Stanley Ellis

On the substrate specificity of cathepsins B1 and B2 including a new fluorogenic substrate for cathepsin B1

Abstract Cathepsin B1 from bovine spleen exhibited its greatest rates of hydrolysis on peptide β-naphthylamide (βNA) derivatives containing paired basic residues, i.e., Cbz-Arg-Arg-βNA, t-Boc-Lys-Lys-βNA, and t-Boc-Lys-Arg-βNA. Internal peptide bonds were not attacked. At its pH 6.5 optimum, cathepsin B1 hydrolyzed Cbz-Arg-Arg-βNA (K m 0.18 mM) 64 times faster than Bz-DL-Arg-βNA (K m 3.3 mM or 1.6 mM for the L isomer) and was therefore chosen to replace the latter as a more soluble and sensitive substrate for the assay of cathepsin B1. Although cathepsin B2 had no action on the β-naphthylamide substrates, it did manifest carboxypeptidase activity by attacking COOH-terminal residues exposed by the action of cathepsin B1. At its pH 5.0 optimum, cathepsin B2 behaved as a SH-dependent, non-specific carboxypeptidase by releasing COOH-terminal amino acids from a variety of Cbz-Gly-X substrates and polypeptides such as glucagon, Val-Leu-Ser-Glu-Gly, and penta-lysine.

[22] Preparation and specificity of dipeptidyl aminopeptidase I.

Publisher Summary The dipeptidyl aminopeptidases (DAP) are enzymes that catalyze the consecutive removal of dipeptide moieties from the unsubstituted NH 2 termini of polypeptide chains. Four such enzymes are characterized and shown to have distinctive substrate specificities. Within this group, dipeptidyl aminopeptidase I (DAP I) exhibits the broadest substrate specificity. When DAP I is observed in bovine pituitary extracts, it is characterized as a sulfhydryl-dependent Ser-Tyr-β-naphthylamidase with an absolute or near-absolute chloride requirement and a pH 4 optimum. The purification serves as the basis for the preparation of DAP I. The practicability of using DAP I as a sequencing reagent is greatly enhanced by the relative ease with which a suitable preparation can be made. The broad substrate specificity of DAP I is first observed with β-corticotropin used as a substrate. DAP I activity has also been measured on Gly-Phe-NH 2 . An analysis of the specific bonds cleaved in polypeptide substrates revealed that DAP I can remove dipeptides having penultimate residues with both hydrophilic and hydrophobic side chains.

Detection of a lysosomal carboxypeptidase and a lysosomal dipeptidase in highly-purified dipeptidyl aminopeptidase I (cathepsin C) and the elimination of their activities from preparations used to sequence peptides.

Abstract The best preparations of dipeptidyl aminopeptidase I (DAP I) from beef spleen and rat liver were found to contain a carboxypeptidase (“catheptic carboxypeptidase C”) and a dipeptidase (“Ser-Met dipeptidase”). Each had a pH optimum near 5.5, a resistance to sulfhydryl inhibitors, and a lysosomal origin. The carboxypeptidase, which was inhibited by diisopropylphosphorofluoridate (DFP), preferentially cleaved COOH-terminal residues adjacent to proline, as in angiotensin II and Z-Pro-Phe. No action was detected on Z-Pro-Phe-NH 2 . The dipeptidase, which was separable by electrofocusing, was most active on Ser-Met, and showed no action on Z-Ser-Met, Ser-Met-NH 2 , Ser-Met-Glu, Gly-Gly or Gly-Leu. Ser-Met dipeptidase was unaffected by DFP, but was strongly inhibited by EDTA. A metal requirement was not apparent, however. A simplified method is described for preparing DAP I as a sequencing reagent free of these contaminating activities.

The hydrolysis of amino acyl-β-naphthylamides by plasma aminopeptidases

Abstract Historically, the study of aminopeptidase activity in mammalian systems has been directed primarily toward tissue extracts. This work culminated in the isolation and characterization of the classical leucine aminopeptidase ( Smith and Hill, 1960 ). Little attempt has been made to characterize the aminopeptidases of normal plasma and serum, and perhaps it is for this reason that the activity has been tacitly identified with tissue leucine aminopeptidase (LAP), and commonly designated serum LAP. Behal et al. (1963) have chromatographically resolved the LAP activity of human blood into a number of aminopeptidase components. In this report, some distinguishing properties are described for a variety of aminopeptidases which can occur in blood plasma. The aminopeptidase activity of normal plasma could not be attributed to the presence of leucine aminopeptidase.

Neurotrophic factor - Characterization and partial purification

Recently published in vivo and in vitro evidence suggests that neurotrophic activity is required for normal proliferation and development of muscle cells. In the present work, two independent and quantitative in vitro assay systems are used to monitor partial purification of neurotrophic activity from adult chicken ischiatic-peroneal nerves. The greatest amount of neurotrophic activity is extracted at pH 8. Progressively less activity is extracted with either increasing or decreasing pH. Aqueous suspensions of neurotrophic activity are labile to changes in pH but not to long-term storage at room temperature or to storage for more than a week at 37°C. Specific activity is doubled upon precipitation of crude extract with ammonium sulfate at 50–75% saturation or after gel filtration on Sephadex G-100 columns. Salt gradient elution from DEAE-cellulose columns yields a single active peak with specific activity increased 4- to 5-fold. Active fraction obtained after gel filtration and rechromatography on DEAE-cellulose yields a single active peak with a specific activity increased 7- to 10-fold. Electrophoresis of the most highly purified active material reveals a greatly concentrated band estimated at 80,000 daltons, whereas other bands disappear or diminish in intensity as a result of purification. Although purification of neurotrophic activity is indicated by an increase in specific activity, maximum activity of more highly purified material is reduced; this leads us to postulate the existence of a cofactor necessary for the expression of maximum activity.

Separation and identification of dipeptides by paper and column chromatography

Abstract The behavior of approximately 140 dipeptides on column chromatography as well as in four different solvent systems on paper chromatography has been reported along with the ninhydrin color values of the dipeptides. These data are useful in predicting the elution times and Rf values of unknown dipeptides. The use of these systems for separation and identification of dipeptides coupled with N-terminal analysis in a few equivocal situations makes possible the separation and identification of practically all dipeptides.

ISOLATION AND PROPERTIES OF RAT AND RABBIT GROWTH HORMONES

The present studies were undertaken with the aim of developing a method for isolating highly purified rodent growth hormones that would be useful in studies on the comparative biochemistry of growth hormone as well as for immunological assays for plasma growth hormone in the rat. The isolation of a highly purified growth hormone from frozen rat pituitaries has been described recently by Reisfeld and colleagues,’ who by means of fractionation with ethanol and zone electrophoresis a t pH 8.6, isolated the hormone having a potency of 1 to 1.5 USP units per mg. Employing acetone-dried pituitaries, Wilhelmi2 has obtained rat growth hormone with a potency of 2 USP units per mg by fractionation with (NH4)2S04 and chromatography on DEAE-cellulose a t pH 8.0. To facilitate the recovery and isolation of other pituitary hormones as well as growth hormone, the sequential extraction procedure, previously employed with bovine and ovine gland^,^ has been applied to the extraction of rat and rabbit glands in our l a b ~ r a t o r y . ~

[23] Sequencing of peptides with dipeptidyl aminopeptidase I

Publisher Summary This chapter presents the methodology by which dipeptidyl aminopeptidase I (DAP I) can be utilized in a new approach to the sequencing of small amounts (0.5 μmole or less) of peptides. The ability of DAP I to remove dipeptides, in sequence, from the NH 2 termini of oligopeptides can be followed, for at least several dipeptide products, by time-course chromatography. Although often this is insufficient for establishing sequence, it is possible to modify the peptide substrate in such a way that DAP I generate the alternate, overlapping dipeptides––an advantage not possible with monoaminoacyl aminopeptidases. Certain advantages over chemical methods of sequencing are inherent in this approach. Asparagine and glutamine are recovered as such (not as aspartic or glutamic acid), and there is no tendency to form pyroglutamic acid, as occurs with chemical methods. The inability of DAP I to remove dipeptides with an NH 2 -terminal arginine or lysine is not a problem if the peptide substrates are derived from tryptic digests wherein most, if not all, lysines and arginines are COOH terminal. A limitation arises from the inability of DAP I to cleave the bond on either side of proline. The chapter focuses on the separation of the dipeptide products that result from the action of DAP I on oligopeptides, their recognition in mixtures when separation of the dipeptides is unnecessary, and the approaches necessary for the reordering of those dipeptide products.

Examination of Carbonic Anhydrase Activities in the Peripheral Nervous Systems of Humans, Rhesus Monkeys, Cats, and Rats

The function of carbonic anhydrase (CA) in the nervous system is not well understood. Biochemical analyses have revealed that CA occurs both as cytosol and membrane-associated forms. On the average, 60% of total activity is in the membrane fraction, and up to 10% of this activity distributes in the myelin fraction.lv2 Our study compares cell-specific localization of CA in different mammals and characterizes intracellular distribution of enzyme activity revealing possible functional relationships. Histochemical CA activity within dorsal and ventral roots and dorsal root ganglia of lumbar spinal nerves was examined in rats, cats, rhesus monkeys, and humans using Hanssons t e~hn ique .~ Axons in the intramuscular nerve bundles of the rat EDL muscle were also studied. Tissues were fixed in 2.5% glutaraldehyde. Normal nerves of the subhuman species were fixed by perfusion. Human material, obtained during therapeutic excision of ganglia injured by compression 2 to 4 weeks earlier, were fixed by i rnmers i~n .~ All of the dorsal root ganglia examined contained subpopulations of sensory neurons with moderate to high CA activity (FIG. 1, A & C-E). Neuronal satellite cells were usually highly reactive. Dorsal roots (FIG. 1, F,H-J) generally possessed a greater percentage of myelinated axons with dark axoplasmic staining than the ventral roots (FIG. 1, K,M-0). Reactive ventral motor axons were in the gamma motor size range. The results for the human material generally agreed, but injured neurons had altered CA activity. Staining was blocked by omission of substrate or by M acetazolamide as illustrated for the rat (FIG. 1, B,G & Lj. Correlative evidence indicates that highly reactive neurons as a group are more tonically (continuously) active, generating more COz and Cat+ fluxes than those with low CA.3 Ultrathin sections of rat tissues were examined electron microscopically. Intensely stained myelinated axons had reaction product distributed throughout the axoplasm, suggestive of cytosol CA trapped by fixation (FIG. 2A). Within moderately reactive axons and sensory cell bodies, clumps of reaction product were localized on the cytoplasmic surfaces of the endoplasmic reticulum and the axolemrna. This localization is indicative of membrane-associated enzyme (FIG. 2, B & Cj. The arrows in 2 B and C point to reaction product on the endoplasmic

Accelerated chromatographic method for determination of hydroxyproline.

Abstract An automated method is described for the determination of urinary hydroxyproline. The method utilizes accelerated column chromatography on spherical sulfonated polystyrene resin, PA-35, and a Beckman/Spinco model 116 amino acid analyzer equipped with sample injection and high sensitivity recording. The effluent is monitored by ninhydrin analysis. The method requires only 30 min per analysis, and allows better than 98% reproducibility and recovery. Moreover, compared to other methods, less handling is required for each sample, and less time is required of the analyst. Eight normal subjects, whose sole source of collagen was derived from a controlled diet containing 10 oz of meat per day, excreted 38.71 mg of hydroxyproline per 24 hr. After correcting for the urinary hydroxyproline excretion arising from dietary collagen, the same urinary excretion of hydroxyproline was obtained as that reported by others for subjects on collagen-free diets.