Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, thein vivo site in brain myelin basic protein

In a previous report [Yanget al., (1987a),J. Biol Chem. 262, 7034–7040], a cyclic-AMP- and calcium-independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation-dependent protein kinase (autokinase) are further determined by two-dimensional electrophoresis/thin-layer chromatography, phosphoamino acid analysis, high-performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation, and direct peptide sequencing. Autokinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C₁₈-reversed phase highper-formance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser¹¹⁵, one of thein vivo phosphorylation sites in MBP, as the major site phosphorylated by autokinase, implicating a physiologically relevant role of autokinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT(p)GILDSLGR (molar ratio 0.9) and TT(p)HYGSLPQK (molar ratio 0.8) as the major phosphorylation site sequences in³²P-MBP phosphorylated by autokinase, further indicating that -Arg-XSer/Thr-(neutral amino acid)₃-(amino acid-containing hydroxyl group such as Ser/Glu/Asp)-(neutral amino acid)₂-may represent a unique consensus sequence motif specifically recognized by this autophosphorylation-dependent multisubstrate/ multifunctional protein kinase in the brain.     

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A₂ activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A₂ activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A₂ activities and arachidonic acid release in cells pretreated with nominal Ca²⁺ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A₂ activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A₂ activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A₂ activity (ii) in addition to the presence of extracellular Ca²⁺, release of Ca²⁺ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A₂ activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A₂ activities does not appear to require new RNA or protein synthesis.Abbreviations A23187 calcium ionophore - AA arachidonic acid - PMSF phenylmethyl sulfonylfuoride - DFP diisopropyl-fluorophosphate - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - PBS phosphate buffered saline - HBPS Hank's buffered physiological saline - PLA₂ phospholipase A₂     

Production of a thermophilic,extracellular alkaline protease by Bacillus stearothermophilus AP-4

A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mm CaCl₂. The enzyme was completely inactivated by PMSF, EDTA and -mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India     

Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.     

Midgut proteinase activities of three keratinolytic larvae,Hofmannophila pseudospretella,Tineola bisselliella,and Anthrenocerus australis,and the effect of proteinase inhibitors on proteolysis

The midgut proteinase activities were characterized from the keratinolytic larvae of two lepidopterans, Hofmannophila pseudospretella (Stainton) (Oecophoridae) and Tineola bisselliella (Hummel) (Tineidae), and one coleopteran, Anthrenocerus australis (Hope) (Dermestidae). The major endopeptidase activities, characterized using specific enzyme inhibitors, were serine proteinases with hydrolytic activity against N-benzoyl-DL-arginine-p-nitroanilide and against N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-leucine-p-nitroanilide. No significant levels of metalloendopeptidase or cysteine endopeptidase activities were detected. Aminopeptidase activity was present in all larvae. The enzyme levels and properties of the two moth larvae were similar to each other and to those of phytophagous lepidopteran larvae but different from those of the beetle larva. Whereas only a limited number of serine proteinase inhibitors inhibited the midgut proteolysis of the lepidopteran larvae, most inhibitors inhibited the midgut proteolysis of the beetle larva. © 1994 Wiley-Liss, Inc.     

Purification and characterization of a thiol-protease induced during senescence of unpollinated ovaries of Pisum sativum

A senescence-specific protease has been purified from senescent unpollinated ovaries of Pisum sativum L. cv. Alaska by acidic extraction. (NH₄)₂SO₄ fractionation, ion exchange chromatography on CM-Sephadex, and affinity chromatography on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-Sepharose. Characterization of the purified protease indicated that it is a thiol-endoprotease (EC 3. 4. 22 class) active over a wide pH range. Purified antibodies against this protease inhibit the degradation of Rubisco in autodigested extracts of senescent ovaries, suggesting that Rubisco might be a substrate for the protease in senescent pea ovaries. The relative levels of the protease were determined by an enzyme-linked immunosorbent assay (ELISA) along the processes of ovary senescence and gibberellic acid (GA)-induced fruit development, indicating its induction at the beginning of senescence and the suppression of its synthesis by GA treatment.     

Effect of agitation speed on the synthesis of Mucor miehei acid protease

Different experiments using Mucor miehei CBS 370.65 were carried out to study the effect of agitation speed on the production of the mold acid protease. The experiments were conducted in shake flasks at a fixed substrate concentration of 58 g l⁻¹ of total carbohydrates and at shaker speeds from 80 to 380 rev min⁻¹. Enzyme production was found to be directly proportional to the shaker speeds, with the highest concentration of enzyme of 1,400 Soxhlet Rennet units (SU) ml⁻¹ obtained at 380 rev min⁻¹. The yield of product to substrate at 380 rev min⁻¹ was determined to be 27,081.0 SU g⁻¹ substrate and the productivity of the process was 221 SU g⁻¹ h⁻¹. Enzyme production was partially growth associated, and glucose supported both cell growth and enzyme production. Product formation and cell concentration were directly related to the rate of substrate consumption. The rate of product formation decreased when product started to accumulate, suggesting that the process was affected by feedback repression.     

Fruit developmental regulation of the kiwifruit actinidin promoter is conserved in transgenic petunia plants

We have examined the expression of actinidin, a cysteine protease found in kiwifruit, over the course of fruit development. Protease activity was first seen in fruit that had reached about half their final weight, and rose to high levels at harvest. The 5-flanking region (nucleotides –1301 to +58) of a kiwifruit actinidin gene was fused to the -glucuronidase (GUS)-coding region, and the chimaeric gene was introduced into transgenic petunia plants. Induction of the GUS gene was observed during the later stages of seed pod development, closely resembling the pattern of actinidin induction in fruit tissues of kiwifruit. Some GUS expression was also detected in the vascular system of the receptacle, leaves, stems and roots. A shorter promoter fragment consisting of nucleotides –115 to +58 conferred similar spatial and temporal regulation in some of the transgenic plants.     

假菠萝麻蛋白酶分离纯化及特性研究

从假菠萝麻叶汁中用乙醇分部沉淀法得到一种蛋白酶,对酪蛋白有强烈的水解活性,经Ｓｅｐｈａｄｅｘ Ｇ－１００柱层析和ＳＰ－Ｓｅｐｈａｄｅｘ Ｃ－５０离子交换柱层析等步骤纯化,可得到六角形结晶。结晶酶液经ＰＡＧＥ圆盘电泳,每条胶柱上只显示一条蛋白带,其活性在ｐＨ４。５－１０、５５℃范围内较稳定,最适ｐＨ８．５最适温度５０℃,Ｋｍ（酪蛋白）值为０．１８５％（Ｗ／Ｖ）。用ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.