Peptidohydrolases of Soybean Root Nodules

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminoptidase, and carboxypeptidase actiities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-LLeu-p8-naphthylamide, while the second hydrolyzed n-benzoyl-L-Arg-/8naphthylamine (Bz-L-Arg-/3NA), n-benzoyl-L-Arg-p-nitroanilide (Bz-L-ArgpNA), and azocasein. The third endopeptidase hydrolyzed Bz-L-Arg-8NA, Bz-L-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. AU of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases. Aminopeptidase activity was measured using amino acyl-p-naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-pi-naphthylamides. The major activity of two of the aminopeptidases was directed toward L-Leuand LMet-p-naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-.-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classiffed as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima. Senescence of soybean root nodules results in a decreased capacity to reduce atmospheric nitrogen, thus potentially limiting the supply of amino nitrogen to the plant. After the decline in nitrogenase activity, total soluble protein per unit fresh weight of nodule decreases rapidly (22), although the fresh weight per nodule shows little change. In addition, the total number of nodules per plant decreases sharply. Thus, nodule senescence can be characterized by a sharp decline in nitrogenase activity coupled with a degeneration ofthe nodule leading to loss of soluble protein and eventual abscission of the nodule from the root. The loss of soluble protein is a characteristic feature of all types of plant senescence, and is generally attributed to increased activity of proteolytic enzymes. The role of proteolytic enzymes in nodule senescence is not as well established as in leaf senescence (7, 8, 19, ' This work was supported by United States Department of Agriculture, Science and Education Administration-CRGO grant 50940410-9-0233 and by a grant from the Nebraska Soybean Development, Utilization and Marketing Board. 2 Published as Paper No. 6089, Journal Series, Nebraska Agricultural Experiment Station. 3To whom all communications should be addressed. 35), although proteolytic activity in alfalfa nodules has been shown to increase as nitrogenase activity and soluble proteins are lost after harvest (32). Studies with animal tissues have demonstrated that many proteolytic enzymes have extremely specialized regulatory roles in cellular processes such as translational polypeptide processing (14), hormone regulation (24), and blood coagulation (6). However, such specialized roles have not been elucidated in plant cells. In most cases, only total proteolytic activity, or activity of a single protease, has been correlated to a physiological change such as plant senescence. Differences in substrate specificity and in catalytic mechanism may reflect functional differences between the various proteolytic enzymes. Since individual proteases probably have distinct roles within the overall process of senescence, the characterization of these enzymes is important in understanding their role in senescence. The purpose of this investigation was to identify and characterize as many of the enzymes responsible for total proteolytic activity in soybean root nodules as possible, in order to investigate their roles in nodule senescence. Proteolytic enzymes unique to the bacteroid fraction will be characterized in a future report. MATERIALS AND METHODS Special Chemicals. Ammonium persulfate and TEMED4 were purchased from Bio-Rad Laboratories. Arcillite was obtained from IMC Chemical Corp. Hemoglobin substrate powder was the product of Worthington Biochemicals. All special reagents, chemicals, and substrates were obtained from Sigma. Acrylamide and bisacrylamide were recrystallized (18) prior to use, and other reagents were used without further purification. Seed Inoculation and Plant Growth. Inoculum was prepared by growing Rhizobium japonicum (strain 3IlbllO) in L-arabinose broth (33) for for 5 to 7 days at 30 C with constant shaking. Soybean seeds (Glycine max var Woodworth) were immersed in inoculum for 30 min, then planted in Arcillite in 6-inch clay pots that had been sterilized for 1 h at 240 C. Four plants were grown in each pot. An additional 20 ml bacterial culture were added to the soil in each pot to ensure adequate nodulation. Plants were grown in a temperature-controlled greenhouse without artificial light from March to July of 1979, and supplied frequently with a nitrogen-free nutrient solution (9). Nodules were harvested during the pod-filling stage of plant development and were stored at -70 C until used. Bacteroid-Free Extracts. Four g nodules were macerated in an ice-cold mortar and pestle with 5 ml 25 mm phosphate buffer (pH 4 Abbreviations: TEMED, N,N,N',N'-tetramethylethylenediamine; DTE, dithioerythritol; Bz-, n-a-benzoyl-; -pNA, -p-nitroanilide; -,BNA, -ft-naphthylamide; Z-, benzoyloxycarbonyl-; PCMB,p-chloromercuribenzoate; DFP, diisopropylphosphorofluoridate; TEA, triethanolamine.