Studies on activation and inhibition of cathepsin B from buffalo liver

Cathepsin B (EC 3.4.22.1) was purified from buffalo liver. The enzyme activity against-benzoyl-dl-arginine-naphthylamme (BANA) was substantially reduced by heat (above 37C) and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-1-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu²⁺ (20–200M) and Ca²⁺ (30–250 mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5-dithiobis(2-nitro-benzoic acid) (DTNB), andp-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity.     

Computational sequence analysis of matrix metalloproteinases

Matrix metalloproteinases (MMP) play a cardinal role in the breakdown of extracellular matrix involved in a variety of biological and pathological processes. Research on MMPs has classified and characterized these enzymes according to their matrix substrate specificity, gene and protein domain structure, and regulation of activity and expression. However, the discovery of new MMPs has introduced a need for a more comprehensive and systematic method of classification and quantitative comparison of known and newly discovered members. This study compiles a sequence alignment, constructs a dendrogram, and calculates physical data and homology percentage assignments in order to obtain further insight into MMP structure-function relationships. Thorough analysis of MMP primary sequence domains, physical data patterns, and statistical analysis of sequence homology yields higher resolution in the similarities and differences that group MMP members.     

Antiprogestagen RU486 prevents the LH-dependent decrease in the serum concentrations of inhibin in the rat

Summary 1. In the rat, the LH-dependent ovarian progesterone rise mediates several actions of the primary surge of LH on the ovary. This experiment was aimed at elucidating the effects of the antiprogestagen RU486 on the LH-dependent decrease in both the serum concentrations and the ovarian content of inhibin.2. All rats in this experiment were treated with an antagonist of LHRH (1 mg/200 µl saline at 0800 h in proestrus) to supress the endogenous release of LH. One group of rats received 32 µg LH/250 µl saline at 1200 h in proestrus. Other group was given 4 mg RU486/200 µl oil at 0800 h in proestrus. The third group was injected with both RU486 and LH. Rats from the control group were injected with 250 µl saline and 200 µl oil. Animals were decapitated at 1700 h in proestrus and trunk blood and ovaries collected to determine the serum concentrations of LH, FSH, progesterone, 17ß-estradiol and inhibin as well as the ovarian content of inhibin.3. The ovulatory dose of LH in LHRHa-treated rats decreased both the serum concentrations and the ovarian content of inhibin and increased the serum concentrations of FSH. The administration of RU486 blocked the effect of LH on the serum concentrations of inhibin but not that on the ovarian content of inhibin.4. Since the antiprogestagen RU486 blocked the effect of LH on the serum concentrations of inhibin, we conclude that ovarian progesterone, besides mediating the effects of the primary LH surge on the ovulatory process and luteinization, participates in the LH-dependent drop in the serum concentrations of inhibin in proestrous afternoon.     

Expression of a 24 kDa GTP-binding protein (Gn24) is increased in lovastatin treated human erythroleukemia cells

A major 27 kDa particulate and a minor 24 kDa cytosolic GTP-binding protein was detected in HEL cells upon incubation with [-³²P]GTP of nitrocellulose blots containing polypeptides separated using SDS-PAGE. Addition of lovastatin (30 M) to HEL cells in culture inhibited protein synthesis by 35%. However, this treatment resulted in a 5-fold increase, as quantitated by [-³²P]GTP binding, in the amount of cytosolic 24 kDa GTP-binding protein. Addition of cycloheximide plus lovastatin to cells in culture abolished the observed increase in 24 kDa GTP-binding protein. Incubation of cells with lovastatin plus [R,S]-[5-³H]mevalonolactone resulted in the incorporation of radioactivity into several polypeptides in both the cytosolic and particulate fractions including a polypeptide of molecular mass of 24 kDa in the cytosol. The mobility of this 24 kDa isoprenylated protein on SDS-PAGE was identical to that of the GTP-binding protein increased in response to lovastatin. However, the 24 kDa protein remained in the cytosol after undergoing isoprenylation. The 24 kDa protein was distinct from the HEL cell, G25K/CDC42Hs GTP-binding protein and the GTP-binding protein that was a substrate for botulinum toxin C3 catalyzed ADP-ribosylation. Results demonstrate that lovastatin specifically increases the expression of a 24 kDa GTP-binding protein in HEL cells and that, isoprenylation of low molecular mass GTP-binding protein(s) may have function(s) in addition to its role in the targetting of these proteins to cell membrane.     

Phospholipase C activation in rat pituitary adenoma (GH) cells

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) G_q and/or G₁₁ has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G_q and/or G₁₁ confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti G_q/11-sera coprecipitated PL-C activity. In essence, only G_q/11 (but neither G_i2, G_i3 nor G_o) seems to mediate the TRH-sensitive PL-C activity, while G_o may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH₃ pituitary cell line PL-C activity. Finally, the steady state levels of G_q/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP).     

Calcium binding to the subunit c ofE. coli ATP-synthase and possible functional implications in energy coupling

The 8-kDa subunit c of theE. coli F₀ ATP-synthase proton channel was tested for Ca⁺⁺ binding activity using a⁴⁵Ca⁺⁺ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds⁴⁵Ca⁺⁺ strongly with Ca⁺⁺ binding properties very similar to those of the 8-kDa CF₀ subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca⁺⁺ binding capacity, shown by loss of Ca⁺⁺ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca⁺⁺ binding, shown by Ca⁺⁺ binding being retained in twoE. coli mutants, Asp61Asn and Asp61Gly. The Ca⁺⁺ binding is pH dependent in both theE. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca⁺⁺ binding affinity to its F₀ binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca⁺⁺ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca⁺⁺ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80–100 nM was obtained using an EGTA-Ca⁺⁺ buffer system to control free Ca⁺⁺ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca⁺⁺ bound to the periplasimic side of theE. coli F₀ proton channel can block H⁺ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca⁺⁺ binding site is on the lumen side of the thylakoid, where Ca⁺⁺ binding can modulate acid-base jump ATP formation. The Ca⁺⁺ binding to the F₀ and CF₀ complexes is consistent with a pH-dependent gating mechanism for control of H⁺ ion flux across the opening of the H⁺ channel.This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia.     

Halcyon days with Bill Arnold

The circumstances that led to the discovery that plants luminesce after they are illuminated are described, as are other discoveries that would not have been possible were it not for the fortuitous association I had with my dear and most admirable friend, W.A. Arnold, to whom this special issue is dedicated.