Neville Marks

On the degradation of enkephalins and endorphins by rat and mouse brain extracts.

Abstract Enkephalins are degraded rapidly by homogenates of rat brain and by ultrafiltrates of mouse brain supernatant with release of N-terminal tyrosine followed later by Met (or Leu), Phe and Gly as measured by amino acid analysis and by microdansylation techniques. Fragments of β-lipotropin (sequences 61–76, α-endorphin and 61–91, β-endorphin) were degraded more slowly with evidence for multiple sites of cleavage by aminopeptidases (N-terminal release of tyrosine), carboxypeptidases, and various endopeptidases active at neutral pH including some with tryptic-like specificity. The slow appearance of Gly and the accumulation of Gly-Gly in digests of enkephalin and fragment 61–76 points to the presence in brain extracts of a glycyl-glycine dipeptidase which is rate-limiting. Rates of degradation observed appear to accord with their duration of action in vivo based on their behavioral and analgesic properties.

Activation of caspase‐3 and apoptosis in cerebellar granule cells

Caspase‐3 activity increased dramatically in cytosolic extracts of rat cerebellar granule cells exposed to apoptotic conditions (basal medium Eagle (BME) containing 5 mM K+ without serum) when assayed with Ac‐DEVD‐amc,but not with Ac‐YVAD‐afc, a preferred substrate for caspase‐1. This provided a basis to examine relationships between enzyme activity and cell viability for purposes of selecting an optimal time for comparing neuroprotective agents or strategies. Exposure of neurons to an apoptotic medium containing 5 mM K+ in absence of serum led to a rapid 5‐ to 10‐fold increase in caspase‐3 within 2‐4 hr but without significant cell loss, or morphological alterations. Exposure to apoptotic medium followed by replacement with maintenance medium containing 25 mM K+and serum led to a rapid fall in caspase‐3 and prevention of cell death. This strategy was not effective after 13 hr exposure despite a large fall in enzyme activity. These temporal changes infer systems for rapid enzyme turnover and/or activation of cytoplasmic components linked to later DNA degradation. The effects of cycloheximide point to requirements for protein synthesis, and those of Glu exclude a caspase‐3 dependent pathway for necrotic cell damage. Brief treatment with 10 μM LIGA20, an anti‐necrotic agent, also attenuated cell loss and caspase‐3 activity, indicating a broad spectrum of neuroprotection. Rapid and long‐lasting effects, together with its biophysical properties, suggest that this semisynthetic ganglioside acted upstream at or near a membrane site. As such, gangliosides provide useful agents to further probe pathways relevant to neuronal death in culture. J. Neurosci. Res. 52:334–341, 1998.

Co-identity of brain angiotensin converting enzyme with a membrane bound dipeptidyl carboxypeptidase inactivating Met-enkephalin

Abstract The distribution in rat brain of angiotensin converting enzyme (EC3.4.15.1) using hippuryl-His-Leu as substrate was identical to a dipeptidyl carboxypeptidase present in membranes assayed with Met-enkephalin as substrate. Highest activity occurred in pituitary, followed by cerebellum, corpus striatum, midbrain, pons-medulla, hypothalamus, cerebral cortex and spinal cord. The ratio of products His-Leu/Tyr-Gly-Gly was identical for all regions but differed from His-Leu/Tyr. Angiotensin converting enzyme purified by immunoaffinity chromatography gave a K m for hippuryl-His-Leu of 0.5mM and for Met-enkephalin of 0.1 mM. In the presence of the specific inhibitor of angiotensin converting enzyme, SQ 14,225, the Ki value was 10 −7 M. Present data point to the co-identity of brain angiotensin converting enzyme with the dipeptidyl carboxypeptidase inactivating enkephalin.

Changes in proteolytic enzymes and proteins during maturation of the brain

(1) Changes during development in the levels of proteinases and peptidases were measured in brain homogenates. At all ages di- and tripeptidase levels were 7-15-fold higher than proteinase activity. (2) Cathepsin A and D and neutral proteinase activity first decreased (during the 5 days before birth) and then increased (primarily during the first 10 days after birth) in development. The total enzyme content per unit weight of brain did not change greatly after 10 days, although specific activity fell owing to an increase in protein in older animals. (3) The developmental pattern of activities or peptidases measured with Leu-Gly and Leu-Gly-Gly and of arylamidases measured with Arg- and Arg-Arg-beta-naphthylamides was similar to that of proteinases. Total and specific activities increased rapidly after birth; then total activity did not change and specific activity decreased. (4) The proteinase content of tissue fractions (nuclear and lysosomal-mitochondrial) similarly reached a maximal peak in the rapid growth phase of the brain. (5) The decrease of hydrolytic activity after 10 days of age seems to parallel a decrease in the rates of protein breakdown in vivo, showing parallel behavior with decreasing protein turnover. In contrast, during the first 10 days of life protein turnover and calculated rate of protein breakdown in vivo decrease while the level of hydrolytic enzymes increases.

Inactivation of somatostatin (GH-RIH) and its analogs by crude and partially purified rat brain extracts

Somatostatin, a cyclic tetradecapeptide, inhibits the release of growth hormone from the pituitary and glucagon and insulin from the pancreas of fasted mammals [1,2]. Its value as a therapeutic agent is severely limited, however, by its extremely short biological half life of approx. 4 min [3, 4]. As in the case of other peptidyl hormones there is considerable interest in the preparation of longer acting derivatives which could have application to the treatment of diabetes and developmental disorders. For the preparation of synthetic analogs a knowledge of the mechanisms of inactivation in brain and tissues could facilitate a more rational approach as illustrated recently for other hypothalamic releasing factors. In the case of LH-RH 0uteinising hormone releasing hormone) it was shown that a simple substitution of an internal residue by D-Ala blocked the action of an endopeptidase with a resultant increase in the biological activity in vivo [5]. This particular study introduced a new concept, namely, that structure as related to biodegradation is equally valid to the traditional approach involving considerations of structure-receptor relationships. Analogs with improved stability in presence of serum or tissue enzymes are more likely to reach the target sites involved in hormonal action and this may account for their longer action. Somatostatin unlike LH-RH is characterised by a free N-terminal grouping and by the presence of a disulfide bridge spanning residues 3 1 4 [ 1 ]. As such, it is a potential substrate for the action of aminopeptidases present in crude extracts but the role of the ring structure in breakdown is unexplored. It has been established in the case of other cyclic hormones, notably oxytocin and vasopressin, that the ring acts as a steric hindrance to aminopeptidases and some endopeptidases; these hormones are inactivated in brain, however, by novel C-terminal cleaving enzymes acting on the tripeptidyl alicyclic tails [6,7]. With the objective of delineating the enzymes involved in breakdown of somatostatin, this hormone and its cyclic and linear analogs were incubated with crude and partially purified enzyme from rat brain since these extracts are known to contain a spectrum of enzymes cleaving a variety of peptidyl hormones [8].

The breakdown of myelin-bound proteins by intra- and extracellular proteases.

Changes in protein components of purified myelin were measured following incubation in vitro with purified intra- and extracellular enzymes. Incubation with calf brain cathepsin D did not result in a significant relese of acid-soluble peptides as measured by ninhydrin analysis but was accompanied by a large loss of myelin proteins as determined on SDS-acrylamide gels. After 5 hr at 37°C there was a loss of about 25% for fast and slow basic proteins and the Agrawal proteolipid, but only a 5–10% loss for the Folch-Lees and Wolfgram components. Rat brain cathepsin D prepared by affinity chromatography gave a 30–60% breakdown of basic proteins and proteolipids. In general, breakdown using lyophilized myelin was increased over two-fold as compared to experiments with fresh myelin. Breakdown induced by cathepsin D was completely inhibited by the pentapeptide pepstatin. Incubation of myelin at physiological pH resulted in an endogenous breakdown of about 12% for basic proteins in freshly prepared, and 50% for lyophilized material. Addition of a soluble neutral proteinase that splits hemoglobin did not induce additional breakdown except for a small change in the Folch-Lees component. The extracellular enzymes pepsin and TPCK-treated trypsin resulted in a larger breakdown of all components as compared to brain enzymes. Present results demonstrate that all protein components of myelin are accessible to hydrolases and vulnerable to breakdown to varying extents by brain enzymes. These facts are consistent with the known rates for myelin protein turnover and may have a bearing on changes associated with demyelinating diseases

Met-enkephalin-Arg6-Phe7 metabolism: conversion to Met-enkephalin by brain and kidney dipeptidyl carboxypeptidases.

Abstract Enzymes degrading Met-enkephalin-Arg6-Phe7, an endogenous brain peptide with enhanced opiate activity in vivo, were isolated from membrane preparations of rabbit kidney and brain, and their specificity compared. A preparation from kidney or brain containing the angiotensin converting enzyme (EC 3.4.15.1) released with time Arg-Phe, Met-enkephalin, Phe-Met and Tyr-Gly-Gly. Kinetic analysis revealed a product precursor relationship with conversion of hepta- to pentapeptide (Met-enkephalin) followed by release of Tyr-Gly-Gly and Phe-Met indicating sequential cleavage at the Met5-Arg6 and Gly3-Phe4 bonds. A second preparation devoid of angiotensin converting enzyme activity released the same products and in addition a tetrapeptide Phe-Met-Arg-Phe. Release of products with time indicated cleavage at Gly3-Phe4 by an endopeptidase and at the Met5-Arg6 and Gly3-Phe4 bonds by a dipeptidyl carboxypeptidase. The dipeptidyl carboxypeptidases thus provide a mechanism for the formation of Met-enkephalin from a potential precursor.

Peptide hydrolases in spinal cord and brain of the rabbit.

Summary Spinal cord and brain fractions from rabbits were compared in terms of their content of free amino acids, the amino acid composition of their proteins, and the level of selected proteases and peptidases. The amino acid composition of poteins in the cervical, thoracic and lumbar areas of the cord were similar, and no major differences were observed between the amino acid composition of cord and brain proteins. Similarity in basic, lipophylic, and acidic amino acids would indicate similarity in gross protein composition. The free amino acid pool lower in cord areas than in brain (the total content in the cord was about two-thirds of that of brain), especially glutamate and GABA. Glycine was higher in the cord. Spinal cord contained the same spectrum of proteases and peptidases as the brain, but the total activities were about half in cord; only neutrral protease was similar. Of the enzymes, highest activity was observed with the amino-peptidase substrate leucyl-glycyl-glycine followed by the dipeptidase substrate lysyl-alanine. Acid proteinase was higher than neutral proteinase; activity with monoacylated arylamides was considerably higher than with dipeptidyl derivatives. Very low activity was observed with the cathepsin C substrate seryl-tyrosyl-β-naphthylamide. Subcellular fractionation showed a similar distribution pattern in cord to that previously observed in brain: more proteinase occured in the particulate fractions; peptide hydrolase were to a larger extent in the soluble fraction. Myelin contained high levels of neutral proteinase; the other enzymes tested at lower level were all present. The presence of proteinases and peptidases in the cord is compatible with the active protein turnover of this area.

DISTRIBUTION OF AMINO ACIDS AND OF EXO- AND ENDOPEPTIDASES ALONG VERTEBRATE AND INVERTEBRATE NERVES

Abstract— The proximo‐distal gradients for representative peptidases, peptidylpeptide hydrolases, and amino acids were measured in segments of peripheral nerve from invertebrates and vertebrates and in the lobster brain and ventral cord.

Pentapeptide (Pepstatin) Inhibition of Brain Acid Proteinase

The pentapeptide pepstatin obtained from culture filtrates of actinomycetes completely inhibited brain acid proteinase (cathepsin D) at exceedingly low concentrations. Among the brain enzymes tested, the effect is specific for acid proteinase because addition of 1000-fold higher concentrations was without effect on neutral proteinase, aminopeptidase, and arylamidases. Pepstatin also inhibits pepsin as tested with hemoglobin or with N-acetylphenylalanyl-L-diiodotyrosine as substrate. Pepstatin must be regarded as the most powerful agent yet described that inhibits intracellular acidic proteolytic enzyme in brain.