Cross-linking of cold-insoluble globulin by fibrin-stabilizing factor.

Cold-insoluble globulin (CI globulin) was purified from human plasma and identified on the basis of its sedimentation coefficient, electrophoretic mobility, and concentration in normal plasma. CI globulin was distinguished from antihemophilic factor (AHF) by amino acid analysis, position of elution from 4% agarose, and electrophoretic migration in polyacrylamide gels in the presence of sodium dodecyl sulfate without prior reduction. CI globulin and AHF could not be distinguished by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction and probably have very similar subunit molecular weights. CI globulin apparently consists of two polypeptide chains, each of molecular weight 2.0 x 10(5), held together by disulfide bonds. CI globulin was a substrate for activated fibrin-stabilizing factor (FSF, blood coagulation factor XIII). FSF catalyzed the incorporation of a fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, into CI globulin and also catalyzed the cross-linking of CI globulin into multimers, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction. In the presence of fibrin, cross-linking of CI globulin by FSF occurred without the formation of CI globulin multimers. Instead, polypeptides with apparent molecular weights of 2.6 x 10(5) and 3.0 x 10(5) were seen. The formation of these polypeptides coincided with the loss of the alpha chain of fibrin and CI globulin. The polypeptides were not seen when fibrin alone was cross-linked. The formation of the polypeptides was greater in fine clots than in coarse clots, and greater in clots incubated at 0 degrees than in clots incubated at 37 degrees. In clots made from purified fibrinogen, CI globulin, and FSF, the concentration of CI globulin in the clot liquor was greater if either FSF or calcium ion was omitted and cross-linking did not take place. These observations suggest that CI globulin is enzymically cross-linked to one of the chains of fibrin, most likely the alpha chain, and is thus covalently incorporated into the fibrin clot. CI globulin is very similar to a protein in the plasma membrane of fibroblasts. The cross-linking of CI globulin to itself and to fibrin may typify reactions also involving the fibroblast membrane protein.     

Galactolipid formation in chloroplast envelopes. I. Evidence for two mechanisms in galactosylation

Bovine and human thyroxine-binding globulin were purified from serum by a three-step purification procedure which comprised affinity chromatography consecutively on thyroxine- and Concanavalin A--Sepharose and finally preparative polyacrylamide gel electrophoresis. The molecular weights of the two proteins were similar (54 000) as well as their carbohydrate contents while some differences in amino acid composition were found. Rabbit antiserum against bovine thyroxine-binding globulin reacted with human thyroxine-binding globulin with no sign of spur formation.     

The solubilization and degradation of pumpkin seed globulin during germination

Pumpkin seed globulin decreased 88% during the first 4 daysof germination. This decrease was concomitant with a 2.5 foldincrease in water soluble protein which arose directly fromthe water insoluble globulin. The sequence of solubilizationand breakdown of the globulin was followed through 12 days ofgermination. Pumpkin seed globulin was determined to have subunitsof 56,000 daltons, while the new water soluble protein consistedof two proteins, as determined by sodium dodecyl sulfate polyacrylamideelectrophoresis; one having a molecular weight of 42,000 daltonsand the other 28,000 daltons. Trypsin mimicked the first stepof the breakdown by solubilizing pumpkin seed globulin to yieldidentical digestion products as were obtained in vitro. Maximumproteolytic acitvity, as measured by the release of ninhydrinpositive, materials occurred at 6 days of germination, at whichtime both the concentration of free amino acids and the incorporationof ¹⁴C into amino acids increased rapidly. A second proteolyticenzyme system which solubilized the pumpkin seed globulin butdid not act on hemoglobin, casein, or bovine serum albumin reachedits maximum activity at two days of germination. (Received April 17, 1978; )     

Effect of specific antibodies on active immunity development in those inoculated with an antirabies vaccine

The study of the effect of gamma globulin introduced in different doses (0.5 and 0.25 ml/mg) in combination with Fermi rabies vaccine (observations on humans were made) and with cerebral rabies vaccine inactivated by UV irradiation (in animal experiments) demonstrated that the injection of the higher doses of gamma globulin resulted in lower geometrical mean of antibody titers. Therefore, in combined administration of rabies vaccine and gamma globulin for postexposure rabies prevention it is advisable to reduce the dose of gamma globulin by one-half.     

蛋白酶抑制剂对含水生菜种子耐冻性的影响

选取生菜(Lactuca sativa)种子作为试材,外源添加蛋白酶抑制剂2-硝基苯甲酸[5,5'-Dithiobis-(2-nitrobenzoic acid),DTNB]对种子吸胀处理,通过程序降温,分析2-硝基苯甲酸对低温下种子发芽率及生理活性的影响。结果表明,低温下含水生菜种子的致死温度为–20 ℃;外源添加2-硝基苯甲酸2 mmol·L–1时种子发芽率最高,即对种子活性的保护效果显著;在此浓度下种子内SOD活性比对照提高1.38倍,羟基自由基清除能力提高1.17倍;与对照组相比产生两种新的蛋白11 S种子贮藏球蛋白Jug r4和11 S种子贮藏球蛋白2,均属于球蛋白家族,可提高含水种子的耐冻性;低温下种子内积累更小分子量的球蛋白多肽,对种子具有低温保护效果。综上,低温条件下生菜种子产生一定的抗冷反应,外源添加2 mmol·L–1 2-硝基苯甲酸可提高含水种子发芽率及生理活性,产生抗冻蛋白,积累更小分子量的球蛋白多肽进而提高种子抗冻性。     

A Component of Wheat Flour Globulin Polymerized at Alkaline Sides and Depolymerized by Reduction Reversibly

1. The SDS-disc electrophoretical analysis of wheat flour globulin revealed that upon incubation with “Kansui,” a mixture of alkali carbonates, one (No. 9) of the components disappeared and another component (No. 11) increased significantly whose molecular weight was estimated to be more than one million. Incubation of flour globulin with “Kansui” resulted in decrease of SH groups of the globulin.

2. However, a mild reduction of the globulin incubated with “Kansui” caused reappearance of component No. 9 and a decrease of component No. 11.

3. Previous treatment of flour globulin with PCMB or AN completely inhibited such effects of “Kansui” as described above.

4. A fraction containing component No. 9 of flour globulin which was the main component responsible to polymerization by “Kansui,” was isolated from “Kansui”-treated globulin by chromatography on columns of Sephadex G200 and Sepharose 4B.

5. The component protein thus isolated showed a typical phenomenon of reversibility in depolymerization to the original component corresponding to No. 9 component by chemical reduction and polymerization to a component corresponding to No. 9 at alkaline sides.

     

Comparison of human thyroxine-binding globulin purification by affinity chromatography procedures

Three procedures for the isolation of thyroxine-binding globulin from human serum, using affinity chromatography on triiodothyronine (T3) linked to Sepharose (A), thyroxine (T4) linked to Sepharose (B) or T3 linked to epoxy-Sepharose (C) as the first purification step, were compared. With the use of additional purification steps, the three procedures yielded pure thyroxine-binding globulin without desialylation. With procedure A, the initial binding of T4-binding globulin to T3-Sepharose was very low, yielding a poor final recovery (17%). Procedure B gave the highest yield (35%) after a three-step purification, with a low T4 content (0.15-0.30 mol/mol). Procedure C also gave a high yield (28%) after only two purification steps, with a T4 content greater than 0.7 mol/mol. The microheterogeneity of T4-binding globulin obtained with these three procedures was demonstrated by isoelectric focusing: five major bands were observed between pH 4.1 and 4.6, and intermediate faint bands (often doublets) in the same pH range. However, with procedures A and C, the most acidic bands (pH 4.10-4.20) were always absent. Thyroxine-binding globulin was preincubated with radioactively labelled T3 or T4 and the hormone-protein complex was analyzed by isoelectric focusing. The binding of T3--compared to that of T4--was reduced in the most acidic protein subspecies. These results suggest differences in the thyroid hormone binding properties of the various subspecies of human T4-binding globulin.     

Effects of Hyaluronic Acid and γ–Globulin Concentrations on the Frictional Response of Human Osteoarthritic Articular Cartilage

Synovial fluid plays an important role in lubricating synovial joints. Its main constituents are hyaluronic acid (HA) and γ–globulin, acting as boundary lubricants for articular cartilage. The aim of the study was to demonstrate the concentration-dependent effect of HA and γ–globulin on the boundary-lubricating ability of human osteoarthritis (OA) cartilage. Normal, early and advance stage articular cartilage samples were obtained from human femoral heads and in presence of either HA or γ–globulin, cartilage frictional coefficient (µ) was measured by atomic force microscopy (AFM). In advanced stage OA, the cartilage superficial layer was observed to be completely removed and the damaged cartilage surface showed a higher µ value (∼0.409) than the normal cartilage surface (∼0.119) in PBS. Adsorbed HA and γ–globulin molecules significantly improved the frictional behavior of advanced OA cartilage, while they were ineffective for normal and early OA cartilage. In advanced-stage OA, the concentration-dependent frictional response of articular cartilage was observed with γ–globulin, but not with HA. Our result suggested that HA and γ–globulin may play a significant role in improving frictional behavior of advanced OA cartilage. During early-stage OA, though HA and γ–globulin had no effect on improving frictional behavior of cartilage, however, they might contribute to disease modifying effects of synovial fluid as observed in clinical settings.     

Subunit structure and functional properties of the predominant globulin of perilla (Perilla frutescens var. frutescens) seeds

Approximately 40% of defatted perilla seeds consists of proteins which are primarily composed of globulin (84%). The amino acid profile of perilla proteins demonstrated balanced amounts of all essential amino acids, except for lysine. The molecular mass of the predominant globulin was estimated to be 340 kDa by gel filtration. This globulin was separated into three intermediary subunits (54, 57 and 59 kDa) by SDS-PAGE. It is suggested from these results that the globulin exists as a hexamer. A treatment with 50 mM dithiothreitol enabled the intermediary subunits to be separated into three acidic subunits (31-34 kDa) and four basic subunits (23-25 kDa). It is interesting that this subunit structure is the same as that of sesame α-globulin, despite them coming from different families. Compared to sesame α-globulin, the heat-induced gel of perilla globulin had better water-holding ability, despite it displaying the same degree of gel hardness.     

Antibodies against the gH/gL/UL128/UL130/UL131 complex comprise the majority of the anti-cytomegalovirus (anti-CMV) neutralizing antibody response in CMV hyperimmune globulin

Anti-cytomegalovirus (anti-CMV) hyperimmune globulin (HIG) has demonstrated efficacy in preventing CMV disease in solid-organ transplant patients as well as congenital disease when administered to pregnant women. To identify the neutralizing component of cytomegalovirus hyperimmune globulin (CMV-HIG), we performed serial depletions of CMV-HIG on cell-surface-expressed CMV antigens as well as purified antigens. Using this approach, we demonstrate that the major neutralizing antibody response is directed at the gH/gL/UL128/UL130/UL131 complex, suggesting little role for anti-gB antibodies in CMV-HIG neutralization.     

Gas Chromatographic Analysis of Higher Alcohols and Ethyl Acetate in Table Wines

Approximately 40% of defatted perilla seeds consists of proteins which are primarily composed of globulin (84%). The amino acid profile of perilla proteins demonstrated balanced amounts of all essential amino acids, except for lysine. The molecular mass of the predominant globulin was estimated to be 340 kDa by gel filtration. This globulin was separated into three intermediary subunits (54, 57 and 59 kDa) by SDS–PAGE. It is suggested from these results that the globulin exists as a hexamer. A treatment with 50 mM dithiothreitol enabled the intermediary subunits to be separated into three acidic subunits (31–34 kDa) and four basic subunits (23–25 kDa). It is interesting that this subunit structure is the same as that of sesame α-globulin, despite them coming from different families. Compared to sesame α-globulin, the heat-induced gel of perilla globulin had better water-holding ability, despite it displaying the same degree of gel hardness.     

Separation and characterization of oat globulin polypeptides

The storage globulin of oat seeds was separated into its acidic (α) and basic (β) polypeptides by ion-exchange chromatography in 6 m urea and further characterized by several electrophoretic techniques. Molecular weights of the α and β polypeptides were 32,500–37,500 and 22,000–24,000, respectively. The unreduced protein existed as disulfide-linked αβ species of molecular weight 53,000–58,000. Isoelectric points were approximately 5.9–7.2 (α) and 8.7–9.2 (β). Two-dimensional electrophoresis showed considerable heterogeneity within both groups of polypeptides. More complete amino acid analyses of the globulin and its polypeptides are presented along with a proposed structure of the native protein based on previous and present data. Similarities were noted between the oat globulin and the legumin (11 S) class of storage proteins in certain legumes.     

Fatty acid requirement of Treponema denticola and Treponema vincentii

Treponema denticola and Treponema vincentii were cultured in a medium supplemented with either 0.2 or 0.4% (w/v) alpha globulin in place of serum. The active factor(s) in alpha globulin was stable at pH 7.0 to autoclaving and was nondialyzable. Extraction of lipids from alpha globulin showed that both protein and lipid, supplied by the alpha globulin, were required for maximal growth of these two oral treponemes. The lipid component was investigated by adding sodium salts of long-chain fatty acids to the basal medium supplemented with 0.4% delipified alpha globulin. The lipid component of alpha globulin was replaced by either oleic acid (cis-18:1(9)) or by elaidic acid (trans- 18:1 (9)0. No other saturated or unsaturated fatty acid tested could support good growth. Tween 80 (polysorbitan monooleate) was the only Tween compound able to support maximal growth of T. denticola. The cellular lipids of T. denticola, grown with oleate in broth supplemented with 0.4% delipified alpha globulin, were extracted and analyzed by gas chromatography. The principle fatty acids were myristic, pentadecanoic, and palmitic acids. Lesser amounts of oleic acid, eicosadienoic acid, and an unidentified fatty acid (retention time, 88 min) were also detected. Treponema denticola appears to be capable of limited synthesis of cellular fatty acids such as myristic, pentadecanoic, and palmitic acids from oleic acid.     

Effects of anti-interferon serum on growth and regression of Moloney sarcoma virus induced tumours in mice

Administration of sheep anti-mouse interferon serum or globulin to weanling BALB/c mice infected with Moloney sarcoma virus (MSV) accelerated the growth of tumours at the site of virus inoculation, increased the number and size of tumours, and inhibited their regression. The outcome of the MSV-induced disease after injection of anti-interferon globulin depended on the age of mice. The antigenic stimulation by normal sheep serum or globulin also enhanced the growth and mortality due to MSV but to a significantly lesser extent than anti-interferon globulin.     

Sex hormone-binding globulin selectively modulates estradiol-regulated genes in MCF-7 cells.

Human sex hormone-binding globulin inhibits the effects of estradiol on proliferation and apoptosis of breast cancer cells. We report here the effect of sex hormone-binding globulin on estradiol regulation of gene expression in MCF-7 breast cancer cells using a selected set of genes. Estradiol upregulates genes that are positive regulators of proliferation (e.g., bcl-2, c-fos, c-myc, cyclin D) or/and related to more aggressive form of breast cancer (e.g. BRCA-1, EGF-R) and downregulates two genes (c-jun and ERalpha). Sex hormone-binding globulin modulates only a selected group of estradiol-controlled genes (inhibiting upregulation of bcl-2, c-myc, EGF-R, PR, and downregulation of ERalpha), starting 48 hours after treatment. Our study demonstrates that in breast cancer cells, sex hormone-binding globulin is effective on few selected genes which are involved in cell growth and apoptosis or related to cell estrogen-dependence and that the protein regulation of estradiol effect is selected and specific. Sex hormone-binding globulin action in estrogen breast cancer cells is strongly associated to cell growth and estrogen-sensitivity.     

Effect of multiple gamma globulin administration on the effectiveness of seroprophylaxis in hepatitis A

The data provided by immunological and epidemiological studies carried out to determine the influence of the multiple injections of 10% commercial gamma globulin on the level of antigamma globulin formation and, in this connection, on the effectiveness of the prophylaxis of hepatitis A are discussed. A sharp increase in the titers of antigamma globulin in persons receiving multiple gamma globulin injections is shown. The increased amount of antibodies to gamma globulin, appearing as the result of the multiple use of this preparation, decreases the effectiveness of the seroprophylaxis of hepatitis A.     

Affinity Chromatography of the Major Seed Protein of the Bean (Phaseolus vulgaris L.)

The major globulin of the French bean (Phaseolus vulgaris L.) undergoes a reversible pH-dependent polymerization. At pH values above 6.5, the monomeric form of the protein predominates; and at pH values below 6.5, the protein occurs as a polymer, probably a tetramer. At extremes of pH, the protein dissociates further into peptides. The reversible pH-dependent interaction between globulin subunits is used in this report as the basis for an affinity chromatography procedure for isolation of the globulin. The major globulin from several genetic variants can be obtained in gram quantities and does not indicate the presence of any impurities on discontinuous sodium dodecyl sulfate gel electrophoresis.