Human placenta S-adenosylmethionine: protein carboxyl O-methyltransferase (protein methylase II). Purification and characterization

1. Protein methylase II was purified from human placenta approx. 8700-fold with a yield of 14%. 2. Unlike protein methylase II from other sources, the activity of human placenta enzyme was completely inhibited by 2 mM Cu2+. Other divalent ions were without effect. 3. Human chorionic gonadotropin (HCG), immunoglobulin A and calf thymus histones served as good in vitro substrates for the enzyme, particularly HCG. 4. The Km for S-adenosyl-L-methionine and Ki for S-adenosyl-L-homocysteine were 2.08 x 10(-6) and 5.8 x 10(-7) M, respectively. 5. The protein methylase II activity in human placenta changed with gestational age, the activity at 1st and 2nd trimester being approximately twice that of term placenta.     

Divalent cation ionophore A23187: a potent protein synthesis inhibitor.

Divalent cation ionophore A23187 has a potent inhibitory effect on protein synthesis in the C6 rat glioma cell line. Treatment with 4 μM A23187 resulted in 93% inhibition of [1-¹⁴C]leucine label incorporation into proteins and a 61% increase in free pool labeling. Total RNA synthesis was not affected. Extracellular ionic calcium or magnesium are not required for these changes to occur. Therefore, these effects of A23187 may be a direct effect on protein synthesis or may result from release of internal stores of divalent cations. By comparison, ionophore X537A (4 μM) has only a slight inhibitory effect on protein synthesis.     

Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis.

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 microM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.     

Sangivamycin, a nucleoside analogue, is a potent inhibitor of protein kinase C

Protein kinase C functions prominently in cell regulation via its pleiotropic role in signal transduction processes. Certain oncogene products resemble elements involved in transmembrane signaling, elevate cellular sn-1,2-diacylglycerol second messenger levels, and activate protein kinase C. Sangivamycin was unique among the nucleoside compounds tested in its ability to potently inhibit protein kinase C activity. Inhibition was competitive with respect to ATP for both protein kinase C and the catalytic fragment of protein kinase C prepared by trypsin digestion. Sangivamycin was a noncompetitive inhibitor with respect to histone and lipid cofactors (phosphatidylserine and diacylglycerol). Sangivamycin inhibited native protein kinase C and the catalytic fragment identically, with apparent Ki values of 11 and 15 microM, respectively. Sangivamycin was an effective an inhibitor of protein kinase C as H-7, an isoquinolinsulfonamide. Sangivamycin did not inhibit [3H]phorbol-12,13-dibutyrate binding to protein kinase C. Sangivamycin did not exert its action through the lipid binding/regulatory domain; inhibition was not affected by the presence of lipid or detergent. Unlike H-7, sangivamycin selectively inhibited protein kinase C compared to cAMP-dependent protein kinase. The discovery that protein kinase C is inhibited by sangivamycin and other antitumor agents suggests that protein kinase C may be a target for rational design of antitumor compounds.     

X-ray crystal structure of sangivamycin, a potent inhibitor of protein kinases

The X-ray crystal structure of sangivamycin, a potent nucleoside inhibitor of protein kinases, has been determined. Sangivamycin crystallizes from water with its purine ring in a conformation anti to its ribose sugar. Such an anti conformation has been detected in solution for sangivamycin and other potent protein kinase inhibitors and appears to correlate with inhibitor potency [(1990) Biochemistry (in press)]. An intramolecular hydrogen bond between purine ring substituents is detected in the X-ray structure and may be an important structural feature of sangivamycin related to its degree of inhibition of rhodopsin kinase and of protein kinases C and A.     

Genistein, a soy isoflavone, is a potent alpha-glucosidase inhibitor.

Genistein is an isoflavone that is known to be contained in soybean. It was proved that genistein plays a pivotal role in homeostasis in the human body. In the course of screening for useful alpha-glucosidase inhibitors, we isolated and identified genistein as a candidate for alpha-glucosidase inhibitor from fermentation broths of a Streptomyces sp. Genistein was shown to be a reversible, slow-binding, non-competitive inhibitor of yeast alpha-glucosidase with a K(i) value of 5.7x10(-8) M when the enzyme mixture was pretreated with genistein. These results show a possibility that genistein could be a useful tool for metabolic disorders.     

Hemin as a generic and potent protein misfolding inhibitor

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.     

Solution structure of Compstatin,a potent complement inhibitor.

The third component of complement, C3, plays a central role in activation of the classical, alternative, and lectin pathways of complement activation. Recently, we have identified a 13-residue cyclic peptide (named Compstatin) that specifically binds to C3 and inhibits complement activation. To investigate the topology and the contribution of each critical residue to the binding of Compstatin to C3, we have now determined the solution structure using 2D NMR techniques; we have also synthesized substitution analogues and used these to study the structure-function relationships involved. Finally, we have generated an ensemble of a family of solution structures of the peptide with a hybrid distance geometry-restrained simulated-annealing methodology, using distance, dihedral angle, and 3J(NH-Halpha)-coupling constant restraints. The Compstatin structure contained a type I beta-turn comprising the segment Gln5-Asp6-Trp7-Gly8. Preference for packing of the hydrophobic side chains of Val3, Val4, and Trp7 was observed. The generated structure was also analyzed for consistency using NMR parameters such as NOE connectivity patterns, 3J(NH-Halpha)-coupling constants, and chemical shifts. Analysis of Ala substitution analogues suggested that Val3, Gln5, Asp6, Trp7, and Gly8 contribute significantly to the inhibitory activity of the peptide. Substitution of Gly8 caused a 100-fold decrease in inhibitory potency. In contrast, substitution of Val4, His9, His10, and Arg11 resulted in minimal change in the activity. These findings indicate that specific side-chain interactions and the beta-turn are critical for preservation of the conformational stability of Compstatin and they might be significant for maintaining the functional activity of Compstatin.     

Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase

Staurosporine, microbial alkaloid which has been known to have antifungal activity was found to inhibit markedly phospholipid/Ca++dependent protein kinase (protein kinase C) from rat brain, with an IC50 value of 2.7 nM. However, it had little effect on the binding of 3H-phorbol-12, 13-dibutyrate (PDBu) to protein kinase C. The inhibition of protein kinase C was not competitive with phospholipid. This compound also showed the strong cytotoxic effect on the growth of HeLa S3 cells, with an IC50 value of 4 X 10(-12)M under the condition of 72 hr-exposure.     

Inherent flexibility in a potent inhibitor of blood coagulation, recombinant nematode anticoagulant protein c2.

Nematode anticoagulant proteins (NAPs) from the hematophagous nematode Ancylostoma caninum inhibit blood coagulation with picomolar inhibition constants, and have been targeted as novel pharmaceutical agents. NAP5 and NAP6 inhibit factor Xa by binding to its active site, whereas NAPc2 binds to factor Xa at a different, as yet unidentified, site and the resultant binary complex inhibits the tissue factor-factor VIIa complex. We have undertaken NMR studies of NAPc2, including the calculation of a solution structure, and found that the protein is folded, with five disulfide bonds, but is extremely flexible, especially in the acidic loop. The Halpha secondary shifts and 3JHNHalpha coupling constants indicate the presence of some beta structure and a short helix, but the intervening loops are highly conformationally heterogeneous. Heteronuclear NOE measurements showed the presence of large amplitude motions on a subnanosecond timescale at the N-terminus and C-terminus and in the substrate-binding loop, indicating that the conformational heterogeneity observed in the NMR structures is due to flexibility of the polypeptide chain in these regions. Flexibility may well be an important factor in the physiological function of NAPc2, because it must interact with other proteins in the inhibition of blood coagulation. We suggest that this inhibitor is likely to become structured on binding to factor Xa, because the inhibition of the tissue factor-factor VIIa complex requires both NAPc2 and factor Xa.     

2-fluorourocanic acid, a potent reversible inhibitor of urocanase.

The K_i for the interaction of 2-fluorourocanic acid with urocanase (from $\text{Pseudomonas fluorescens}$) is 1000 times as great as K_m for the natural substrate, urocanic acid, whereas enzymatic hydration of the fluoro analog occurs $\text{ca}$. 100 times more slowly. Inhibition is competive and is eventually overcome by utilization of the analog. By contrast, 4-fluoro- and 2-amino-urocanic acid are neither significant inhibitors nor substrates for the enzyme. 2-Fluorourocanic acid may prove a useful tool for blocking the utilization of histidine as a one-carbon source in metabolism.     

Proctolin: a potent inhibitor of aminoenkephalinase

Proctolin is a potent selective inhibitor of aminoenkephalinase. The specificity of its inhibition of various aminopeptidases is similar to that of puromycin; it inhibits aminoenkephalinase, but not leucine aminopeptidase or aminopeptidase M. Enkephalin breakdown by synaptic plasma membrane, but not by brain slices, is sensitive to proctolin. The inhibition by proctolin is partially caused by its resistance to enzymatic breakdown. The inhibition is of mixed type and is concentration dependent, and the two amino acids at the N-terminal are important for its action. The minimal structure for inhibition is a dipeptide with a basic amino acid at the N-terminal and a basic or an aromatic amino acid at the C-terminal.     

Synthesis and characterization of a potent and selective protein tyrosine phosphatase inhibitor, 2

The synthesis and biological activity of a series of 2-[(4-methylthiopyridin-2-yl)methylsulfinyl]benzimidazoles are described. These compounds have potent inhibitory effects against the protein tyrosine phosphatase activity of CD45. Enzymatic analysis with several phosphatases revealed that compound 5a had high specificity for CD45 compared with serine/threonine phosphatases (PP1, PP2A), tyrosine phosphatases (LAR, PTP1B and PTP-S2) and dual phosphatase (VHR).     

Cremophor EL, a widely used parenteral vehicle, is a potent inhibitor of protein kinase C

Cremophor EL, a castor oil derivative, has been considered a non-toxic solubilizer for lipophilic drugs and vitamins. Protein kinase C, a phospholipid/Ca++-dependent protein kinase, is known to phosphorylate, in response to extracellular stimuli, a variety of proteins for cellular functions. The present study shows that Cremophor EL selectively inhibits the activity of protein kinase C in vitro. The potency of this selective inhibition is greater than that of other protein kinase C-specific inhibitor thus far reported. Cremophor EL acts primarily on the enzyme activator diacylglycerol (or the phorbol ester) and prevents the latter from both interacting with the phospholipid and binding to protein kinase C. This is the first report of a significant biological activity induced by this widely used substituted castor oil solubilizer.     

The effect of K-252a, a potent microbial inhibitor of protein kinase, on activated cyclic nucleotide phosphodiesterase.

K-252a, an indole carbazol compound of microbial origin, inhibited activation of bovine brain phosphodiesterase induced by calmodulin (CaM), sodium oleate, or limited proteolysis with almost equal potency. Kinetic analysis revealed that the CaM-activated phosphodiesterase (CaM-PDE) was competitively inhibited by K-252a with respect to CaM. On the other hand, inhibition of the trypsin-activated phosphodiesterase was competitive with respect to cyclic AMP. Addition of a lower amount of phosphatidylserine or sodium oleate to the reaction medium was efficacious in attenuating the inhibition of the CaM-PDE by W-7, compound 48/80, or calmidazolium but, in contrast, had no effect on the inhibition by K-252a. Furthermore, CaM-independent systems such as [3H]nitrendipine receptor binding or Na+ + K+-ATPase were influenced less by K-252a compared with W-7, compound 48/80 and calmidazolium. In conclusion, K-252a is an inhibitor of CaM-dependent cyclic nucleotide phosphodiesterase and it appears that it inhibits the enzyme not only via CaM antagonism but possibly also by interfering with the enzyme.     

Reactive blue 2 is a potent inhibitor of a thylakoid protein kinase

The anthraquinone dye reactive blue 2 was found to be a potent inhibitor of a protein kinase isolated and purified from thylakoids. This enzyme was also inhibited in situ, with corresponding inhibition of ATP-dependent quenching of the chlorophyll fluorescence. The mode of inhibition was noncompetitive, with a Ki of 8 microM for the membrane-bound kinase, and 6 microM for the purified kinase. The inhibitor did not modify the substrate preference of the endogenous kinase and could be removed from the membrane by washing. Unlike reactive blue 2, the enzyme did not partition into detergent micelles and is therefore presumably not a hydrophobic, intrinsic membrane protein.     

Chelerythrine is a potent and specific inhibitor of protein kinase C

The benzophenanthridine alkaloid chelerythrine is a potent, selective antagonist of the Ca++/phospholopid-dependent protein kinase (Protein kinase C: PKC) from the rat brain. Half-maximal inhibition of the kinase occurs at 0.66 microM. Chelerythrine interacted with the catalytic domain of PKC, was a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) (Ki = 0.7 microM) and a non-competitive inhibitor with respect to ATP. This effect was further evidenced by the fact that chelerythrine inhibited native PKC and its catalytic fragment identically and did not affect [3H]- phorbol 12,13 dibutyrate binding to PKC. Chelerythrine selectively inhibited PKC compared to tyrosine protein kinase, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. The potent antitumoral activity of celerythrine measured in vitro might be due at least in part to inhibition of PKC and thus suggests that PKC may be a model for rational design of antitumor drugs.     

A potent antifungal protein from Helianthus annuus flowersis a trypsin inhibitor

A 16-kDa protein was isolated from Helianthus annuus flowers by its ability to inhibit the germination of fungal spores. This protein, SAP16, displays an associated activity of trypsin inhibitor and was further purified to apparent homogeneity by affinity chromatography on trypsin-agarose. SAP16 causes the complete inhibition of Sclerotinia sclerotiorum ascospores germination at a concentration of 5 μg·mL^–1 (0.31 μM) and a clear reduction of mycelial growth at lower concentrations, indicating a strong antifungal potency against this natural pathogen of sunflower. Our data suggest that the antifungal ability of SAP16 would not be the result of the inhibition of a fungal protease. This study contributes to the characterization of the emerging family of antifungal proteins with an associated activity of trypsin inhibition and emphasizes their role in plant resistance against fungal attack.