Changes occurring during aging and senescence in a submerged aquatic angiosperm (Potamogeton pectinatus)

Changes occurring during aging and senescence of leaves of a submerged aquatic angiosperm ( Potamogeton pectinatus L.) were studied. Total chlorophyll and chlorophylls a and b were maximal in mature, and minimal in old leaves. The chlorophyll a to b ratio was highest in mature leaves. During senescence, the chlorophyll content and the ratio of chlorophyll a to b decreased. The content of DNA, RNA, protein and dry weight, and the activity of alkaline pyrophosphatase decreased while free amino acids, the activity of protease, RNase and acid pyrophosphatase, and the ratio of acid to alkaline pyrophosphatase activity increased during aging and senescence. Kinetin (0.23 m M ) deferred leaf senescence by delaying the loss of chlorophyll, protein, nucleic acids and dry weight, and reducing the rise in free amino acids, the activity of protease, RNase and acid pyrophosphatase and the ratio of acid to alkaline pyrophosphatase activity; while both 0.69 m M ethrel and 0.075 m M ABA hastened senescence. Kinetin pretreatment for an optimum period (12 h) followed by ethrel or ABA treatment partially erased the senescence-promoting effect of the latter. But treatments in a reverse order markedly reduced the delaying effect of kinetin on senescence.     

交联琼脂糖珠固定化蛋白酶在纯化牛肺Kunitz抑制剂中的应用

本文介绍了珠状交联琼脂糖及以此作为载体,经氯代环氧丙烷活化后与蛋白酶(胰蛋白酶或糜蛋白酶)结合,制成固定化蛋白酶亲和吸附剂,进而用以亲和层析牛肺提取液中的Kunitz抑制剂的方法。纯化出的抑制剂在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带,与参照物Trasytol(商品Kunitz抑制剂)具有相对应的电泳迁移率,其分子量也相符。纯化产品每毫克蛋白的抑制活力相当于16 000胰蛋白酶BAEE单位。纯化效果为90倍,收率约85％。     

Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction

The egg jelly-induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA), but not by Suc-Ala-Ala-Pro-Phe-MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE-Sephacel and hydroxylapatite chromatographies, Sephacryl S-300 gel filtration, and heparin-Sepharose CL-6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc-Ala-Ala-Pro-Phe-MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22-33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin-type and the other trypsin-type. The former activity was enhanced by poly-L-lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin-like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255-266, 1989). Bringing these findings together, we suggest that the chymotrypsin-like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.     

Calcium-free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2

Calcium-free calmodulin-(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X-ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV-1 and HIV-2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV-1 protease is the Asn-Tyr linkage in the sequence, -N-I-D-G-D-G-Q-V-N-Y-E-E-, found in the fourth calcium binding loop. In contrast, it is an Ala-Ala bond in the third calcium loop, -D-K-D-G-N-G-Y-I-S-A-A-E-, that is first hydrolyzed by the HIV-2 enzyme, followed in short order by cleavage of the same Asn-Tyr linkage described above. Thereafter, both enzymes proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

Effect of an extract of nereis virens on mammalian spermatozoa

Epididymal sperm of the mouse, rat, and guinea pig and ejaculated sperm of rabbits are cleaved at the head-tail junction by an extract of Nereis virens. Annelids are extracted with water and the extract is purified by ion exchange chromatography. Electron microscopy shows that the extract acts on the filaments connecting the capitulum of the tail with the basal plate lining the nuclear envelope. Following detachment, the basal plate remains with the head. The extract contains proteases as indicated by hydrolysis of tosyl arginine methyl ester (TAME), benzoyl arginine ethyl ester (BAEE), and Azocoll, a general protease substrate. The hydrolysis of TAME is inhibited by tosyl lysine chloromethyl ketone (TLCK), a trypsin inhibitor, but TLCK does not prevent head-tail separation by the Nereis extract. Similarly tosyl phenylalanine chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and phosphoramidon and leucyltryptophan, both thermolysin and acrolysin inhibitors — singly or in combination — do not prevent hydrolysis of Azocoll. Head-tail separation activity of the extract was inhibited by dithiothreitol, which reduces disulfide bonds, and phenylmethyl sulfonyl fluoride, an inhibitor of serine proteases. These results indicate that the extract is a mixture of proteases, one being a serine protease similar to trypsin. Digestion of the connecting filaments with the pure proteases, trypsin and Staphylococcus aureus V8 protease, has yielded the following information on the proteins of the filaments. The accessibility of arginine and/or lysine peptide bonds to enzyme action is highest in rat sperm filaments, whereas those in the filaments of mouse, rabbit, and guinea pig sperm are less accessible than in the rat. Another possibility is that the total content of arginine and/or lysine varies between the species. The most dramatic difference is the enzymatic action on glutamyl peptide bonds of the filaments, the order being: mouse 〉 rat 〉 rabbit, with guinea pig sperm filaments completely resistant over the time course of the experiment.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.