Robert M. Metrione

The activities of thiol proteases in the rat visceral yolk sac increase during late gestation

While the rat YVS has been shown to possess an active lysosomal proteolytic system, there are no published reports on the identity of these proteases nor on their changes in activity during the latter half of gestation. We have used specific synthetic substrates to show that cathepsins B, L and H are present in this organ from days 12.5 to 20.5 of gestation. Cathepsins B and L exhibit a marked increase in activity beginning on day 15.5 of gestation. By days 19.5-20.5, cathepsin B activity is increased tenfold over that observed on day 12.5. The activity of cathepsin L may be elevated on day 12.5, decreases more than half by day 14.5 and then increases fourfold by day 20.5. The activity of cathepsin H does not change throughout this period nor do the cathepsins exhibit marked changes in activity in the placenta during this same period or in the PYS from days 12.5 to 14.5 of gestation. These results indicate a specific increase in VYS cathepsin B and L activities late in gestation. These enzymes may be involved in meeting the nutritional needs of the embryo and/or in the degenerative changes which may occur in the VYS and PYS prior to parturition. Studies on the degradation of rat serum albumin by extracts of day 19.5 VYS indicate that cathepsin L may be the quantitatively most important protease in late gestation.

Purification and properties of rat liver cysteine sulfinate decarboxylase

Abstract Rat liver l -cysteine sulfinate (2-amino-3-sulfinopropionate) decarboxylase ( l -cysteine sulfinate carboxy-lyase, EC 4.1.1.29) was purified 300-fold. The purification was achieved by the use of (NH 4 ) 2 SO 4 fractionation between 35–45% satn., gel filtration on Sephadex G-100, and DEAE-cellulose column chromatography. This is the most active and stable cysteine sulfinate decarboxylase reported to date. Observations made by polyacrylamide-gel electrophoresis, molecular sieving on Sephadex G-100, and analytical ultracentrifugation appear to show that the purified enzyme was approaching homogeneity. The sedimentation coefficient and the molecular weight were estimated to be approx. 4.76 S and 66 000, respectively. The cysteine sulfinate decarboxylase activity was measured in Tris-maleate, imidazole, and phosphate buffers at pH 6.8, and the maximal activity was observed in the phosphate buffer. The purified cysteine sulfinate decarboxylase has an apparent K m of 0.2 mM and does not catalyze the decarboxylation of cysteate (2-amino-3-sulfonopropionate). Cupric and mercuric ions are the only metal ions tested which showed some inhibition of the enzyme activity. Dithiothreitol and 2-mercaptoethanol were found as effective as glutathione as activators for cysteine sulfinate decarboxylase.

The thiolesterase activity of leucine aminopeptidase

Abstract 1. 1. Leucine aminopeptidase ( l -leucyl-peptide hydrolase, EC 3.4.1.1) has been shown to be capable of hydrolyzing leucine thiol ethyl ester at a rate 50 times greater than the rate for l -leucine-p-nitroanilide. 2. 2. The pH optimum for the reaction is 8.6 and the Km is 1.3·10−3 M. 3. 3. Both products of the reaction are capable of product inhibition. Alcohols also inhibit the thiolesterase activity of leucine aminopeptidase, but are less effective than mercaptans. (NH4)2SO4 protects leucine aminopeptidase from inhibition by 2-mercaptoethylamine. This effect is not seen in combinations of ammonium sulfate and other mercaptans. 4. 4. A new assay method has been developed which is useful for the measurement of thiolesterase activity of proteolytic enzymes. This method is based on the development of color by the reaction of liberated mercaptan with 5,5′-dithiobis-(2-nitrobenzoic acid). 5. 5. These results extend the types of proteolytic enzymes which are capable of thiolesterase activity to include exopeptidases and metalloenzymes.

DNA replication by isolated rat trophoblast nuclei: Characterization of the system and the product

Abstract DNA biosynthesis by a system containing giant nuclei isolated from rat trophoblast cells at Day 13 of pregnancy has been studied. A method for the isolation of giant nuclei in good yield has been described. These nuclei were capable of incorporating [3H]dTTP into DNA for 2 hr and the incorporation was proportional to the amount of DNA template (nuclei). The system was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg2+ and was stimulated by monovalent ions such as K+. The optimum pH was 8.6. The product of the reaction was insensitive to RNase, sensitive to DNase, and banded at 1.710 g/ml in neutral CsCl together with bulk rat trophoblast DNA. Pulse-chase and density labeling experiments utilizing bromodeoxyuridine have indicated that replicative, discontinuous synthesis was taking place at sites previously active in vivo. DNA polymerases α, β, and γ were shown to be present in the nuclei. Experiments utilizing selective inhibitors of polymerases have demonstrated that DNA replication by trophoblast nuclei in vitro was insensitive to the specific α-polymerase inhibitor, aphidicolin, but almost completely inhibited by 2′, 3′-dideoxythymidine 5′-triphosphate as well as by N-ethylmaleimide suggesting that DNA replication observed in these trophoblast nuclei in vitro may be carried out by DNA polymerase γ.

Separation of lissamine rhodamine B sulfonyl derivative of amino acids by high-performance liquid chromatography and thin-layer chromatography

Abstract The effectiveness of laryl chloride (Lissamine rhodamine B sulfonyl chloride) as a reagent for labeling free amino groups of amino acids, peptides and proteins has been demonstrated. Laryl amino acids are bright red compounds which absorb light with a maximum at a wavelength of 560 nm and emit light with a maximum at a wavelength of 595 nm. On thin-layer plates, fluorescence is about 125 times more sensitive than visual observation of the colored spots and it was possible to observe laryl amino acids in amounts as low as 400 fmol, about half the amount which is needed for dansyl amino acids. The colorimetric thin-layer methods are as sensitive as the dabsyl chloride method and do not require development with hydrochloric acid fumes. A high-performance liquid chromatographic method was developed for the separation of the laryl amino acids and the usefulness of the technique was demonstrated on small peptides.

Bromoacetyl Sepharose: a solid-phase inhibitor of sulfhydryl enzymes.

Bromoacetyl-Sepharose, a solid-phase irreversible inhibitor, has been tested on a variety of enzymes and found to rapidly inactivate the sulfhydryl proteolytic enzymes papain, ficin, cathepsin B, and dipeptidyl aminopeptidase 1. A number of enzymes which do not have activesite sulfhydryls were unaffected by the inhibitor. These enzymes included trypsin, chymotrypsin, amino acid arylamidase, monoamine oxidase, cytochrome b, and alkaline phosphatase. The papain-acetyl-Sepharose conjugate was shown to be susceptible to hydrolysis by pepsin. Thin-layer chromatography of the supernatant fluid from peptic hydrolysates separates at least seven peptides. Amino acid analysis before and after pepsin treatment shows a loss of amino acid content due to proteolysis.

The thiolesterase activity of sulfhydryl-activated enzymes: A new assay for thiol proteases based on activation by Sepharose-bound mercaptan

Abstract It has been shown that sulfhydryl enzymes can be activated in a two-phase system by mercaptans which are coupled to Sepharose beads. Such an activator permits the use of thiol esters as substrates for enzymes requiring mercaptan activators, since the activator mercaptan and product mercaptan are easily separated by centrifugation or filtration before analysis. An assay for cathepsin B using benzyloxycarbony-Lys-thiobenzyl ester as a substrate and glutathione coupled to Sepharose as an activator, has been shown to be 275 times as sensitive as a standard assay using benzoyl- dl -arginine- P -nitroanilide. For papain, the thiol esterase assay is 162 times as sensitive as the benzoyl- dl -arginine- P -nitroanilide assay. A comparison of the advantages and disadvantages of this assay is included.

Variation of DNA polymerase activities of rat giant trophoblast cells in mid-gestation

The activities of the DNA polymerases alpha, beta, and gamma were determined in rat giant trophoblast cells during mid-gestation. Trophoblasts at this period of time have ceased to divide but continue to carry out DNA endoreduplication resulting in polyploidy. DNA polymerase-alpha activity in extracts was found to drop sharply from a high level at Day 11 to 1/10 of that level at Day 12 and to continue at a constant level thereafter. A similar pattern of activity was observed for polymerase-gamma, however, in this case the drop represented 50% of the activity at 11 days. Polymerase-beta showed no significant change in activity during this period of development. Endoreduplication (polyploidy) continued during this period as measured by a linear increase in chromosomal DNA content. The sharp drop in alpha-polymerase activity from Day 11 to Day 12 appears to result from the loss of a protein(s) which specifically stimulate(s) alpha-polymerase.

A protein stimulatory factor for DNA polymerase α in rat giant trophoblast cells

Summary Rat giant trophoblast cells contain a factor which stimulates DNA polymerase α derived from trophoblast cells as well as from calf thymus. The factor, which has been partially purified on a glycerol gradient, has an approximate M r of 85,000 and is non-dialyzable, heat-labile, sensitive to trypsin and insensitive to N-ethylmaleimide. With activated DNA as the preferred template/primer, the factor stimulates both the initial rate and the extent of DNA synthesis as a linear function of the concentration of the factor. The stimulation is abolished by aphidicol in but is unaffected by 2′, 3′ dideoxythymidine triphosphate.

Chromatography of dipeptidyl aminopeptidase I on inhibitor-Sepharose columns.

A number of affinity materials for the purification of dipeptidyl amino-peptidase (dipeptidylpeptide hydrolase, EC 3.4.14.1) have been prepared and tested. These material include peptide and amino acid inhibitors bound to agarose and reversible sulfhydryl adsorbents. Several of these materials are effective affinity adsorbents. The most useful material to be employed in combination with earlier purification methods is acetoxy-anilinomercuri-Sepharose. This removes proteins which are contaminants of some preparations and yields consistently high specific activities. Results with affinity and hydrophobic columns indicate that the primary interactions of the enzyme with amino acid and peptide derivative inhibitors are ionic in nature. This results is in agreement with the conclusions reached in studies of the interactions of these inhibitors with dipeptidyl aminopeptidase in solution.