Structural characterization of the 9-10S estradiol receptor from calf uterus

An affinity chromatography-based method has been developed for estrogen receptor isolation which requires the inclusion of sodium molybdate in purification buffers for maintaining the large 9-10S form of the receptor. The protein products obtained from affinity chromatography of calf uterine receptor extracts or from extracts presaturated with estradiol have been analyzed by gel electrophoresis under denaturing conditions. Major estrogen sensitive proteins were peptides with Mr approximately 90,000, 65,000 and 50,000. Two additional proteins (60,000 and 53,000) of lower abundance and with demonstrated estrogen sensitivity were also observed. Affinity labeling with [3H]tamoxifen aziridine identified the Mr 65,000 protein as the estrogen receptor and suggested that the Mr 60,000, 53,000 and 50,000 peptide components were derived proteolytically from this parent unit. The 90,000 mol. wt component was readily dissociated from heparin-sepharose immobilized estrogen receptor by elution with low salt buffers without molybdate. Peptide mapping experiments indicated that the 90,000 mol. wt component was not related to the Mr 65,000 and 50,000 estrogen receptors, but confirmed the smaller binding unit to be a proteolytic fragment of the 65,000 mol. wt receptor. The results suggest that the 90K protein associates non-covalently with the Mr 65,000 estrogen binding unit as a nonhormone binding component of the 9-10S receptor.     

Selective cleavage of variant surface glycoproteins from Trypanosoma brucei.

Two conformationally distinct regions were revealed by tryptic cleavage of six undenatured variant surface glycoproteins purified from clones of Trypanosoma brucei. Within 5 min, the native glycoproteins (65,000 mol.wt.) were cleaved, yielding a large N-terminal fragment (48,000-55,000 mol.wt. depending on the variant) together with one or more C-terminal fragments. After 30-60 min incubation, further breakdown of the large fragment occurred in some variants. The ultimate large product (40,000-52,000 mol.wt.) was very resistant to further degradation by trypsin (in the absence of denaturation). The distinction between N-terminal and C-terminal domains may be significant in relation to the organization and function of these glycoproteins on the trypanosome surface.     

Bovine serum hemopexin: properties of the protein from a single animal

1. Hemopexin was isolated from bovine serum of a single animal in a yield of 0.5 mg/ml. 2. Bovine hemopexin was found to exist in two isoforms of mol. wt 68,000 and 65,000. 3. Treatment of hemopexin with glycopeptidase F yields a single band corresponding to a mol. wt of 51,000. 4. The protein binds heme on an equimolar ratio and shows a single component in reverse-phase high performance liquid chromatography. 5. The amino acid composition of bovine hemopexin compares with that of hemopexin isolated form other animals.     

A comparison of the major surfactant-associated proteins in different species

We examined the major surfactant-associated proteins in a number of species by using two-dimensional gel electrophoresis, protein blotting and immunostaining. All species have a 30,000 to 35,000 mol. wt protein group consisting of multiple isoforms with isoelectric points ranging from pH 4.4 to 5.6. The techniques used in this study have resolved three component subgroups within the 35 K group. A group of proteins at 60,000-65,000 mol. wt has also been consistently identified. We conclude that remarkable similarity exists among the major surfactant-associated proteins from various mammals with regard to isoelectric points, molecular weights and antigenic sites.     

The molecular size of nematode acetylcholinesterases and their separation from nematode allergens

The molecular size of nematode acetylcholinesterases, and their separation from nematode allergens. International Journal for Parasitology 3: 735–741. Acetylcholinesterases and allergens were derived from two parasitic nematodes, Nippostrongylus brasiliensis which parasitises rats and Trichostrongylus colubriformis which infects sheep and guinea pigs.

Chromatographic studies indicated that the mol. wt of nematode acetylcholinesterases lies between 65,000 and 75,000. The acetylcholinesterases of both species were separated from nematode allergens whose mol. wt is in the range of 10,000–50,000.

γG binding antigens of T. colubriformis were located in fractions with a mol. wt range of 30,000–150,000. γE binding activity was confined to allergenic material with a mol. wt of less than 70,000.     

Characterization of domains obtained from a mollusc haemocyanin by limited proteolytic digestion.

The haemocyanin from the freshwater gastropod Lymnaea stagnalis was digested with proteolytic enzymes under conditions where it existed as whole (native) molecules (mol.wt. approx. 9 X 10(6)), or as one-tenth molecules. Digestion of whole molecules yielded a fragment of mol.wt. approx. 110,000 believed to correspond to the 'collar' of the molecule, and an aggregate some 20--30 times the size of the original native molecule formed by end-to-end polymerization of the molecule after removal of the collar. Digestion of one-tenth molecules yielded a mixture of products that could be separated into three fractions by gel filtration. Analysis of these by sodium dodecylsulphate/polyacrylamide-gel electrophoresis revealed that they typically contained two or three components. The collar fragment was present as a component of the intermediate-molecular-weight fraction, and it dissociated on sodium dodecyl sulphate/polyacrylamide gels to give two bands corresponding to apparent mol.wts. 65,000 and 60,000. The c.d. spectra of the separated fractions were recorded and fitted with Gaussian curves by a computer procedure. The fractions each possessed distinct c.d. spectra, by which they could be identified: the collar-fragment c.d. and absorption spectra showed the most striking differences compared with those of the other fragments. The results were interpreted in terms of the postulated existence, within the haemocyanin molecule, of multi-domain structures, each comprising a single polypeptide chain of mol.wt. 200,000--300,000.     

Oat seed globulin: subunit characterization and demonstration of its synthesis as a precursor

The predominant storage protein of oat (Avena sativa L.) seeds is a saline-soluble globulin with a mol wt of 320,000 which is composed of six large (M_r = 35,000 to 40,000) and six small (M_r = 20,000 to 25,000) subunits. Experiments were conducted to further describe the subunit polypeptides and to identify the initial translation products of globulin mRNAs. Approximately 20 large subunits and 10 small subunits were resolved by two-dimensional gel analysis. The large and small subunits had acidic and basic isoelectric points, respectively. Disulfide-linked complexes of one large and one small subunit were isolated by extraction in buffer lacking a reducing agent. The NH₂-terminal sequence of the small subunits was homologous to a small subunit of soybean glycinin. Immunoprecipitation of in vitro translation products of poly(A)⁺ RNA with anti-oat globulin sera yielded M_r = 60,000 to 68,000 polypeptides. In vivo labeling of spikelets with radioactive amino acids resulted in high amounts of incorporation into polypeptides with M_r = 65,000 to 68,000 which were immunoprecipitated with anti-globulin sera. These two results suggest oat globulin is synthesized as a higher mol wt precursor which is subsequently processed to yield the large and small subunit polypeptides.     

Properties of a series of tegumental membrane-bound phosphohydrolase activities of Schistosoma mansoni.

1. Incubation of Schistosoma mansoni for 5 min in a phosphate-buffered medium, pH 7.4, released tegumental material containing the following phosphohydrolase activities: alkaline phosphatase, 5'-nucleotidase, glycerol-2-phosphatase, glucose 6-phosphatase, phosphodiesterase and ATPase. 2. Maximum activity of these enzymes was measured at pH 9.5; however, the phosphodiesterase and ATPase activities were also appreciable at pH 7.0. 3. Solubilization of the released tegumental material in 1% Triton X-100 followed by gel filtration distinguished three peaks of enzyme activity: an ATPase (mol.wt. greater than 1000 000), a phosphodiesterase (mol.wt. 1 000 000) and an alkaline phosphomonoesterase with broad specificity (mol.wt. 232 000). 4. The ATPase activity was highly activated by 10 mM-Mg2+ or 1 mM-Ca2+ and was inhibited by chelating agents. Ouabain, Na+ and K+ had little effect on enzyme activity, whereas activity was increased by 50% in the presence of calmodulin. The phosphodiesterase activity was highest in the presence of 100 mM-Na+ or -K+, and 10 mM-Mg2+ or -Ca2+. Alkaline phosphatase activity was also stimulated by 100 mM-Na+ or -K+, and 10 mM-Mg2+; however Ca2+ inhibited at greater than 1 mM. 5. Surface iodination of parasites followed by detergent solubilization and gel filtration of the released tegumental membranes indicated that these enzymes were not accessible. A major surface component, apparent mol.wt. 80 000, was iodinated. 6. Rabbit anti-(mouse liver 5'-nucleotidase) antibodies did not inhibit the phosphohydrolase activities. However, an immunoglobulin G fraction from sera of mice chronically infected with S. mansoni partially inhibited alkaline phosphatase activity, but was without effect on the phosphodiesterase and ATPase activities. 7. The location of the enzymes in the double membrane of the tegument and their significance in host-parasite interactions is discussed.     

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.     

Soluble interferon-alpha receptor molecules are present in body fluids.

Soluble forms of the interferon-alpha receptor (sIFN-alpha R) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-alpha R, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-alpha R and [125I]IFN-alpha with anti IFN-alpha MAb. Elevated levels of sIFN-alpha R were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS-PAGE, and, as expected, the cross-linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross-linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN-alpha R prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be as high as 70,000 Da. It is therefore concluded that sIFN-alpha R molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-alpha R, are present in human serum and in normal human urine.     

A gene encoding for an alpha-amylase from thermophilic Bacillus sp. strain TS-23 and its expression in Escherichia coli

An alpha-amylase gene from Bacillus sp. strain TS-23 was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli. A cell fractionation experiment revealed that approximately 60% of the amylase activity was in the periplasmic space. Analysis and activity staining of the concentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a mol. wt of approximately 65,000. The amylase gene (amyA) consisted of an open reading frame of 1,845 bp encoding a protein of 613 amino acids with a calculated mol. wt of 69,543. The predicted amino acid sequence showed high homology with Bacillus species, E. coli and Salmonella typhimurium alpha-amylases. Deletion of 96 amino acids from the C-terminal portion of the amylase did not result in the loss of amylolytic activity. The truncated amylase, deletion of the first 50 amino acids from the N-terminus, was overexpressed in E. coli system and refolded to yield an activable enzyme.     

Isolation of a basophilic membrane protein binding the anti-allergic drug cromolyn.

The membrane protein component in basophils, responsible for the specific, Ca2+-dependent, binding of the anti-allergic drug cromolyn [disodium cromoglycate, DSCG; the disodium salt of 1,2 bis(2- carboxychromon -5- yloxy )-2-hydroxy propane] was isolated by two procedures based on affinity for the drug. In the first procedure, involving immunoprecipitation, rat basophilic leukemia cells (RBL-2H3), surface labeled by 125I were reacted with a polyvalent conjugate of DSCG and bovine serum albumin and then subjected to solubilization by the non-ionic detergent Nonidet P-40 (NP-40). From these lysates, precipitation was specifically attained by subsequent addition of rabbit anti-DSCG antibodies. In an SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single radioactive band was observed, having an apparent mol. wt. of 60 000 daltons. Competitive inhibition of the immunoprecipitation in the presence of free drug or excess of EDTA demonstrated the specificity of the isolation. Furthermore, this particular membrane component could not be isolated from several other cell types examined. The second isolation from several other cell types examined. The second isolation procedure employed affinity chromatography on DSCG immobilized on polyacryl- hydrazido agarose beads. The DSCG-binding protein was eluted from the affinity column with either DSCG or with EDTA and also migrated on SDS-PAGE as a single band of 60 000 mol. wt., similar to that obtained by the immunoprecipitation procedure. These and other results suggest that this newly isolated protein is the one responsible for the Ca2+-dependent binding of the drug to the basophil membrane.     

Identification of a nuclear antigen with molecular weight of 48,000 differentially expressed in tumour and normal cells

A non-histone protein with mol. wt of 48,000 differentially expressed in normal and tumour cells was identified using immunological criteria. Antibodies were raised against a component specific for Kirkman-Robbins hepatoma of mol. wt about 48,000 separated from hepatoma non-histone proteins by preparative electrophoresis in polyacrylamide gel. It was demonstrated by immunoblotting that Morris hepatoma 7777 and Ehrlich ascites cells share an antigenic non-histone protein with Kirman-Robbins hepatoma. Tumour cells when compared with normal cells, i.e. hamster and rat liver, are characterized by significant enrichment of this component. Intracellular distribution of the polypeptide with mol. wt 48,000 suggests that this component may be a structural protein the biosynthesis of which increases or the antigenic determinants of which change in tumour cells.     

Two-dimensinal gel electrophoresis of rat liver nuclear washes,nuclear matrix,and hnRNA proteins

The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis. The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction. In the nuclear washes and chromatin, we observed five classes of proteins: (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet; (c) proteins of 94,000, 25,000, and 20,500 mol wt specific to the nuclear washes; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix; and (e) two proteins of 68,000 mol wt present only in the final chromatin. The major 65,000- 75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group. A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus. Actin was the second major nuclear membrane-lamina protein. Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP. The concept of NHP being a distinct set of DNA- bound proteins is unnecessarily limiting. Many are derived from the nuclear matrix or hnRNp particles and vary in the degree to which they share different intracellular compartments.     

The quaternary structure of bovine brain kinesin.

In the present work we have studied the subunit composition of kinesin, the microtubule-activated, mechanochemical ATPase, isolated from bovine brain. Polypeptides with mol. wts of 120 and 62 kd are the major components of the kinesin preparation. These polypeptides could not be separated by electrophoresis under nondenaturing conditions or by FPLC on a MonoQ column, and are therefore assumed to form a tight complex. As shown by immunoblotting with polyclonal and monoclonal antibodies to the 120-kd polypeptide and by one-dimensional peptide mapping, the 62-kd polypeptide does not appear to be a proteolytic product of the 120-kd component. Densitometric scanning of polyacrylamide-SDS gels shows that these polypeptides are present in a complex in a 1:1 molar ratio. The mol. wt of native kinesin was studied by sedimentation equilibrium and was found to be 386 +/- 14 kd. A comparison of the mol. wts of individual polypeptides with the mol. wt of the intact molecule indicates that the native molecule contains two 120-kd subunits and two 62-kd subunits.     

Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes.

Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.     

Detection of lymphokines in patients' blood serum]

A factor suppressing the migration of donor leukocytes and macrophages of guinea pigs in vitro was revealed in the blood serum of patients suffering from chronic inflammatory diseases (pneumonia, rheumatism, tuberculosis) and carcinoma. A factor stimulating the leukocyte migration was sometimes revealed in the blood sera of the patients. In chromatography of the blood sera on sephadex G-100 the activity of both factors proved to localize in fractions with the mol wt of 15000--45000 dalton. Depression of stimulation of leukocyte migration could be also caused by immunoglobulin fractions (mol wt--150000 dalton) of the blood sera of patients suffering from acute pneumonia, apparently on account of the presence in them of the antigen-antibody complex; however, these sera contained no migration suppression factor. The blood serum fractions with the mol wt of 15000--45000 dalton, including those containing the migration suppression factor inhibited the inhibited the spontaneous and induced by phytohemagglutinin blast transformation lymphocytes, and the immunoglobulin ones--the latter only. Apparently the migration suppression factor of the blood serum served as the product of activated lymphocytes.     

Characterization of the delta-endotoxin of a Bacillus thuringiensis Isolate Active Against Tsetse, Glossina morsitans, and a Stem Borer, Chilo partellus

The delta-endotoxin crystals of a Bacillus thuringiensis isolate active against the tsetse fly, Glossina morsitans, were isolated from a nutrient broth culture by low speed centrifugation. Analysis of these crystals by denaturing gel electrophoresis revealed that the major component of the crystal delta-endotoxin was a protein of mol. wt ~ 120000. Upon solubilization under alkaline pH and reducing conditions, the crystal yielded a toxin of mol. wt ~ 64 000. Treatment of the toxin with bovine trypsin resulted in a shift in the mol. wt to a toxin of ~ 62000, while treatment with bovine chymotrypsin gave a toxin of ~ 60 000. Methyl green staining revealed that the endotoxin was phosphorylated, while staining with periodic acid schiff reagent showed that it was glycosylated. The carbohydrate moiety was of the high mannose type as shown by staining with fluorescein isothiocyanate conjugated to concanavalin A. Following gel permeation chromatography on a Superose 12 column, the solubilized toxin resolved into six main protein peaks, two of which had trypsin-like activity. The delta-endotoxin caused mortalities in the tsetse, G. morsitans morsitans (LC50 of 42.4mug ml-1) and 4th instar Chilo partellus larvae (LC50 of 53.8 mug ml-1), but had no effect on 3rd instar Aedes aegypti larvae.     

Albumin associated with purified pig lymphocyte plasma membrane.

Plasma-membrane preparations purified from pig lymphocytes contained a major polypeptide component of mol.wt. about 68 000. This component was identified as pig albumin by the following comparisons with authentic pig serum albumin: (a) co-migration when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions; (b) identical isoelectric points; (c) similar "fingerprints" of arginine-containing tryptic peptides; (d) reactivity with anti-(pig albumin) serum. The albumin was bound tightly to the plasma membrane. Biosynthetic labelling of pig lymphocytes under a variety of conditions failed to provide evidence that albumin was synthesized by lymphocytes, suggesting that the plasma-membrane-associated albumin was of extraneous origin. Radiolabelled pig serum albumin, however, failed to bind to the plasma-membrane fraction when added before cell disruption. Although lymphocyte plasma membrane preparations from other species possessed a polypeptide of about 68 000 mol.wt., this was judged not to be albumin on the basis of electrophoretic mobility under non-reducing conditions; also, no polypeptide was precipitated by anti-albumin sera. It is concluded that pig lymphocyte plasma-membrane preparations possess albumin which, although firmly attached, was probably of extraneous origin. This association appeared not to be common to lymphocytes from other species.     

Mechanism of the Increase in Succinate Dehydrogenase Activity in Wounded Sweet Potato Root Tissue

The large subunit (mol wt: 65,000) of sweet potato succinatedehydrogenase was isolated by SDS-polyacrylamide gel electrophoresisof a succinate dehydrogenase preparation, which had been partiallypurified from root mitochondria by solubilizing the enzyme withEmulgen 810, DEAE-cellulose column chromatography, and polyacrylamidegel electrophoresis. Antibody to the purified large subunitwas produced in a rabbit, and the antiserum obtained was judgedto be specific to the large subunit based on the results ofdouble immunodiffusion tests and immunoelectrophoresis. Rocketimmunoelectrophoresis with the antiserum showed that the increasein succinate dehydrogenase activity during the ageing of sliced,sweet potato root tissue was due to an increase in the amountof enzyme protein. Both the increases in the activity of succinatedehydrogenase and in the amount of the large subunit proteinwere inhibited by cycloheximide or chloramphenicol. We proposethat synthesis of the large subunit of succinate dehydrogenaseon cytoplasmic ribosomes is controlled by a mitochondrial translationproduct(s). ¹ This work was supported in part by a research fund from TheIshida Foundation, Nagoya, Japan. (Received November 28, 1981; Accepted February 17, 1982)