Molecular anatomy: Phyletic relationships derived from three-dimensional structures of proteins

Summary A distance measure that reflects the dissimilarity among structures has been developed on the basis of the three-dimensional structures of similar proteins, this being totally independent of sequence in the sense that only the relative spatial positions of mainchain alpha-carbon atoms need be known. This procedure leads to phyletic relationships that are in general correlated with the sequence phylogenies based on residue type. Such relationships among known protein three-dimensional structures are also a useful aid to their classification and selection in knowledge-based modeling using homologous structures. We have applied this approach to six homologous sets of proteins: immunoglobulin fragments, globins, cytochromesc, serine proteinases, eye-lens gamma crystallins, and dinucleotide-binding domains.     

Amino acid and peptide derivatives of dimethyl 5-aminoisophthalate as fluorogenic substrates for proteinases

A number of amino acid and peptide derivatives of the fluorophore, dimethyl 5-aminoisophthalate have been synthesized, characterized and tested as substrates for the plant cysteine proteinases papain, ficin and bromelain. In every case, replacement of alanine by citrulline, in the position adjacent to the dimethyl 5-aminoisophthalate resulted in a higher rate of hydrolysis. The partly deprotected dipeptide derivative dimethyl phenylalanylcitrulline-5-aminoisophthalate was hydrolysed most rapidly of all the compounds tested, and on this basis may provide a useful substrate for the detection and quantitative assay of these enzymes.     

Proteinase A-like enzyme from germinated kidney bean seeds. Its action on phaseolin and vicilin

A cysteine proteinase that possibly participates in the degradation of phaseolin, the main storage protein of kidney bean ( Phaseolus vulgaris L. cv. Moldavian) was isolated from germinating kidney bean seeds and partially characterized. According to its properties it may be classified as a member of a group of homologous cysteine proteinases A, also present in germinating seeds of a number of other plants. The proteinase of this group hydrolyze storage proteins to short peptides. Similarly, the kidney bean proteinase hydrolyzes vicilin, the reserve protein of vetch ( Vicia sativa ). However, its action on phaseolin is limited to the cleavage of subunits into two approximately equal parts and to the splitting off a small number of short peptides. An explanation of phaseolin resistance to the action of this proteinase is proposed on the basis of the differences of its structure from that of other homologous 7S proteins.     

Amino acid sequences of ovomucoid third domain from 25 additional species of birds

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowskiet al. (1987,Biochemistry 26, 202–221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro¹⁴ Ser¹⁴ replacement in weka, an Ala¹⁴Thr¹⁵ replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile¹⁸ and Gly¹⁸ at the P₁ reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu³⁴) rather than positive (Lys³⁴ or Arg³⁴) amino acid residue at the NH₂ terminus of the helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genusGallus, in the five species genusSyrmaticus, and in the two species generaAix andPavo.     

Suppression of larval Colorado potato beetle growth and development by digestive proteinase inhibitors

Evidence is presented that the Colorado potato beetle (CPB) (Leptinotarsa decemlineata (Say)) depends, at least partially, on cysteine proteinases for protein digestion. Midgut homogenates of CPB larvae have a mildly acidic pH and exhibit major proteolytic activity in the mildly acidic pH range. This proteolytic activity is activated by reducing agents, is inhibited by E-64 (a specific cysteine proteinase inhibitor), and is not inhibited by serine proteinase inhibitors. In addition, consumption of E-64 treated potato leaves by CPB larvae at rates as low as 0.8 g/cm² of leaf tissue has a deleterious effect on larval growth and development.

---

Résumé Les protéinases intestinales des larves de Leptinotarsa decemlineata sont en partie caractérisées par les pH limitant leur activité, et par l'effet d'inhibiteurs de protéinases de spécificités connues. Les protéinases ont été testées avec la méthémoglobine tritiée comme substrat et nous avons déterminé les taux relatifs de peptides radioactives TCA solubles, libérées, dans des conditions précises, pour des pH compris entre 2,0 et 12,0. Le taux le plus élevé d'activité protéolytique a été observé pour des pH 5,0 à 6,0, bien qu'il y ait eu une activité appréciable aux pH 4,0 et 8,0. Parmi les inhibiteurs examinés, le E-64, inhibiteur protéinase cystéine très sélectif, a été le plus efficace sur l'activité protéinase. La pepstatine, inhibiteur protéinase aspartique, a été actif, mais seulement dans une gamme plus moin de pH. Les inhibiteurs protéinase sérine, ont été pratiquement inactifs sur l'activité protéinase.Chez des larves de doryphores nourries de feuilles de pommes de terre traitées avec différentes concentrations de E-64 ou de pepstatine, la consommation de E-64 a retardé fortement la croissance larvaire et le développement, la pepstatine a provoqué un retard du développement plus limitée.Nos résultats suggèrent que le doryphore dépend, tout au moins partiellement, des protéinases cystéine pour l'assimilation des protéines.

     

Keratinase of an anaerobic thermophilic bacterium <Emphasis Type="Italic">Thermoanaerobacter</Emphasis> sp. Strain 1004-09 isolated from a hot spring in the Baikal rift zone

A thermophilic anaerobic bacterial strain 1004-09 belonging to the genus Thermoanaerobacter and capable of growth on protein substrates such as albumin, gelatin, casein, and α- and β-keratins was isolated from the Urinskii hot spring (Barguzin river valley, Republic of Buryatia, Russia). A 150-kDa serine proteinase was revealed in the strain supernatant; it exhibited optimal activity at 60°C and pH 9.3 and was capable of keratin hydrolysis. A number of characteristics for the strain 1004-09 keratinase were established including activation by SDS and NaCl and residual activity (15% to the activity of the intact protein) in the presence of 10% ethanol and acetone.     

中性粒细胞蛋白酶抑制剂elafin毕赤酵母表达系统的建立及活性检测

中性粒细胞蛋白酶抑制剂elafin是中性粒细胞弹性蛋白酶特异性抑制剂。为建立中性粒细胞蛋白酶抑制剂elafin毕赤酵母表达系统,通过逆转录PCR扩增获取elafin cDNA,双酶切后定向克隆至穿梭质粒pPIC9K上。SalI酶切线性化后,电穿孔法转至毕赤酵母GS115菌株中,PCR筛选出阳性克隆,接种至最小甘油缓冲培养基中,甲醇诱导表达后,取上清阳离子交换层析纯化靶标蛋白。分别以western-blot、弹力纤维蛋白-聚丙烯酰胺凝胶电泳检测重组elafin免疫原性、抗蛋白酶活性。重组elafin在毕赤酵母系统中呈分泌性表达,产量及活性高,分离纯化简便。此表达系统的建立为其后续研究及临床应用奠定了基础。     

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.     

Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate

High-resolution crystallographic analysis of a complex of the serine-carboxyl proteinase sedolisin with pseudo-iodotyrostatin revealed two molecules of this inhibitor bound in the active site of the enzyme, marking subsites from S3 to S3('). The mode of binding represents two products of the proteolytic reaction. Substrate specificity of sedolisin was investigated using peptide libraries and a new peptide substrate for sedolisin, MCA-Lys-Pro-Pro-Leu-Glu#Tyr-Arg-Leu-Gly-Lys(DNP)-Gly, was synthesized based on the results of the enzymatic and crystallographic studies and was shown to be efficiently cleaved by the enzyme. The kinetic parameters for the substrate, measured by the increase in fluorescence upon relief of quenching, were: k(cat)=73+/-5 s(-1), K(m)=0.12+/-0.011 microM, and k(cat)/K(m)=608+/-85 s(-1)microM(-1).