THE EFFECT OF THE CONCENTRATION OF ENZYME ON THE RATE OF DIGESTION OF PROTEINS BY PEPSIN

1. In certain cases the rate of digestion of proteins by pepsin is not proportional to the total concentration of pepsin. 2. It is suggested that this is due to the fact that the enzyme in solution is in equilibrium with another substance (called peptone for convenience) and that the equilibrium is quantitatively expressed by the law of mass action, according to the following equation. See PDF for Equation It is assumed that only the uncombined pepsin affects the hydrolysis of the protein. 3. The hypothesis has been put in the form of a differential equation and found to agree quantitatively with the experimental results when the concentration of pepsin, peptone, or both is varied. 4. Pepsin inactivated with alkali enters the equilibrium to the same extent as active pepsin. 5. Under certain conditions (concentration of peptone large with respect to pepsin, and concentration of substrate relatively constant) the relative change in the amount of active pepsin is inversely proportional to the concentration of peptone and the equation simplifies to Schütz''s rule. 6. An integral equation is obtained which holds for the entire course of the digestion (except for the first few minutes) with varying enzyme concentration. This equation is identical in form with the one derived by Arrhenius for the action of ammonia on ethyl acetate. 7. It is pointed out that there are many analogies between the action of pepsin on albumin solutions and the action of toxins on an organism.     

THE MECHANISM OF THE INFLUENCE OF ACIDS AND ALKALIES ON THE DIGESTION OF PROTEINS BY PEPSIN OR TRYPSIN

1. The effect of the addition of acid on the amount of ionized protein has been compared with the effect on the rate of digestion of gelatin, casein, and hemoglobin by pepsin. 2. A similar comparison has been made of the addition of alkali in the case of trypsin with gelatin, casein, hemoglobin, globin, and edestin. 3. In general, the rate of digestion may be predicted from the amount of ionized protein as determined by the titration curve or conductivity. The rate of digestion is a minimum at the isoelectric point of the protein and a maximum at that pH at which the protein is completely combined with acid or alkali to form a salt. 4. The physical properties of the protein solution have little or no effect on the rate of digestion.     

THE SIGNIFICANCE OF THE HYDROGEN ION CONCENTRATION FOR THE DIGESTION OF PROTEINS BY PEPSIN

The experiments described above show that the rate of digestion and the conductivity of protein solutions are very closely parallel. If the isoelectric point of a protein is at a lower hydrogen ion concentration than that of another, the conductivity and also the rate of digestion of the first protein extends further to the alkaline side. The optimum hydrogen ion concentration for the rate of digestion and the degree of ionization (conductivity) of gelatin solutions is the same, and the curves for the ionization and rate of digestion as plotted against the pH are nearly parallel throughout. The addition of a salt with the same anion as the acid to a solution of protein already containing the optimum amount of the acid has the same depressing effect on the digestion as has the addition of the equivalent amount of acid. These facts are in quantitative agreement with the hypothesis that the determining factor in the digestion of proteins by pepsin is the amount of ionized protein present in the solution. It was shown in a previous paper that this would also account for the peculiar relation between the rate of digestion and the concentration of protein. The amount of ionized protein in the solution depends on the amount of salt formed between the protein (a weak base) and the acid. This quantity, in turn, according to the hydrolysis theory of the salts of weak bases and strong acids, is a function of the hydrogen ion concentration, up to the point at which all the protein is combined with the acid as a salt. This point is the optimum hydrogen ion concentration for digestion, since the solution now contains the maximum concentration of protein ions. The hydrogen ion concentration in this range therefore is merely a convenient indicator of the amount of ionized protein present in the solution and takes no active part in the hydrolysis. After sufficient acid has been added to combine with all the protein, i.e. at pH of about 2.0, the further addition of acid serves to depress the ionization of the protein salt by increasing the concentration of the common anion. The hydrogen ion concentration is, therefore, no longer an indicator of the amount of ionized protein present, since this quantity is now determined by the anion concentration. Hence on the acid side of the optimum the addition of the same concentration of anion should have the same influence on the rate of digestion irrespective of whether it is combined with hydrogen or some other ion (provided, of course, that there is no other secondary effect of the other ion). The proposed mechanism is very similar to that suggested by Stieglitz and his coworkers for the hydrolysis of the imido esters. Pekelharing and Ringer have shown that pure pepsin in acid solution is always negatively charged; i.e., it is an anion. The experiments described above show further that it behaves just as would be expected of any anion in the presence of a salt containing the protein ion as the cation and as has been shown by Loeb to be the case with inorganic anions. Nothing has been said in regard to the quantitative agreement between the increasing amounts of ionized protein found in the solution (as shown by the conductivity values) and the amount predicted by the hydrolysis theory of the formation of salts of weak bases and strong acids. There is little doubt that the values are in qualitative agreement with such a theory. In order to make a quantitative comparison, however, it would be necessary to know the ionization constant of the protein and of the protein salt and also the number of hydroxyl (or amino) groups in the protein molecule as well as the molecular weight of the protein. Since these values are not known with any degree of certainty there appears to be no value at present in attempting to apply the hydrolysis equations to the data obtained. It it clear that the hypothesis as outlined above for the hydrolysis of proteins by pepsin cannot be extended directly to enzymes in general, since in many cases the substrate is not known to exist in an ionized condition at all. It is possible, however, that ionization is really present or that the equilibrium instead of being ionic is between two tautomeric forms of the substrate, only one of which is attacked by the enzyme. Furthermore, it is clear that even in the case of proteins there are difficulties in the way since the pepsin obtained from young animals, or a similar enzyme preparation from yeast or other microorganisms, is said to have a different optimum hydrogen ion concentration than that found for the pepsin used in these experiments. The activity of these enzyme preparations therefore would not be found to depend on the ionization of the protein. It is possible of course that the enzyme preparations mentioned may contain several proteolytic enzymes and that the action observed is a combination of the action of several enzymes. Dernby has shown that this is a very probable explanation of the action of the autolytic enzymes. The optimum hydrogen ion concentration for the activity of the pepsin used in these experiments agrees very closely with that found by Ringer for pepsin prepared by him directly from gastric juice and very carefully purified. Ringer''s pepsin probably represents as pure an enzyme preparation as it is possible to prepare. There is every reason to suppose therefore that the enzyme used in this work was not a mixture of several enzymes.     

THE INFLUENCE OF THE SUBSTRATE CONCENTRATION ON THE RATE OF HYDROLYSIS OF PROTEINS BY PEPSIN

1. It is pointed out that the apparent exceptions to the law of mass action found in enzyme reactions may be found in catalytic reactions in strictly homogeneous solutions. 2. These deviations in the rate of reaction from the law of mass action may be explained by the hypothesis that the active mass of the reacting substances is not directly proportional to the total concentration of substance taken. 3. In support of this suggestion it is shown that for any given concentration of pepsin the relative rate of digestion of concentrated and of dilute protein solutions is always the same. If the rate of digestion depended on the saturation of the surface of the enzyme by substrate the relative rate of digestion of concentrated protein solutions should increase more rapidly with the concentration of enzyme than that of dilute solutions. This was found not to be true, even when the enzyme could not be considered saturated in the dilute protein solutions. 4. The rate of digestion and the conductivity of egg albumin solutions of different concentration were found to be approximately proportional at the same pH. This agrees with the hypothesis first expressed by Pauli that the ionized protein is largely or entirely the form which is attacked by the enzyme. 5. The rate of digestion is diminished by a very large increase in the viscosity of the protein solution. This effect is probably a mechanical one due to the retardation of the diffusion of the enzyme.     

EFFECT OF PEPSIN PRETREATMENT ON PULSE-CYTOPHOTOMETRIC DNA HISTOGRAMS

Samples of mitotic L-cells were investigated after different preparation and staining procedures using the technique of pulse-cytophotometry. It is shown that most mitotic cells which should appear in the second peak of the DNA histogram are disintegrated or separated into halves by pepsin pretreatment. Hence, the designation‘G₂+ M’for the second peak is not correct for this preparative method. This should be taken into account in cell kinetic investigations performed after pepsin pretreatment.     

INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN

In the presence of iodine at pH 5.0–6.0 a solution of pepsin absorbs iodine and the specific proteolytic activity of the solution decreases. The activity is less than 1 per cent of the original activity when the number of iodine atoms per mol of pepsin is 35–40. If the pH is 4.5 or less, iodine reacts very slowly and there is a correspondingly slower loss in activity. Glycyl tyrosine reacts with iodine in a manner similar to pepsin. Experiments were performed to determine the extent to which oxidation of pepsin by iodine occurs during iodination, and if such oxidation were responsible for the loss in enzymatic activity. Although the results were not absolutely decisive, there seems to be no appreciable oxidation taking place during iodination and no relationship between the slight oxidation and loss in peptic activity. From a dialyzed preparation of completely iodinated pepsin which was inactive and contained 13.4 per cent bound iodine, 82 per cent of the iodine was obtained in a solution which analyzed as a solution of diiodo-tyrosine. Because of the presence of a material which contained no iodine and prevented quantitative crystallization, only 53 per cent of the iodine containing substance could be crystallized. This 53 per cent was, however, identified as diiodo-tyrosine. The part of the titration curve which in pepsin and most proteins represents the phenolic group of tyrosine was, in the curve for iodinated pepsin, shifted toward the acid region as expected. From these results, it appears that the loss in proteolytic activity of pepsin, when treated with iodine under the specified conditions, is due to the reaction of the iodine with the tyrosine in pepsin.     

THE ABSORPTION OF ULTRA-VIOLET RADIATION BY CRYSTALLINE PEPSIN

Determination of the absorption spectra of pure preparations of Northrop''s crystalline pepsin inactivated by irradiation with ultra-violet light shows that the total absorption in the ultra-violet region of the spectrum increases with the degree of inactivation. This increase is especially marked between 2400 and 2750 Å.u. The rate of photoinactivation is shown to be sensitive to changes in pH, increasing with lower values, and evidently bears a one-quantum relationship to the energy flux. Tests of the rate of inactivation of pepsin exposed to several different bands of the ultra-violet spectrum, in relation to the absorbed energy, indicate that the destruction spectrum of the enzyme agrees essentially with its absorption spectrum and is similar to that of urease.     

双歧杆菌培养滤液中核酸的提取及对肠癌细胞cAMP,cGMP的影响

实验观察了对数期长双歧杆菌、青春双歧杆菌培养滤液中提取的总核酸对肠癌细胞ｃＡＭＰ、ｃＧＭＰ的影响。结果发现,双歧杆菌培养中滤液中存在大量核酸,将双歧杆菌培养滤中的核提取纯化作用于大肠癌细胞ＣＣＬ１８７,ｃＡＭＰ增高,ＣＧＭＰ没有变化,提示核酸可能作为细胞膜外的第一信使物质腺苷环化酶活性。     

EFFECT OF HAEMORRHAGE ON THE RAPID AXONAL TRANSPORT OF NEUROHYPOPHYSIAL PROTEINS OF THE RAT

Following injection of [³⁵S]cysteine into the region of the supraoptic nucleus male rats were subjected to haemorrhage and the radioactivity of the supraoptic nucleus and neurohypophysial proteins was measured at various time intervals after injection. Following haemorrhage the incorporation of [³⁵S]cysteine into supraoptic nucleus proteins increased. Evidence was obtained for a lag period of 1 to 2 h for the supraoptic nucleus proteins to become available for axonal transport. As judged from the time of arrival of labelled material in the neurohypophysis, haemorrhage did not change the rapid rate of axonal transport (190 mm/day). At 15 min following bleeding, the radioactivity in fraction A (a neurophysin) of the neurohypophysis was reduced, which indicated a release of this rapidly transported protein. During the following 15 min an increase in the protein-bound radioactivity of the neural lobe occurred which exceeded that in controls. This is taken as evidence for increased axonal transport in response to haemorrhage.     

人参茎叶皂甙对不同年龄大白鼠心肌细胞的影响

实验用3—5月龄青年鼠11只,18月龄老年鼠13只,各分对照与实验两组,实验组在基础饲料中加入参皂甙。三个月后取心肌制超薄切片,电镜观察。青年对照组与实验组心肌结构未见明显差别。老年对照组心肌细胞间胶元纤维增多。细胞内脂褐素数量增多,电子密度致密。核内异染色质在核边缘堆积且核边缘不规则,多凹陷。线粒体在肌原纤维间排列不整齐,线粒体嵴破裂以致消失。实验组细胞结构有所改善,表现在核边缘比较完整,异染色质较少,而且大部分线粒体嵴结构较清晰,脂褐素电子密度较低,文中井讨论了脂褐素的来源。     

THE FLOCCULATION OF BACTERIA BY PROTEINS

1. The effect of adding pure proteins to bacterial suspensions at different H ion concentrations has been studied. 2. The zone of flocculation of protein-treated bacteria bears a significant relationship to the isoelectric point of the protein used. With the higher concentration of protein, agglutination occurs at or near the isoelectric point of that protein; at reactions acid to this, the bacteria carry a positive charge and are not agglutinated. With diminishing concentration of protein, the zone of flocculation shifts toward and goes beyond that characteristic of the untreated bacteria. This occurs both in the presence and absence of salts. 3. A diversity of other suspensions, such as sols of gold, mastic, cellulose nitrate, cellulose acetate, Fe(OH)₃, oil emulsions, and erythrocytes, have been found by ourselves and others to exhibit a similar altered stability when treated with proteins in the same way.     

不同波长的激光对水稻胚芽中可溶性蛋白质及同工酶的作用

用波长0.3371、0.6328μm和1.06μm的激光器,用不同剂量(5、15、50min和3、7、9个脉冲),照射水稻干胚,使其萌动生长72hr后,再用聚丙烯酰胺凝胶电泳法,分析胚芽中可溶性蛋白质,酯酶同工酶和过氧化物酶同工酶。结果证明,波长0.3371～1.06μm的激光辐照水稻种胚,都能使同一生物大分子的活性和分子结构产生变异。由此认为,不能用辐照离体制备液所得结果去预言在活体上的反应。