SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS : I. METHODS OF ESTIMATION

1. Methods have been described for reducing protein S-S groups, for oxidizing protein SH groups, and for estimating protein S-S and SH groups. 2. It has been found necessary in estimating the cystine content of proteins by the Folin-Marenzi method to take into account any cysteine that may be present. 3. A method for estimating the cysteine content of proteins has been described. 4. With these methods, estimations have been made of the S-S and SH groups and of the cystine and cysteine contents of a number of proteins. 5. In a denatured, but unhydrolyzed protein, the number of S-S and SH groups is equivalent to the quantity of cystine and cysteine found in the protein after hydrolysis.     

RELATION BETWEEN WATER PERMEABILITY AND INTEGRITY OF SULFHYDRYL GROUPS IN MALIGNANT AND NORMAL CELLS

When malignant cells, animal and human, were exposed in vitro to solutions of heavy metals or other selected compounds, three types of cell blebs were produced: (1) acentric blebs, arising from one side of the cell, e. g., by chlormerodrin, meralluride sodium, mercuric chloride; (2) symmetrical blebs; which completely enveloped the cell, e. g., by strong silver protein, auric chloride, p-chloromercuribenzoate; (3) scallop blebs, numerous small spherical elevations which completely covered the cell, e.g., by N-ethyl-maleimide, trivalent arsenicals, iodoacetamide. As indicated by vital stains and morphologic appearance, the blebs arose in healthy cells. They also can be made to appear in vivo in ascites tumor cells by intraperitoneal administration of a blebbing agent. All the bleb-producing chemicals have the property of reacting with protein-sulfhydryl groups by alkylation, oxidation or mercaptide formation. The three bleb types have been induced in 8 mouse and 2 rat ascites tumor cells; in 4 human and 1 mouse malignant cell lines; and in 3 normal cell lines grown in tissue culture. In contrast, cells from normal solid tissues of liver, lung, spleen, kidney, testis and brain from mouse, rat and rabbit failed to produce blebs. A possible interpretation for these observations is presented.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : II. THE RELATION BETWEEN THE SOLUBILITY OF CASEIN AND ITS CAPACITY TO COMBINE WITH BASE. THE SOLUBILITY OF CASEIN IN SYSTEMS CONTAINING THE PROTEIN AND SODIUM HYDROXIDE.

1. The solubility in water of purified, uncombined casein has previously been reported to be 0.11 gm. in 1 liter at 25°C. This solubility represents the sum of the concentrations of the casein molecule and of the soluble ions into which it dissociates. 2. The solubility of casein has now been studied in systems containing the protein and varying amounts of sodium hydroxide. It was found that casein forms a well defined soluble disodium compound, and that solubility was completely determined by (a) the solubility of the casein molecule, and (b) the concentration of the disodium casein compound. 3. In our experiments each mol of sodium hydroxide combined with approximately 2,100 gm. of casein. 4. The equivalent combining weight of casein for this base is just half the minimal molecular weight as calculated from the sulfur and phosphorus content, and one-sixth the minimal molecular weight calculated from the tryptophane content of casein. 5. From the study of systems containing the protein and very small amounts of sodium hydroxide it was possible to determine the solubility of the casein molecule, and also the degree to which it dissociated as a divalent acid and combined with base. 6. Solubility in such systems increased in direct proportion to the amount of sodium hydroxide they contained. 7. The concentration of the soluble casein compound varied inversely as the square of the hydrogen ion concentration, directly as the solubility of the casein molecule, Su, and as the constants Ka₁ and Ka₂ defining its acid dissociation. 8. The product of the solubility of the casein molecule and its acid dissociation constants yields the solubility product constant, Su·Ka₁·Ka₂ = 2.2 x 10^–12 gm. casein per liter at 25°C. 9. The solubility of the casein molecule has been estimated from this constant, and also from the relation between the solubility of the casein and the sodium hydroxide concentration, to be approximately 0.09 gm. per liter at 25°C. 10. The product of the acid dissociation constants, Ka₁ and Ka₂, must therefore be 24 x 10^–12N. 11. It is believed that these constants completely characterize the solubility of casein in systems containing the protein and small amounts of sodium hydroxide.     

l-THREONINE DEAMINASE IN MARINE PLANKTONIC ALGAE. II. DISULFIDE AND SULFHYDRYL GROUP REQUIREMENTS OF ENZYME ACTIVITY IN TWO CRYPTOPHYTES1

The “biosynthetic”l threonine (deaminating) dehydratase of 2 cryptophytes (Chroomonas salina and Hemiselmis virescens) showed sensitive inhibition from all thiols tested (dithiothreitol, cysteine, etc.) but no effect from ascorbic acid or reduced NAD. By contrast, the enzyme activities from 5 noncryptophyceaen unicellular algae (2 cyanophytes, 1 rhodophyte, 1 diatom, 1 chlorophyte) were generally not affected by any of these reagents. The thiol reagent inhibition of the cryptophyte enzymes (1) achieved saturation with 60–70% reduction in activity, (2) was considerably reduced by pretreatment of the enzymes with l -threonine and l -isoleucine, and (3) was partially reversed by subsequent treatment with arsenite and exposure to air. It was deduced that such inhibitions were caused by thiol-specific reduction of enzyme-protein disulfide groups essential for the full expression of activity and that these groups were susceptible to ready reductive cleavage and oxidative restoration. This disulfide requirement, unique to the cryptophytes, may be the first recorded case of such a property of threonine dehydratase from all forms of life hitherto studied. The additional activity requirement of the cryptophyte enzymes for sulfhydryl groups (which requirement was common to all the algal enzymes) was confirmed (1) by the study of their sensitivity to inhibition from mercurials and disulfide-sulfhydryl exchanging reagents, and (2) by the partial reversal of these inhibitions from subsequent treatment with dithio-threitol. Both cryptophyte enzymes were densitized to feedback inhibition from l -isoleucine by prior exposure to subinhibitory concentrations of HgCl₂ or dithiodipyridine.     

五步蛇主要数量性状与排毒量的相关分析

本文首次报道了五步蛇身体主要部位的数量性状与其排毒量的关系。其中我们发现与排毒量有关的部位是头积、体粗、体重、佛指长。     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : III. THE RELATION BETWEEN THE AMINO ACID COMPOSITION OF CASEIN AND ITS CAPACITY TO COMBINE WITH BASE.

1. The methods of measuring the base-combining capacities of proteins have been considered, and the constants and corrections that are employed in their calculation have been critically examined. 2. The base-combining capacities of ten casein preparations have been determined. These differed from each other to a far greater extent than can be attributed to the experimental errors involved in their measurement and calculation. The variations were, moreover, systematic in manner, and can be explained as dependent upon the method employed in the preparation of the casein. 3. Casein that had never been exposed to greater alkalinities than those in which it exists in nature combined with approximately 0.0014 mols of sodium hydroxide per gm., while casein prepared nach Hammarsten, and casein that was saturated with base during its preparation, combined with approximately 0.0018 mols of sodium hydroxide per gm. 4. 1 mol of sodium hydroxide, therefore, combined with 735 gm. of casein that had not previously been exposed to alkaline reactions, or with 535 gm. of casein that had previously been saturated with base. 5. If the minimal molecular weight of casein, based upon its tryptophane content, is placed at 12,800, the native protein must, therefore, contain approximately eighteen acid groups, and in addition six acid groups that are released in alkaline solutions, and presumably represent internally bound groups. The total base-combining capacity therefore represents that of a substance with a molecular weight of 12,800 and containing twenty-four acid valences. 6. This base-combining capacity is no greater than can be accounted for on the basis of our knowledge of the structure and composition of casein. On the basis of a molecular weight of 12,800 casein contains at least 19 molecules of glutamic acid, 4 of aspartic, and 8 of hydroxyglutamic acid. If the amino acids in the protein molecule are bound to each other in polypeptide linkage, each of these thirty-one dicarboxylic acids should yield terminal groups. The ammonia in casein suggests that twelve of these groups are bound as amides. As many as nineteen carboxyl groups may, therefore, be free in the protein molecule. 7. Casein contains phosphorus. If this phosphorus represents phosphoric acid, and if we consider that all of the valences of this acid are either themselves free, or that they have liberated carboxyl groups by entering into the structure of the protein molecule, casein should contain nine additional acid groups. 8. Recent analytical results, therefore, indicate that casein contains at least nineteen, and possibly twenty-eight, free acid groups. The physicochemical measurements presented suggest that casein combines with base as though it contained twenty-four acid groups, of which six, or one-fourth, appear to be bound in the native protein. These experimental results are therefore in close agreement with the expectation on the basis of the classical theory of protein structure.     

THE RELATIONSHIP BETWEEN DIURNAL VARIATION OF THE NUMBER OF CELLS IN MITOSIS AND OF THE NUMBER OF CELLS SYNTHESIZING DNA IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH

1. The numbers of cells in mitosis and in DNA synthesis in the epithelium of the hamster cheek pouch have been studied at different times of the day and night. 2. By accumulation of mitotic cells using colcemid, both the rate of entry of cells into mitosis and the duration of mitosis have been estimated at two different times of day. 3. A diurnal variation has been demonstrated in both the mitotic index and in the tritiated thymidine labelling index. Although these variations are of different amplitude and timing, the experimental data fit closely to the hypothesis that the diurnal mitotic variation is the result of a partially synchronous population moving through the DNA synthetic period. No direct action on the mitotic process need be postulated. 4. From the results of mitotic accumulation, it is clear that the rate of entry of cells into mitosis depends on the time of day at which this is studied. There is also the possibility that the duration of mitosis is slightly longer when the mitotic index is high. 5. It is concluded that, at least in the epithelium of the hamster cheek pouch, the diurnal rhythm in the number of mitoses present is a reflection of the diurnal variation in the number of cells synthesizing DNA at some time earlier. Small fluctuations in the mitotic pattern imposed by this partially synchronous population moving from S into mitosis, could be caused by slight variations in the duration of mitosis.     

胃癌survivin基因mRNA和蛋白的表达与临床病理关系

目的　研究胃癌组织中survivin基因的mRNA和蛋白表达情况及其与临床病理参数的关系。方法　应用原位杂交方法和免疫组化SP法检测 5 4例胃癌 ,34例胃良性病变 ,2 0例胃正常组织标本中survivin基因mRNA及蛋白的表达。并对其与临床病理因素和二者的关系进行分析。结果　Survivin蛋白表达阳性率在胃癌、良性疾病组织和正常组织分别为 87 0 % (47/ 5 4 )、 2 3 5 % (8/ 34)和 15 % (3/ 2 0 ) ;SurvivinmRNA阳性率为 79 6 % (43/ 5 4 )、 2 3 5 % (8/ 34)和2 0 % (4/ 2 0 )。胃癌组远大于正常胃组织和良性疾病组 ,而正常胃组织与胃良性疾病组之间差异无显著性。SurvivinmRNA与蛋白在胃癌中的表达呈正相关 (rs=0 6 79,P <0 0 5 )。且其阳性率高低与性别、年龄、组织学类型无关 ;而与淋巴结转移、TNM分期、组织学分级有关。结论　SurvivinmRNA与蛋白在胃癌中表达较高 ,且与淋巴结转移、TNM分期、组织学分级有关 ,它可作为评估胃癌生物学行为和判断预后的生物学指标。     

PERIODIC CHANGES IN THE CONTENT OF PROTEIN-BOUND SULFHYDRYL GROUPS AND TENSION AT THE SURFACE OF STARFISH OOCYTES IN CORRELATION WITH THE MEIOTIC DIVISION CYCLE*

The sulfhydryl content of protein and the tension at the surface were measured for starfish oocytes from the first meiotic division to the cleavage stage. A cyclic change in both the protein-SH and the tension at the surface was found to accompany the division cycle, including the first and second meiotic divisions. It is concluded that the unequal meiotic divisions share the same character with the equal divisions of cleavage, with respect to changes both in the protein-SH and the tension at the surface.     

THE INACTIVATION OF TRYPSIN : II. THE EQUILIBRIUM BETWEEN TRYPSIN AND THE INHIBITING SUBSTANCE FORMED BY ITS ACTION ON PROTEINS.

1. A study has been made of the equilibrium existing between trypsin and the substances formed in the digestion of proteins which inhibit its action. 2. This substance could not be obtained by the hydrolysis of the proteins by acid or alkali. It is dialyzable. 3. The equilibrium between this substance (inhibitor) and trypsin is found to agree with the equation, trypsin + inhibitor ⇌ trypsin-inhibitor The equilibrium is reached instantaneously and is independent of the substrate concentration. If it be further assumed that the rate of hydrolysis is proportional to the concentration of the free trypsin and that the equilibrium conforms to the law of mass action, it is possible to calculate the experimental results by the application of the law of mass action. 4. The equilibrium has been studied by varying (a) the concentration of the inhibiting substance, (b) the concentration of trypsin, (c) the concentration of gelatin, and (d) the concentration of trypsin and inhibitor (the relative concentration of the two remaining the same). In all cases the results agree quantitatively with those predicted by the law of mass action. 5. It was found that the percentage retarding effect of the inhibiting substance on the rate of hydrolysis is independent of the hydrogen ion concentration between pH 6.3 and 10.0. 6. The fact that the experimental results agree with the mechanism outlined under 3, is contrary to the assumption that any appreciable amount of trypsin is combined with the gelatin at any one time; i.e., the velocity of the hydrolysis must depend on the time required for such a compound to form rather than for it to decompose. 7. The experiments may be considered as experimental proof of the validity of Arrhenius'' explanation of Schütz''s rule as applied to trypsin digestion. 8. Inactivated trypsin does not enter into the equilibrium.     

VALENCY RULE AND ALLEGED HOFMEISTER SERIES IN THE COLLOIDAL BEHAVIOR OF PROTEINS : II. THE INFLUENCE OF SALTS.

It is shown by the older experiments by Loeb and by the experiments reported in this paper that the effect of salts on the membrane potentials, osmotic pressure, swelling of gelatin chloride, and that type of viscosity which is due to the swelling of protein particles, depends only on the valency but not on the chemical nature of the anion of the salt, and that the cation of the salt has no effect on these properties, if the pH of the protein solution or protein gel is not altered by the salt. The so called Hofmeister series of salt effects on these four properties are purely fictitious and due to the failure of the former authors to measure the hydrogen ion concentration of their protein solutions or gels and to compare the effects of salts at the same pH of the protein solution or the protein gel. These results confirm the older experiments of Loeb and together they furnish a further proof for the correctness of the idea that the influence of electrolytes on the four properties of proteins is determined by membrane equilibria. Such properties of proteins which do not depend on membrane equilibria, such as solubility or cohesion, may be affected not only by the valency but also by the chemical nature of the ions of a salt.     

THE RELATIONSHIP BETWEEN DNA SYNTHESIS AND NUCLEAR PROTEINS IN THE CEREBRUM AND CEREBELLUM OF 8-DAY-OLD RATS

In-8-day-old rats the higher rate of DNA replication in cerebellum than in cerebrum is accompanied by an enhanced synthesis of nuclear proteins. The greatest difference between the incorporation of tritiated leucine into proteins of cerebral and cerebellar cell nuclei occurs in the acid-soluble deoxyribonucleoproteins. However, the specific radioactivity of the acidic deoxyribonucleoproteins is similar in both tissues. The relative content of these proteins and the activity of the RNA polymerase is higher in cerebrum than in cerebellum. The results suggest that in the cerebrum of young rats these proteins are mainly concerned with the regulation of RNA synthesis.     

STUDIES ON THE ANOMALOUS VISCOSITY AND FLOW-BIREFRINGENCE OF PROTEIN SOLUTIONS : II. ON DILUTE SOLUTIONS OF PROTEINS FROM EMBRYONIC AND OTHER TISSUES

1. An extensive investigation has been made of protein particle shape using the methods of flow-birefringence and anomalous viscosity measurement in the coaxial cell. 2. As a result of investigations on a number of proteins, it is concluded that they may be divided into four groups. Group A consists of those which show flow-anomaly both in the bulk phase and in the surface film. These also show flow-birefringence in the bulk phase. Examples: tobacco mosaic disease virus nucleoprotein; myosin. Though corpuscular proteins, they have elongated particles before denaturation. Group B consists of those which show flow-anomaly only (in the first instance) in the surface film, and no flow-birefringence in the bulk phase. They are probably close to spherical in shape in solution, but form elongated particles as they denature in the surface film. After this process has been completed, they may show flow-anomaly also in the bulk phase. Some proteins show flow-anomaly in the surface film immediately it forms, others only show it after a certain time has elapsed for the building up of the film. We designate the former as group B₁ and the latter as group B₂. Group B₁, immediate surface film flow-anomaly. Examples: serum euglobulin, amphibian embryo euglobulin b. Group B₂, slowly appearing surface film flow-anomaly. After the film has once been fully formed and then dispersed by shaking, the solution may have the properties of that of a protein in group B₁; i.e., anomalous flow in the film may occur immediately on testing in the viscosimeter. Examples: avian ovalbumin, amphibian embryo pseudoglobulin. Group C consists of those proteins which show flow-anomaly neither in the bulk phase nor in the surface film, under the conditions used by us. They are probably close to spherical in shape. Examples: insulin, methaemoglobin, amphibian embryo euglobulin c, mucoproteins. 3. The theoretical significance of protein fibre molecules, whether native or formed by denaturation in the living cell, is discussed, especially in relation to experimental morphology and cytology.     

三种寄生蜂虫体可溶性蛋白组成及蝶蛹金小蜂卵黄蛋白的分子特性分析(英文)

聚丙烯凝胶电泳PAGE和毛细管电泳分析结果表明 ,蝶蛹金小蜂雌蜂个体和卵巢中明显存在雌性特异蛋白 ,即卵黄蛋白。但茧蜂科的螟长距茧蜂Macrocentruslinears和菜蛾盘绒茧蜂Cotesiaplutella雌雄虫体可溶性蛋白间无明显差异。SDS PAGE梯度电泳结果表明 ,蝶蛹金小蜂卵黄原蛋白 (Vg)和卵黄磷蛋白 (Vt)均由二个分子量接近的亚基组成 ,分子量各为 74 4和 5 2 8KD。双向免疫扩散和PAGE梯度电泳都显示该蜂隐成蜂的雌性虫体及雌蜂血淋巴、脂肪体和卵巢中都有Vg或Vt,且卵黄原蛋白是由雌蜂脂肪体合成     

THE EQUILIBRIA BETWEEN NATIVE AND DENATURED HEMOGLOBIN IN SALICYLATE SOLUTIONS AND THE THEORETICAL CONSEQUENCES OF THE EQUILIBRIUM BETWEEN NATIVE AND DENATURED PROTEIN

The denaturation of hemoglobin by salicylate in neutral solution is completely reversible. There is a mobile equilibrium between native and denatured hemoglobin in neutral salicylate solution. The higher the salicylate concentration the greater is the percentage denaturation. When there is a mobile equilibrium between the native and denatured forms of a protein, denaturation is caused by the addition of any substance which has a greater affinity for the denatured than for the native form. Theoretically the heat of denaturation must vary with the denaturing agent and must depend on the heat of combination of the denaturing agent with the protein.