Partial purification and characterization of endoproteinases from senescing barley leaves

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP₁) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP₂) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP₂ contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [¹⁴C]ribulose-1,5-bisphosphate carboxylase ([¹⁴C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [¹⁴C]RuBPCase biphasically. A third endoproteinase (EP₃), not detected by standard proteolytic assays, was observed when [¹⁴C]RuBPCase was the substrate.     

Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells

The localization of two previously characterized endoproteinases (EP₁ and EP₂) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP₁ and partially purified EP₂. It is therefore concluded that EP₁ and EP₂ are localized in the vacuoles of mesophyll cells.     

Relationship of Ribulose-1,5-bisphosphate Carboxylase-Oxygenase Specific Activity to Subunit Composition

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase, EC 4.1.1.39) was isolated from Nicotiana sylvestris and from two cultivars and three nuclear substitution lines of Nicotiana tabacum. Isoelectric focusing patterns, supported by amino acid analyses and tryptic peptide mapping, were used to divide these enzymes into two categories: (a) RuBPCase with variable large subunits and identical small subunits; and (b) RuBPCase with identical large but different small subunits. Specific activities for both the carboxylation and oxygenation reactions were determined for all six RuBPCase enzymes under standard conditions of activation and assay. High, intermediate, and low levels of carboxylase (880, 530, and 340 nanomoles HCO₃⁻ per milligram per minute) and oxygenase (66, 45, and 35 nanomoles O₂ per milligram per minute) activity were noted. The carboxylase to oxygenase ratios ranged from 9 to 14.     

Relationship between the Kinetic Properties and the Small Subunit Composition of Nicotiana Ribulose-1, 5-bisphosphate Carboxylase

Genetic variability in the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in several Nicotiana species has been characterized by isoelectric focusing patterns. This heritable variation provides an opportunity to examine the functional role of each of these subunits. In this study, specifically designed RuBPCase enzymes composed of identical large subunits but different small subunits were constructed in vivo by interspecific hybridization between the species N. sylvestris, N. tabacum, N. glauca, N. glutinosa, N. plumbaginifolia, and N. tomentosiformis. Small subunit polypeptides were combined to form a sequence of one, two, three, and four polypeptides with the large subunit of N. sylvestris. Kinetic properties of these hybrid enzymes were compared. No differences in the specific activity of either carboxylation or oxygenation nor in K_m values for ribulose 1,5-bisphosphate, CO₂, or O₂ were detected among the RuBPCase enzymes from the various interspecific hybrids. Likewise, the ratio of carboxylation to oxygenation was constant.     

Structural, Functional, and Evolutionary Analysis of Ribulose-1,5-Bisphosphate Carboxylase from the Chromophytic Alga Olisthodiscus luteus

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified from the marine chromophyte Olisthodiscus luteus. This study represents the first extensive analysis of RuBPCase from a chromophytic plant species as well as from an organism where both subunits of the enzyme are encoded on the chloroplast genome. The size of the purified holoenzyme (17.9 Svedberg units, 588 kilodaltons) was determined by sedimentation analysis and the size of the subunits (55 kilodaltons, 15 kilodaltons) ascertained by analytical sodium dodecyl sulfate gel electrophoresis. This data predicts either an 8:9 or 8:8 ratio of the large to small subunits in the holoenzyme. Amino acid analyses demonstrate that the O. luteus RuBPCase large subunit is highly conserved and the small subunit much less so when compared with the chlorophytic plant peptides. The catalytic optima of pH and Mg²⁺ have been determined as well as the response of enzyme catalysis to temperature. The requirements of NaHCO₃ and Mg²⁺ for enzyme activation have also been analyzed. The Michaelis constants for the substrates of the carboxylation reaction (CO₂ and ribulose bisphosphate) were shown to be 45 and 48 micromolar, respectively. Competitive inhibition by oxygen of RuBPCase-catalyzed CO₂ fixation was also demonstrated. These data demonstrate that a high degree of RuBPCase conservation occurs among widely divergent photoautotrophs regardless of small subunit coding site.     

Partial Isolation and Degradation of Caseins by Cell Wall Proteinase(s) of Streptococcus cremoris HP

The cell wall proteinase fraction of Streptococcus cremoris HP has been isolated. This preparation did not exhibit any activity due to either specific peptidases known to be located near the outside surface of and in the membrane or intracellular proteolytic enzymes. By using thin-layer chromatography for the detection of relatively small hydrolysis products which remain soluble at pH 4.6, it was shown that β-casein is preferentially attacked by the cell wall proteinase. This was also the case when whole casein or micelles were used as the substrate. κ-casein hydrolysis is a relatively slow process, and α_s-casein degradation appeared to proceed at an extremely low rate. These results could be confirmed by using ¹⁴CH₃-labeled caseins. A relatively fast and linear initial progress of ¹⁴CH₃-labeled β-casein degradation is not inhibited by α_s-casein and only slightly by κ-casein at concentrations of these components which reflect their stoichiometry in the micelles. Possible implications of β-casein degradation for growth of the organism in milk are discussed.     

Ribulose Bisphosphate Carboxylase from Three Chlorophyll c-Containing Algae : Physical and Immunological Characterizations

Distinctive properties are identified in the molecular structure of ribulose, 1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in chlorophyll c-containing algae (i.e., chromophytes). Using purified enzyme from Cryptomonas sp., Coccolithophora sp., and Cylindrotheca fusiformis, we have determined that the RuBPCase holoenzyme of each species has a molecular weight, subunit composition, and isoelectric points of its subunits similar to the purified enzymes from pea and Chlamydomonas reinhardtii. The large subunits from chromophytes exhibit microheterogeneity in their isoelectric points, whereas two to four well-resolved isoelectric variants of the small subunit were observed in each RuBPCase preparation. In spite of the high degree of similarity in terms of physical properties, both the small and large RuBPCase subunits of the chromophytes are structurally different from those of chlorophytes; immunological studies demonstrate that RuBPCase subunits of these two groups have few antigenic determinants in common.     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052     

XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

“Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (V_max of 10 μmol min⁻¹ mg⁻¹ protein, K_m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.     

Enhancement of CO(2) Uptake in Avena Coleoptiles by Fusicoccin

When Avena coleoptile segments are immersed in a solution containing H¹⁴CO₃⁻, the appearance of label in the tissue is stimulated approximately 3-fold by fusicoccin application. This effect is rapid (1-2 minutes lag time), dependent upon respiratory energy, inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not appreciably altered by cycloheximide treatment. A large percentage of the cellular radioactivity is found in the form of malate. Preliminary experiments indicate that CO₂, as opposed to HCO₃⁻, is the favored species of “CO₂” taken up by the segments. These results are consistent with the notion that CO₂, presumably by virtue of its fixation and conversion to malic acid, participates in the early events associated with fusicoccin-enhanced acidification of the cell wall region leading to the stimulation of cell extension growth.     

Carbon-14 Distribution in Carbohydrates of Immature Zea mays. Kernels Following CO(2) Treatment of Intact Plants

Shortly after Zea mays L. plants were exposed to ¹⁴CO₂, most of the radioactivity in the kernel occurred in the free monosaccharides, glucose and fructose. Later the proportion of ¹⁴C in sucrose increased and that in the monosaccharides declined. These data have been interpreted as showing that the translocated sugar is hydrolyzed prior to or during its movement into the storage cells of the endosperm. This hydrolysis appears to occur in the “pedicel region” of the kernel. After entry into the endosperm tissue, sucrose was rapidly resynthesized from the monosaccharides prior to its utilization in starch synthesis.     

'LeishMan' topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani

The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme.     

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K_m s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

Terpene Biosynthesis in Cell-free Extracts and Excised Shoots from Wedgwood Iris

Excised shoots and cell-free extracts prepared from Wedgwood iris (Iris hollandica Hoog. “Wedgwood”) shoots metabolized ¹⁴C-labeled mevalonic acid (MVA). By using cell-free extracts, the ¹⁴C from MVA-1-¹⁴C was recovered as ¹⁴CO₂, while that from MVA-2-¹⁴C was recovered as neutral terpenes, acid-hydrolyzable terpenes, or ¹⁴CO₂. Also, under optimal incubation conditions, 12.8 nanomoles R-MVA-2-¹⁴C was incorporated into neutral terpenes per milligram fresh weight per hour. In contrast, excised shoots incorporated only 0.58 nanomoles R-MVA-2-¹⁴C per mg fresh weight per hour. Labeled products identified from the cell-free system were squalene, farnesol, geranylgeraniol, and compounds that are converted to farnesol and geranylgeraniol after alkaline hydrolysis. Squalene and a 4,4-dimethylsterol were identified as products from excised shoots but not the terpene alcohols or the alkaline-hydrolyzable compounds.     

The specific activity of ribulose-1,5-bisphosphate carboxylase in relation to genotype in wheat

The specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) in crude extracts of leaves from euploid, amphiploid and alloplasmic lines of wheat fell into high or low categories (3.75 or 2.70 mol·mg^–1·min^–1, 30°C). For the alloplasmic lines, where the same hexaploid nuclear genome was substituted into different cytoplasms, the specific activity of RuBPCase was consistent with the type of cytoplasm (high for the B and S cytoplasms and low for the A and D cytoplasms). There was no evidence from the euploid and amphiploid lines that small subunits encoded in different nuclear genomes influenced the specific activity. High specific activity was conferred by possession of the chloroplast genome of the B-type cytoplasm which encodes the large subunit of RuBPCase. All lines with a cytoplasm derived from the Sitopsis section of wheat, with the exception of Aegilops longissima and A. speltoides 18940, had RuBPCase with high specific activity. In contrast with the euploid lines of A. longissima, the alloplasmic line containing A. longissima cytoplasm from a different source had RuBPCase with high specific activity. The difference in specific activity found here in-vitro was not apparent in-vivo when leaf gas exchange was measured.Abbreviation RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

Phosphorylation of small subunit plays a crucial role in the regulation of RuBPCase in moss and spinach

RuBPCase has been purified to electrophoretic homogeneity from moss and spinach. On denaturing SDS-polyacrylamide gels the purified enzyme revealed two discrete bands, thereby indicating the presence of large and small subunits. The phosphoprotein nature of RuBPCase was proved by in vivo labelling of enzyme with [³²P]orthophosphate. Autoradiographic analysis of ³²P-labelled RuBPCase on SDS-PAG demonstrated that phosphorylation was restricted to the small subunit. Dephosphorylation of purified RuBPCase with alkaline phosphatase resulted in a dramatic decline (70% decrease) in the biological activity of the enzyme. Fractionation of the dephosphorylated enzyme on denaturing gels revealed only the presence of large subunits of RuBPCase. Thus it became evident that dephosphorylation of RuBPCase brings about the dissociation of small subunits from the catalytic large subunits (octamer). The dephosphorylated small subunits were isolated as dimers. These results clearly indicate that phosphorylation of small subunits is mandatory for the reconstitution of holoenzyme and hence crucial for the activation of RuBPCase.     

Mechanism of Inhibition by C-terminal α-Helices of the ? Subunit of Escherichia coli FoF1-ATP Synthase

The ϵ subunit of bacterial F_oF₁-ATP synthase (F_oF₁), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal α-helices of ϵ, and the “extended” but not “hairpin-folded” state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of ϵ has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli F_oF₁ (EF_oF₁) mutant in which the ϵ subunit lacked the C-terminal domain (F_oF₁^ϵΔC), and ATP synthesis driven by acid-base transition (ΔpH) and the K⁺-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (F_oF₁^ϵWT). The turnover numbers (k_cat) of F_oF₁^ϵWT were severalfold lower than those of F_oF₁^ϵΔC. F_oF₁^ϵWT showed higher Michaelis constants (K_m). The dependence of the activities of F_oF₁^ϵWT and F_oF₁^ϵΔC on various combinations of ΔpH and ΔΨ was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of ϵ. Solubilized F_oF₁^ϵWT also showed lower k_cat and higher K_m values for ATP hydrolysis than the corresponding values of F_oF₁^ϵΔC. These results suggest that the C-terminal domain of the ϵ subunit of EF_oF₁ slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit.     

Purification and Partial Characterization of Ribulose Bisphosphate Carboxylase Holoenzyme and Its Subunits from Chlorella sorokiniana and Use of Its Antigen Affinity-Purified Antibodies in Specific Immunoprecipitation and Immunoadsorption Procedures

Chlorella sorokiniana ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified to homogeneity with yields of 35 to 40%. Molecular weights of the holoenzyme and its large subunit (LS) and small subunit (SS) were estimated to be 562,000, 55,000, and 15,800, respectively. Amino acid compositions of LS from C. sorokiniana and spinach were similar, whereas the compositions of their SS were very different. Antisera prepared against holoenzyme, LS, and SS were purified by antigen-affinity column chromatography. Purified anti-holoenzyme immunoglobulin G (IgG) and anti-LS IgG cross-reacted with holoenzyme and LS but not with SS. Anti-SS IgG reacted neither with holoenzyme nor with LS. Because purified anti-holoenzyme IgG or the anti-LS IgG inhibited RuBPCase activity, antibody preparations were titered by the amount of ³⁵S-labeled RuBPCase immunoprecipitated. Approximately 40% of the total RuBPCase activity in cell homogenates was tightly particulate-bound and was solubilized with 0.5% Nonidet P-40 without inhibition of enzyme activity. Direct-immunoprecipitation and indirect-immunoadsorption procedures, with affinity-purified anti-holoenzyme IgG, gave specific and quantitative recovery of ³⁵S-labeled RuBPCase from cell extracts containing Nonidet P-40. Affinity-purified anti-LS IgG and anti-SS IgG were used to immunoprecipitate either the LS or SS antigens synthesized in vitro in a mRNA-dependent in vitro translation assay system. Rocket immunoelectrophoresis was used to quantify as little as 50 nanograms of RuBPCase antigen in cell extracts.