Limited proteolysis ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Incubation ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments. Cleavage sites determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and sequence analyses identified protease-sensitive peptide bonds between amino acid residues at positions 9–10 and 76–77. Additional fragmentation sites were also detected in a region approximately 70–80 amino acids before the carboxyl end of the protein. These results suggest that the enzyme is formed by a central compact domain comprising more than two thirds of the whole protein structure. From proteolysis experiments carried out in the presence of substrates, it could be inferred that CO₂ binding specifically protects position 76–77 from trypsin action. Intrinsic fluorescence measurements demonstrated that CO₂ binding induces a protein conformational change, and a dissociation constant for the enzyme CO₂ complex of 8.2±0.6 mM was determined     

Site-Directed Mutagenesis in Basic Amino Acid Residues of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V _max/K _mADP. These results indicate that Arg76 is not involved in CO₂ binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.     

Effects of elevated CO2 on the nutrient compositions and enzymes activities of Nilaparvata lugens nymphs fed on rice plants

Elevated CO 2 may reduce the tolerance of Nilaparvata lugen (N. lugens) to adverse environmental factors through the biological and physiological degeneration of N. lugens. In an artificial climate box, under 375 and 750 μL L 1 CO 2 levels, the rice stems nutrient content, the nutrient content and enzyme activities of N. lugens nymph fed on rice seedlings exposed to ambient and elevated CO 2 were studied. The results showed that rice stems had significantly higher protein and total amino acid levels under ambient than elevated CO 2 levels. Nymphs had significantly higher protein levels in the ambient CO 2 treatment, while their glucose levels were significantly lower under ambient CO 2 conditions. Significantly higher trypsin activity was observed in nymphs grown in elevated CO 2 . Significantly lower activities of the protective enzymes total superoxide dismutase and catalase were observed in the nymphs under ambient CO 2 . Meanwhile, the activity of the detoxification enzyme glutathione S-transferase was significantly higher in the ambient CO 2 treatment. Measuring how energy and resources were allocated to enzymes in N. lugens nymphs under elevated CO 2 conditions can provide a more meaningful evaluation of their metabolic tolerances to adverse climatic conditions.     

The role of effector molecules in regulating ribulosebisphosphate carboxylase activity

Ribulosebisphosphate carboxylase can exist in two forms having different kinetic properties. The fraction of enzyme present in each of the two forms is determined by both the absolute and the relative amounts of substrates and other effector molecules in solution with the enzyme. High CO₂ levels induce formation of an active CO₂ form of the enzyme while high ribulosebisphosphate (RuBP) levels cause formation of a much less active form. The CO₂ form is characterized by a high K_m(RuBP), low K_m(CO2), and relatively high V. The ribulosebisphosphate form has comparatively lower K_m(RuBP) and V and higher K_m(CO2). CO₂ appears to bind before RuBP in the reaction sequence catalyzed by the CO₂ form; this catalytic binding order is apparently reversed in the RuBP form. Steady state rates of enzyme reaction reflect the contributions of both these forms. A brief model, based on the cooperative effects of binding at a small number of catalytic or activator sites in the multimeric enzyme, is presented to account for the changes in enzyme activity with varying substrate and effector molecule concentrations.     

Trypsin cleavage of the α-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities

Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable) nucleotide binding in beef heart mitochondrial F₁-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103–7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the α-subunit of F₁ is altered by the mild trypsin treatment, whereas no change was detected in β-, γ-, δ- or ?-subunits. Binding of ADP to the trypsin-treated F₁ was compared to binding to control enzyme over a range of 0–40 μM ADP in a 30 min incubation period. There was no difference between the two enzymes, K^ADP_d in Mg²⁺-containing buffer was about 2 μM in each. Since the tight (nonexchangeable) sites are abolished in trypsin-treated F₁, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the α-subunit on β-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the α-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F₁ but leaves partial functions intact.     

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman–Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P4_1, with R_work/R_free values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme–inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.     

Identification of two binding sites of the D-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach for D-ribulose 1,5-bisphosphate and effectors of the carboxylation reaction.

Fluorimetric studies of the binding of d-ribulose 1,5-bisphosphate (RuP₂) and the effectors 6-phosphogluconate and fructose 1,6-bisphosphate to the d-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach were correlated with the functions of these sugar phosphates in the carboxylation reaction. These agents compete for two binding sites of the enzyme. At relatively low concentrations they bind to an allosteric site, where 6-phosphogluconate and fructose 1,6-bisphosphate display their stimulating effect on the fixation of CO₂. At higher concentrations these compounds inhibit the carboxylation reaction and compete with RuP₂ for the reaction center of the carboxylase. Preincubation of the enzyme with low concentrations of RuP₂ (0.1–5 μm) inhibits the activity of these effectors as well as the effector-induced fluorescence changes of the enzyme-2-p-toluidinonapthalene-6-sulfonate (TNS) complex by competition for the regulatory center which could be identified as the high affinity binding site of the enzyme for RuP₂ with a K_D = 0.6 μm. The deactivation of the carboxylase which is observed on preincubation of the enzyme with RuP₂ in the absence of bicarbonate and Mg²⁺ cannot be correlated to the binding of RuP₂ to the effector site. The deactivation process occurs in an RuP₂ concentration range similar to that for CO₂ fixation.     

A new esteroproteinase (proteinase F) from the submandibular glands of female mice

One of the esteroproteinases present in the submandibular glands of female mice was purified and characterized. The enzyme, designated proteinase F in this report, had a pI value of 4.6 and a molecular weight of 27 600, being comprised of two subunits of 10 000 and 18 000 daltons. The amino acid composition of proteinase F resembled that of the epidermal growth factor-binding protein, but antiserum against proteinase F only reacted weakly against the binding protein. Proteinase F had an optimum pH at around 9.0 and was strongly inhibited by Cu²⁺ and Hg²⁺ (42 and 76% inhibition, respectively, at a concentration of 4·10^?6 M). It was also inhibited by aprotinin, phenylmethylsulfonylfluoride, iodoacetamide, leupeptin, antipain, and benzamidine but neither by trypsin inhibitors from pancreas, soybean or ovomucoid, nor by TLCK, TPCK, and ?-amino-n-caproic acid. Although its actual physiological function has yet to be determined, these properties indicate that proteinase F is a new enzyme, being distinguished from known proteinases, kallikrein, plasmin, trypsin, chymotrypsin, tonin, angiotensin-converting enzyme, proteinase A (β-nerve growth factor endopeptidase), proteinase D (epidermal growth factor-binding protein), P-esterase, renin A, and renin C. Proteinase F was present in the submandibular glands of female mice more abundantly than in those of males, but it increased in males following castration. Thus, proteinase F appears to be affected by male hormones in vivo.     

α2-Macroglobulin-protease reactions: Relationship of covalent bond formation,methylamine reactivity,and specific proteolysis

Experiments were performed to define the relation between covalent binding of enzymes to β₂-macroglobulin (α₂M), the specific proteolysis of α₂M subunits to 85K fragments, and the reactivity of the methylamine site on α₂M. We studied the reaction of α₂M with native trypsin, anhydrotrypsin, and two active lysyl-blocked derivatives, methyl-trypsin and dimethylmaleyl-trypsin, the last with reversibly modified amino groups that can be regenerated at low pH. The results were: (1) All enzymes tested reacted with α₂M but only native trypsin formed covalent complexes (not dissociable by sodium dodecyl sulfate). Trypsin and the lysyl-blocked enzymes caused complete proteolysis of the α₂M subunits, in agreement with previous studies. (2) The dimethyl-maleyl-trypsin became covalently bound to α₂M only after removing the blocking groups of the bound enzyme, indicating that sequential proteolysis and covalent bond formation is possible. Under the conditions used for deblocking, there was no change in the covalent/noncovalent binding ratio of native trypsin, anhydrotrypsin, or the other lysyl-blocked derivative, methyl-trypsin. (3) Native trypsin or anhydrotrypsin displaced methyl- or dimethylmaleyl-trypsin from their α₂M complexes but the newly bound enzymes with free amino groups did not form covalent bonds indicating that enzymes must remain in association with the inhibitor for the bond to form. (4) Methylamine reacts with noncovalent α₂M complexes but not with covalent complexes. (5) Methylamine-treated α₂M can still form complexes with trypsin but at a drastically reduced rate and only noncovalent complexes are formed. In summary, sequential proteolysis and covalent bond formation is possible under certain conditions, and there is a strong correlation between covalent binding and loss of methylamine reactivity. The latter observation is suggestive evidence for the identity of the covalent binding site of α₂M and the putative thiol ester of the methylamine site. The enzyme lysyl amino groups, are likewise possible candidates for attacking nucleophile at that site.     

Purification and Characterization of a Proteinase Inhibitor from Field Bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^?1 and 3.1 × 10⁷ M^?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

CONVERSION OF TRYPSIN TO A COPPER ENZYME: TYROSINASE/CATECHOL OXIDASE BY CHEMICAL MODIFICATION

New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase. The introduction of a coordination complex into a substrate binding pocket of trypsin could probably also be extended to various enzymes of significant therapeutic and biotechnological importance.

The aim of this study is the conversion of trypsin into a copper enzyme: tyrosinase by chemical modification. Tyrosinase is a biocatalyst (EC.1.14.18.1) containing two atoms of copper per active site with monooxygenase activity. The active site of trypsin (EC 3.4.21.4), a serine protease was chemically modified by copper (Cu⁺²) introduced p-aminobenzamidine (pABA- Cu⁺²: guanidine containing schiff base metal chelate) which exhibits affinity for the carboxylate group in the active site as trypsin-like inhibitor. Trypsin and the resultant semisynthetic enzyme preparation was analysed by means of its trypsin and catechol oxidase/tyrosinase activity. After chemical modification, trypsin-pABA-Cu⁺² preparation lost 63% of its trypsin activity and gained tyrosinase/catechol oxidase activity. The kinetic properties (K_cat, K_m, K_cat/K_m), optimum pH and temperature of the trypsin-pABA-Cu⁺² complex was also investigated.     

Site-Directed Mutagenesis in Basic Amino Acid Residues of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V _max/K _mADP. These results indicate that Arg76 is not involved in CO₂ binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.     

Protective effect of the pteroylpolyglutamates and phosphate on the proteolytic inactivation of thymidylate synthetase

Thymidylate synthetase is readily inactivated by trypsin, chymotrypsin, and carboxypeptidase A when incubated in 10–20 mm potassium phosphate buffer (pH 7.0). The loss is activity produced by trypsin and chymotrypsin is accomplished by extensive protein degradation, while inactivation by carboxypeptidase A is accompanied by release of the carboxyl-terminal valine only (Aull et al., 1974, J. Biol. Chem., 249, 1167–1172). In contrast, when the incubations are conducted in 100–200 mm potassium phosphate buffer (pH 7.0), the synthetase is not inactivated by any of the three enzymes and the results of amino acid analysis and sodium dodecyl sulfate disc gel electrophoresis demonstrate that proteolysis is prevented. The resistance of thymidylate synthetase to inactivation was shown not to be due to the inhibition of the proteolytic enzymes by the buffer. The inactivation is not prevented either by pteroylmonoglutamates or by 2′-deoxyuridine 5′-phosphate (dUMP) alone, but the presence of both is partially protective. The pteroylpolyglutamates, however, offer limited protection against carboxypeptidase A and chymotrypsin; in combination with dUMP, proteolytic inactivation of the snythetase by all three enzymes is prevented. Characterization of the properties of carboxypeptidase A-inactivated thymidylate synthetase reveals the following, (i) The binding of deoxynucleotides is unaltered, but the binding of a variety of pteroylpolyglutamate derivatives is reduced or abolished, (ii) Pteroylpolyglutamates are bound provided dUMP or an analog such as 5-fluorodUMP is present, (iii) Ternary complex formation between carboxypeptidase A-inactivated enzyme and (+)5,10-methylenetetrahydropteroyltetraglutamate plus 5-fluorodUMP occurs in the same molar binding ratio (1:2:2) at saturation as with the native enzyme, but differs from the native enzyme ternary complex in that the dissociation constant for 5-fluorodUMP is increased by approximately 10⁵. In addition, there is no evidence for the formation of covalent linkages between the ligands and enzyme, (iv) The treated enzyme cannot catalyze tritium release from [³H₅]dUMP in the presence of either (+)5,10-methylenepteroylmonoglutamate or (+)5,10-methylenetetrahydropteroyltetraglutamate.     

A structural model for across membrane coupling between the Qo and Qi active sites of cytochrome bc1

The two spatially distant quinone-binding sites of the ubihydroquinone: cytochrome c oxidoreductase (cyt bc₁) complex have been shown to influence one another in some fashion. This transmembrane communication alters cofactor and redox partner binding interactions and could potentially influence the timing or ‘concerted’ steps involved in the steady-state turnover of the homodimeric enzymes. Yet, despite several lines of evidence corroborating the coupling of the quinone binding active sites to one another, little to no testable hypothesis has been offered to explain how such a “signal” might be transmitted across the presumably rigid hydrophobic domain of the enzyme. Recently, it has been shown that this interquinone binding sites communication influences the steady-state position of the mobile [2Fe–2S] cluster containing iron sulfur protein (Sarewicz M., Dutka M., Froncisz W., Osyczka A. (2009) Biochemistry 48, 5708–5720) as mediated by at least one transmembrane helix of the b-type cyt containing subunit (Cooley, J. W., Lee, D. W., and Daldal, F. (2009) Biochemistry 48, 1988–1999). Here we provide an overview of the evidence supporting the structural coupling of these sites and provide a theoretical framework for how the redox state of a quinone at one cofactor binding site might influence the cofactor–, inhibitor–, and/or protein–protein interactions at the structurally distant opposing Q binding site.     

Electron paramagnetic resonance evidence of metal-ion binding sites in Micrococcus lysodeikticus (M. luteus) F1-ATPase

Evidence is presented for the presence of divalent cation binding sites in purified F₁-ATPase from Micrococcus lysodeikticus (Micrococcus luteus). Electron paramagnetic resonance studies of native F₁-ATPase indicate that the enzyme binds Mn²⁺ and Cu²⁺. Scatchard-type plot for Mn²⁺ binding to the enzyme indicates the presence of 3–4 independent and identical sites with a dissociation constant of 18.3 · 10⁻⁶ M. Cu²⁺ binds to the enzyme at only one kind of site(s). This Cu²⁺ binding site(s) is characterized by a moderately ionic ligand field provided by the protein and by a tetragonal symmetry of nitrogen and/or oxigen ligands. Competition studies indicate that Mg²⁺ binds at these Mn²⁺ and Cu²⁺ binding sites.     

Modeling and mutagenesis of amino acid residues critical for CO2 hydration by specialized NDH-1 complexes in cyanobacteria

The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO₂-concentrating mechanism (CCM). This includes specialized Type-1 NDH complexes that function to couple photosynthetic redox energy to CO₂ hydration forming the bicarbonate that accumulates to high cytoplasmic concentrations during the operation of the CCM, required for effective carbon fixation. Here we used a Synechococcus PCC7942 expression system to investigate the role of conserved histidine and cysteine residues in the CupB (also designated, ChpX) protein, which has been hypothesized to participate in a vectoral CO₂ hydration reaction near the interface between CupB protein and the proton-pumping subunits of the NDH-1 complex. A homology model has been constructed and most of the targeted conserved residues are in the vicinity of a Zn ion modeled to form the catalytic site of deprotonation and CO₂ hydration. Growth and CO₂ uptake assays show that the most severe defects in activity among the targeted residues are due to a substitution of the predicted Zn ligand, CupB-His86. Mutations at other sites produced intermediate effects. Proteomic analysis revealed that some amino acid substitution mutations of CupB caused the induction of bicarbonate uptake proteins to a greater extent than complete deletion of CupB, despite growth under CO₂-enriched conditions. The results are discussed in terms of hypotheses on the catalytic function of this unusual enzyme.     

Binding of pyridine coenzymes to the β-subunit of the voltage sensitive potassium channels

The β-subunit of the voltage-sensitive K⁺ channels shares 15–30% amino acid identity with the sequences of aldo–keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of K_vβ2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The K_D values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1±0.007 and 0.05±0.006 M, respectively — indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the α-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the K_D for NADH and NAD were between 1–3 μM whereas the K_D for FAD was >10 μM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2′ phosphate at the binding site. The selectivity of K_vβ2.1 for NADPH over NADP may be significant in regulating the K⁺ channels as a function of the cellular redox state.     

Characterization of cold-adapted Atlantic cod (Gadus morhua) trypsin I — Kinetic parameters,autolysis and thermal stability

Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k_cat/K_m) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 °C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.     

Partial purification and properties of a protein kinase C type enzyme from plants

Partial purification of a protein kinase with a dependence on micromolar concentrations of free calcium has been achieved from seedlings of Amaranthus tricolor. The enzyme has a M_r of 77 600 as determined by gel filtration and 84 500 by SDS-PAGE analysis. Interaction of the enzyme with membranes (inside-out erythrocyte vesicles) is regulated by calcium, a characteristic of animal protein kinase C. Phospholipid and diolein activation of the enzyme is markedly dependent on the phospholipid used and on both calcium and phospholipid concentration. K_m values for Ca²⁺ in the absence of phospholipid was 20–40 μM and in the presence of phosphatidylserine 5–10 μM. Diolein plus phosphatidylserine lowered the K_m to < 1.5 μM. The best activation was achieved at 1OOμM calcium with 40μg/ml phosphatidylserine and 8μg/ml diolein. These properties indicate a protein kinase C type enzyme. The plant enzyme reacted with antiserum directed against the regulatory domain of bovine brain protein kinase C in an immunoblot experiment.