Limited Proteolysis of Disulfide-reduced Ovalbumin by Subtilisin

Our previous study [Takahashiet al., J. Biochem., 109, 846–851 (1991)] has shown that the disulfide-reduced form of ovalbumin was proteolyzed by subtilisin into three major fragments. It was investigated whether or not these three fragments would be folded into one molecule. Gel permeation and ion-exchange chromatography indicated that the three fragments were eluted in a single peak. The proteolyzed protein had a CD spectrum that was almost indistinguishable from the disulfide-reduced, non-proteolyzed, form of ovalbumin. Differential scanning calorimetry, however, revealed, that the proteolyzed ovalbumin was denatured at a lower temperature than that of the disulfide-reduced, non-proteolyzed. protein. Thus, it is concluded that the three fragments were folded into a native-like conformation with decreased stability. Chemical analyses of the fragments purified by reverse-phase HPLC revealed that there was a cleavage site in the disulfide-reduced form of ovalbumin, at least at the amino-terminal side of Cys⁷³, in addition to the well-known cleavage sites in plakalbumin.     

Binding of Pepsin Inhibitor (S-PI) and Pepsin

Relation between cellular phospholipids and L-glutamic acid excretion was investigated using Corynebacterium alkanolyticum GL–21 (a glycerol auxotroph).

When strain GL–21 was cultured in glycerol-limited medium which contained n-hexadecane, acetic acid or fructose as carbon source, there occurred the limitation of cellular phospholipid content and the over-accumulation of L-glutamic acid in the broth. Two-dimensional thin-layer chromatograms provided evidence that both the parent and the mutant strains contained the same phospholipids such as cardiolipin, phosphatidylethanolamine, phosphadityl-glycerol, phosphatidylinositol and phosphatidic acid. Limited supply of glycerol to the mutant did not greatly alter the proportions of the individual phospholipids.     

Cleavage of Rabbit Myelin Basic Protein by Pepsin

Rapid cleavage of bovine and guinea pig myelin basic proteins by pepsin at pH 6.0 is limited to the Phe-Phe bond in the middle of the molecule. In the rabbit protein, however, rapid cleavages occur elsewhere in addition to the Phe⁸⁷-Phe⁸⁸ bond in regions in which there are amino acid substitutions. Rapid cleavage occurs at the Leu¹⁵¹-Phe¹⁵² bond, at which Ile-151 has been replaced by Leu, the residue that actually contributes the scissile bond. Rapid cleavages occur at the Phe⁴⁴-Phe⁴⁵ and Leu¹⁰⁹-Ser¹¹⁰ bonds, which in the bovine and guinea pig proteins are relatively resistant under the experimental conditions (pH 6.0). The increased susceptibility of these bonds in the rabbit protein appears to be related to the replacement of Gly-46 by Ser and the change in the sequence immediately NH₂-terminal to Leu-109, from Leu-Ser to Thr-Val. These cleavages of the rabbit protein at the four very susceptible bonds have permitted us to isolate peptides (1-44), (45-87), (88-109), (110-151), and (152-168) in high yield. We have also isolated peptides (88-151), (1-14), and (15-44) in low yield; the latter two result from limited cleavage at the relatively resistant Tyr¹⁴Leu¹⁵ bond. Peptide (88-109) has been chromatographically resolved into species differing in the degree of methylation of Arg-105; this resolution is thought to result from differences in hydrogen bonding ability of the guanidinium groups.     

Immunization by Irradiated Eimeria acervulina

SYNOPSIS. In 5 experiments the infectivity and immunogenicity for chickens of sporulated oocysts of Eimeria acervulina that had been previously subjected to different levels of gamma radiation was tested. It was found that the irradiation doses can be divided into three groups: a) High dosage levels of over 16,000 rad impair the reproduction of the parasites so that few or no oocysts are discharged and no immunity is produced. b) Low dosage levels of 7,000 rad and less permit reproduction and development of immunity not perceptibly different from that resulting from unirradiated oocysts. c) An intermediate range of levels with the optimum from 9,100–13,700 rad induces attenuation while at the same time assuring sufficient immunogenic response.
A dose of 100,000 oocysts or more per bird is required to produce immunity. The optimum irradiation dose increases with the immunizing dose. It was concluded that the development of an irradiated vaccine is possible.     

Pepsinogen and Pepsin

Evidence relating to the structure and properties of swine pepsinogen and pepsin has been reviewed and used to suggest a tentative two dimensional picture of the skeleton of these two proteins. When pepsinogen, a folded single peptide chain, is converted to pepsin, there is a profound change in the physical and chemical properties of the protein. In an as yet unknown manner, except that it is initiated by a peptic cleavage of the protein chain, a single enzymic site is formed. This site is made up, quite probably, of the secondary carboxyl group of glutamic acid or of aspartic acid and a tyrosine phenol group in close proximity so that they can form hydrogen or hydrophobic bonds with the substrate in some unique manner that permits hydrolysis to occur at an accelerated rate.     

人卵清蛋白抑丝酶家族

人ov-抑丝酶家族为抑丝酶家族亚系。多数ov－抑丝酶存在细胞内,作为丝氨酸蛋白酶和半胱氨酸蛋白酶抑制剂参与蛋白质加工处理,细胞凋亡,细胞外基质重构,以及保护细胞免疫损伤等。除了蛋白酶抑制作用之外,人ov-抑丝酶家族还是有其它生物学功能。     

鸡卵清蛋白表达调控

简要介绍鸡卵清蛋白,综述其基因结构和表达调控,表达调控从染色质水平、转录水平和翻译水平三个方面进行综述。     

Das Pepsin verschiedener Vertebraten

Ohne Zusammenfassung     

Ovalbumin is an elastase substrate

Ovalbumin is partially homologous in sequence with the proteinase inhibitors alpha 1-proteinase inhibitor and anti-thrombin III. The region of sequence in ovalbumin which corresponds to the reactive sites of these proteinase inhibitors is susceptible to attack by subtilisin, elastase, thermolysin, bromelain, and Bacillus cereus protease. The esterase activity of elastase is not inhibited by ovalbumin, but ovalbumin is efficiently cleaved by elastase. In contrast with these proteases, trypsin does not cleave ovalbumin.     

云芝发酵产胃蛋白酶抑制剂的反应机理探讨

目的:对一种从云芝发酵液中提取得到的胃蛋白酶抑制剂的动力学性质和反应机理进行研究.方法:采用Lineweaver-Bunk双倒数法和UltroGelACA-54凝胶过滤法:结果:实验证明抑制剂为胞内产物.其成分是蛋白质,分子量为23 100Da,K值为0.996 μmol/L.结论:抑制剂对胃酶的抑制类型属于非竞争和反竞争混合型抑制模式,它与胃酶结合作用的方式可能是在酶的活性基同形成氢键并封锁酶与底物的结合部位.     

Pepsin and the esophagus

Esophagitis results from excessive exposure of the esophagus to gastric juice through an ineffective or dysfunctional lower esophageal sphincter mechanism. A possible role of pepsin in damaging the esophageal mucosa with consequent esophagitis may be examined directly by testing pepsin under various conditions in experimental models of esophagitis. Since gastric juice contains both acid and pepsin, all experiments examine separately effects of perfusion of the esophagus by acid without and with pepsin in various combinations. Acid perfusion alone at concentrations represented by pH 1.3 or above does not produce esophagitis. The addition of pepsin to acid between pH 1 and 3.5 causes considerable acute esophageal damage. Outside the proteolytic range, i.e., higher than pH 3.5, pepsin does not damage the esophagus. The damage caused by acidified pepsin may be made much worse by the further addition of aspirin or other NSAIDs, presumably by further breaking down mucosal barriers.     

Ovalbumin digestion by human pepsins 1, 3 and 5.

1. Of the three major human pepsins, pepsin 1 has greater proteolytic activity towards ovalbumin than has pepsin 3. Pepsin 5 has low activity towards this substrate. 2. Proteolytic pH-activity curves show only on pH maximum, about pH 1.4 for pepsin 1, pH 1.4--1.5 for pepsin 3 and pH 1.2--1.4 for pepsin 5. The curve for pepsin 3 has a shoulder between pH 2.4 and 3.4. 3. The rate of digestion of ovalbumin by pepsin 1 is approximately three times slower than are those of bovine haemoglobin or human globin. 4. The results suggest that there may be a physiological advantage in having more than one pepsin.     

Hydrolysis of poly-L-lysine by pronase

Pronase E is able to hydrolyze poly-L -lysine in 100% yield into the monomer L -lysine. The experiments show an intermediate production of tri-L -lysine and di-L -lysine that are clipped off from the polymeric peptide. During the hydrolytic process a number of characteristic amino acids are released from the enzyme that may indicate its activation. Amino acids as well as mono-L -lysine are decomposed by pronase E after a period of several days.     

Inhibition of Acid Proteases by a Pepsin Inhibitor (S-PI)

S–PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.

S–PI made an equimolar complex with these acid proteases. A part of the enzyme-S–PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S–PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S–PI free) enzyme, each of the enzyme-S–PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10^?10M, Rh. glutinis acid protease; 1.8×10^?7M, Cladosporium acid protease; 3.0×10^?6M.

These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S–PI complex. S–PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.

Binding between these acid proteases and S–PI dissociated at around neutral pH. S–PI was separated from enzyme-S–PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S–PI complex by DEAE cellulose column chromatography as a native form.