclusion, our data suggest that (1) SpIII, the N-terminal portion of vWF which binds to platelet membrane glycoprotein Ib, functionally substitutes for vWF in supporting platelet adhesion to collagen; (2) SpII, the C-terminal portion which binds to glycoprotein IIb/IIIa, has no such effect; (3) in addition to its platelet binding domain, SpIII contains another site for binding to collagen; and (4) the multimeric structure of vWF is not required for platelet adhesion to collagen.

Detection of a subunit of cellular Pol II within highly purified preparations of RNA polymerase isolated from rabbit poxvirus virions

A large (∼200 kd) subunit of cellular RNA polymerase II (Pol II) and a virus-encoded subunit of rabbit poxvirus (RPV) RNA polymerase (137 kd) react with common monoclonal antibodies. Hybridization studies with viral and cellular DNA clones confirm that the viral and cellular proteins are related. Following RPV infection, the Pol II subunit is translocated from the nucleus to the cytoplasmic virosomes, then packaged into mature virus, and is found associated with the viral RNA polymerase purified from virions. The results suggest that the cellular Pol II subunit may be directly involved in the transcription of viral genes.

Age-dependent changes in the heat-stable crystallin, beta Bp, of the human lens.

The present study examined the effects of aging and cataractogenesis on the biochemical properties of the uniquely heat-stable lens crystallin, beta basic principle polypeptide (beta Bp). Using the techniques of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot immunoassay, we analyzed cortical and nuclear lens sections from normal lenses of individuals aged 0 to 91 years for beta Bp content in the soluble fraction, and retention of heat stability with aging. In addition, we compared the characteristics of beta Bp in cataractous lenses with those of normal lenses of approximately the same age. While beta Bp is synthesized in new cortical cells throughout life, the beta Bp of the nucleus, which had been laid down early in life, decreased significantly in both absolute concentration and in its proportion of the total soluble protein fraction during the normal aging process. In addition, posttranslational changes in the nuclear soluble beta Bp result in a gradual loss of approximately 3000 d in the apparent mass of the beta Bp molecule; i.e., as a result of the aging process, the single heat-stable band of an apparent mass of 26 kd on SDS-PAGE of the young lens nucleus becomes two heat-stable bands, one at 26 kd and one at 23 kd. Normal lenses up to 91 years of age always retain some of the 26 kd subunit, whereas lenses with severe nuclear cataracts had only the 23 kd subunit in the soluble fraction.

An immunohistochemical and quantitative examination of dorsal root ganglion neuronal subpopulations

Sensory neurons of adult rat lumbar dorsal root ganglia were labeled in cryostat sections with antisera against tyrosine hydroxylase (TH), substance P (SP), and somatostatin (SOM), and with a monoclonal antibody (RT97) that labels the 145- and 200-kilodalton (kd) subunits of neurofilaments. These neurons were also histochemically stained for fluoride-resistant acid phosphatase (FRAP), and the size and distribution of each population were determined. In addition, the double-label immunoperoxidase technique of Sternberger and Joseph (Sternberger, L.A., and S.A. Joseph (1979) J. Histochem. Cytochem. 27: 1424-1429) was employed to determine whether these antibodies labeled distinct or overlapping populations of neurons. The results indicate that the dopaminergic (TH+) cells constitute a separate population from the SP+ and SOM+ neurons and that the size distributions of the SP+, SOM+, TH+, and FRAP+ cells are all different despite the fact that all of these subpopulations are part of the "small dark" subpopulation as indicated by their size and by the fact that they are RT97-. RT97 is a putative marker for the "large light" population (Anderton, B., H.B. Coakham, J. A. Garson, A.A. Harper, and S.N. Lawson (1982) J. Physiol. (Lond.) 334: 97-98P). Furthermore, the distribution data indicate that all of the "small dark" cell subpopulations are not evenly distributed within the ganglion, and the staining with RT97 and with another antibody which recognizes the 68-kd neurofilament subunit indicates heterogeneity among the "large light" population. These results are discussed in terms of the significance of the "small dark"-"large light" classification.

Covalent association between human thymus leukemia-like antigens and CD8(Tp32) molecules.

Several TL-like molecules have been identified on human thymocytes. Some of these TL-like molecules are disulfide-bonded to CD8(Tp32), on which they form the thymus-specific 45 kilodalton (Kd) subunit of the CD8 complex. TL-like molecules are associated with beta 2 microglobulin. However, beta 2 microglobulin is not co-precipitated with the CD8-TL complexes, suggesting that CD8 and beta 2 microglobulin are alternative association structures for TL-like molecules. CD8 is present as high m.w. complexes of 72 and 135 Kd that are composed of 32 Kd subunits only, and as high m.w. complexes of 120 and greater than 150 Kd that are composed of both 32 and 45 Kd (TL-like) subunits. A third subunit of CD8 with an apparent m.w. of 52 Kd was seen as part of the 72 Kd complex, and may represent an incompletely reduced dimer of the 32 Kd subunit.

On the Composition, Deposition and Mobilization of Proteins in the Cotyledons of Castor Bean (Ricinus communisL. cv. Hale) Seeds: Their Role as Storage Proteins

Proteins in the soluble and insoluble fractions, extracted from mature castor bean cv. Hale seed cotyledons, differ quantitatively and qualitatively from their counterparts extracted from the endosperm. The soluble fraction contains no glycoproteins, and the lectins RCA1 and ricin D are absent. While the insoluble proteins are electrophoretically and immunologically similar to those in the endosperm, they do not form the 100 kD subunit dimers which characterize some of the endosperm insoluble crystalloid proteins. Rapid rates of deposition of all of the soluble and insoluble proteins present in the mature seed cotyledons commences 30–35 d after pollination (DAP) and continues until 45 DAP. These proteins are mobilized rapidly beginning 1–2 d after seed imbibition and this coincides with an increase in specific activity, in the cotyledons, of two aminopeptidases and a carboxypeptidase. The soluble and insoluble proteins in the cotyledons of the mature seed probably function as storage proteins and support the growth of the germinated seed prior to the mobilization of the major protein storage reserves of the endosperm.

Nucleotide sequence of the clustered genes for apocytochrome b6 and subunit 4 of the cytochrome b/f complex in the spinach plastid chromosome

A 2.4 kilobase-pair segment of the spinach plastid chromosome carrying the genes for apocytochrome b6 and subunit 4 of the thylakoid membrane cytochrome b/f complex has been analysed by DNA sequencing and Northern blot analysis. The nucleotide sequence reveals two uninterrupted open reading frames of 211 and 139 triplets coding for two hydrophobic proteins of 23.7 kd (cytochrome b6) and 15.2 kd (subunit 4). The genes are located on the same strand and are separated from each other by 1018 untranslated base pairs. They map adjacent to the gene for the P680 chlorophyll α apoprotein of the photosystem II reaction center. The three genes appear to be under common transcriptional control and the transcripts post-transcriptionally modified. The deduced amino acid sequences of cytochrome b6 and subunit 4 both exhibit significant homology with published sequences from mitochondrial b cytochromes (42 kd) suggesting that these functionally equivalent polypeptides in photosynthetic and respiratory electron transport chains arose monophyletically.

Undecagold labelling of glutamine synthetase from E. coli

Glutamine synthetase (GS) from E. coli consists of 12 identical 50 kD subunits arranged in a double hexagon with one six-fold axis of rotational symmetry and six two-fold axes. The divalent cations Mn2+ or Co2+ induce the formation of linear face-to-face aggregates of individual GS molecules, resulting in the formation of long linear arrays, GSn. This aggregation is retarded and eventually reversed to the native form (GS) as the salt concentration increases. We have used an undecagold (11-Au) cluster ion, ((p-H2NCH2C6H4)3) - P)7Au113+, which induces similar aggregation of native GS, resulting in a wide distribution of ordered linear arrays of GSn (where n=2-10, or more). These aggregates show the highly electron dense labels located exclusively at the face-to-face interaction surface. Specimens of the GS aggregates have been studied with the Brookhaven STEM and molecular weight values have been determined with the Brookhaven STEM mass measurement computer system.

Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: Basal cells with properties of terminally differentiating cells

The keratin pattern of newborn mouse epidermis was investigated during terminal differentiation. In highly pure fractions of basal and suprabasal cells, obtained by Percoll density gradient centrifugation, we identified two sets of mRNA-encoded proteins: a basal set of 58.5, 52, and 47 kd subunits and a suprabasal set of 67 and 60 kd subunits. The large subunits of each set were alkaline to neutral, while the small subunits were acidic. Polyclonal antibodies against the suprabasal, acidic 60 kd protein and the basal, alkaline 58.5 kd protein selectively recognized their antigens in immunoblots of NEPHGE-resolved keratins and decorated the corresponding epidermal compartments in frozen sections. The antibody to the suprabasal 60 kd protein also recognized distinct cells in the basal cell layer. Quantification of this cell population revealed a 10% cell fraction, morphologically indistinguishable from the total cell population, that, in addition to expressing basal keratin proteins, was already synthesizing suprabasal keratin subunits.

Differentiation of neurons and radial glia in the spinal cord of the teleost Brachydanio rerio (the zebrafish): An immunocytochemical study

The differentiation of neurons and glial cells in the spinal cord of the zebrafish was studied by means of immunohistochemistry, using antisera against the 68 kD subunit of neurofilament (anti-NFP68) and against glial fibrillary acidic protein (anti-GFAP), both isolated from the bovine brain. Anti-NFP68 and anti-GFAP reactivity appear in the spinal cord at about 60 h after fertilization. At that time the anti-NFP68 reactivity is detectable in the dorsal Rohon-Beard neurons. About 12 h later, NFP68 positive neurons appear in the prospective motor column. In this respect the differentiation of the primary sensory system precedes that of the spinal motor system. During development the configuration of the glial cell processes changes from a horizontal arrangement in embryos to a radial frame work in larvae and in adults. From these observations together with data on the organization of the adult spinal motor column28 we conclude that the motoneurons of the white and those of the red myotomal muscle fibers may have different origins in the neuroepithelial germinal layer. The anti-NFP68 serum recognizes a 120 and a 94 kD component of fish neurofilaments. Thus the subunit composition of neurofilament in fishes differs from that in mammals.

A GABA/benzodiazepine receptor complex from bovine brain: purification, reconstitution and immunological characterization.

A GABA/benzodiazepine receptor complex was purified from bovine cerebral cortex. The receptor fraction displayed binding sites for benzodiazepines as well as high and low affinity binding sites for GABA which are characteristics of the membrane-bound receptor. Two monoclonal antibodies of which one was directed against the 50 kd and the other against the 55 kd subunit were used for immunoprecipitation studies. Both of them were shown to quantitatively precipitate the entire receptor population. These results indicate that the binding sites for benzodiazepines and GABA (high and low affinity sites) reside on the same receptor complex containing a mixture of 50 kd and 55 kd subunits. Reconstitution of the receptor in phospholipid vesicles was achieved.

Interactions between subunit polypeptides of the crystalloid protein complex of castor bean endosperm

Proteins forming the 11S crystalloid protein complex (CP c) are the major storage proteins deposited in the endosperm of castor bean ( Ricinus communis L. cv. Hale). There are at least six of these proteins and they aggregate to form highly insoluble crystalloid protein body inclusions. Each of these proteins is a hexamer (CP c 1–6) and the M t 50 kD subunits of these hexamers comprise a M r 30 kD acidic and a M r 20 kD basic polypeptide linked by disulphide bridges. Unlike other 11S dicot storage proteins, the acidic and basic polypeptides of the crystalloid proteins can form tetrameric associations with an approx. M r 100 kD. Tetramers are formed from polypeptides derived from CP c 3–6 subunits only. Polypeptides from CP c 1 and 2 subunits are never present in the M r 100 kD proteins. There is a correlation between the amount of radiolabel derived from methionine which is incorporated into the polypeptides and their ability to form tetramers.

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a1c1 was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.

Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli.

The terminal electron transfer enzyme fumarate reductase has been shown to be composed of a membrane-extrinsic catalytic dimer of 69- and 27-kilodalton (kd) subunits and a membrane-intrinsic anchor portion of 15- and 13-kd subunits. We prepared inverted membrane vesicles from a strain carrying the frd operon on a multicopy plasmid. When grown anaerobically on fumarate-containing medium, the membranes of this strain are highly enriched in fumarate reductase. When negatively stained preparations of these vesicles were examined with an electron microscope, they appeared to be covered with knob-like structures about 4 nm in diameter attached to the membrane by short stalks. Treatment of the membranes with chymotrypsin destroyed the 69-kd subunit, leaving the 27-, 15-, and 13-kd subunits bound to the membrane; these membranes appeared to retain remnants of the structure. Treatment of the membranes with 6 M urea removed the 69- and 27-kd subunits, leaving the anchor polypeptides intact. These vesicles appeared smooth and structureless. A functional four-subunit enzyme and the knob-like structure could be reconstituted by the addition of soluble catalytic subunits to the urea-stripped membranes. In addition to the vesicular structures, we observed unusual tubular structures which were covered with a helical array of fumarate reductase knobs.

Transducin and the cyclic GMP phosphodiesterase: amplifier proteins in vision.

Our experiments have delineated the flow of information in the cyclic nucleotide cascade of vision of ROS. A single, photoexcited rhodopsin molecule activates several hundred phosphodiesterase molecules in two stages. First, photoexcited rhodopsin (R*) interacts with transducin (T), a peripheral membrane protein consisting of alpha- (39 kD), beta- (36 kD), and gamma- (approximately 10 kD) subunits. R* catalyzes the exchange of GTP for GDP bound to the subunit of transducin. About 500 T alpha- GTPs are produced per photoexcited rhodopsin at low light levels. T alpha-GTP, released from the beta- and gamma-subunits of transducin, then interacts with the phosphodiesterase to relieve the inhibitory constraint imposed by its gamma-subunit. Hydrolysis of GTP bound to T alpha serves to restore the system to the dark state. Transducin is the amplified signal carrier in this light-triggered cascade. The formation of hundreds of T alpha- GTPs is likely to be the first stage of amplification in visual excitation. The photoactivation of the phosphodiesterase in ROS closely resembles the activation of adenylate cyclase in hormone-sensitive cells. Our cholera toxin labeling studies have shown that transducin is akin to the signal-coupling G protein of the adenylate cyclase system. Cholera toxin specifically ADP- ribosylates and inactivates the GTPase activity of T alpha, just as it does with Gs. The action of pertussis toxin on ROS further underscores the homology of the photoreceptor and hormone-responsive systems. It seems likely that transducin, the stimulatory G protein, and the inhibitory G protein are members of the same family of signal-amplifying proteins. The study of the cyclic nucleotide cascade of vision is proving to be rewarding in affording a view of a recurring motif of signal amplification in nature in addition to providing insight into the mechanism of vision.

Subunit composition of proteins from seeds of Lupinus albus

All the main globulins in the seeds ofLupinus Albus are oligomeric glycoproteins. Legumins (33%) consist of two similar protein molecules which contain protomers linked by disulphide bridges. They result from a partial proteolytic breakdown of an original polypeptide chain. Vicilins (44%) consist of four similar protein molecules with several protomers linked together by non-covalent bonds. Globulin 1 (6%) has a native M.W. of 199 kd and is formed by four 45.0 kd subunits consisting of two smaller protomers (28.0 and 16.0 kd) linked by -S-S- bonds. Globulin 9b (12.5%) has the lowest M.W. (44.0 kd) and is made up of three protomers, two of which are linked by disulphide bonds.