Joyce W. Margolis

Calf lens neutral proteinase activity using calf lens crystallin substrates

Activity of the calf lens neutral proteinase was measured with the calf lens crystallin fractions and purified α-crystallin A and B chains. For all substrates, rates of hydrolysis were linear for at least 1 hr. With α-crystallin, the rate of hydrolysis began to level off after 1 hr, but continued for at least 48 hr at a slower rate. The β L -crystallin fraction was hydrolyzed at the greatest rate, followed in decreasing order by α and β H -crystallin. ψ-Crystallin was not hydrolyzed. The purified B chains of α-crystallin were hydrolyzed at a greater rate than the A chains or unfractionated α 2 -crystallin. In unfractionated α 2 -crystallin a higher rate of degradation of B chain compared to A chain was shown by SDS gel was not extensive, polypeptide products of hydrolysis were identified

The bovine lens neutral proteinase comprises a family of cysteine-dependent proteolytic activities

Inhibitor studies with peptide substrates demonstrate that bovine lens neutral proteinase comprises three distinct activities. Diisopropylfluorophosphate distinguishes the activity hydrolyzing carbobenzoxy-Gly-Gly-Leu-p-nitroanilide (inhibited) from that hydrolyzing carbobenzoxy-Leu-Leu-Glu-2-naphthylamide (not inhibited). Leupeptin inhibits hydrolysis of the substrate carbobenzoxy-Leu-Leu-Arg-2-naphthylamide, but not hydrolysis of carbobenzoxy-Gly-Gly-Leu-p-nitroanilide or carbobenzoxy-Leu-Leu-Glu-2-naphthylamide, demonstrating the presence of the third activity. Inhibition of the three activities by thiol reagents suggests that each activity may be dependent on an active-site cysteine residue.

Age changes in bovine lens endopeptidase activity

Lens endopeptidase activity and thermal stability have been determined as a function of cell development, cell age, and animal age. Lenses from animals aged 3 months to 15 years (lens weights 1.15-2.80 g) were divided into epithelial (outermost), cortical (peripheral), and nuclear (central) regions. Changes accompanying cell development were determined by measuring specific activity in epithelial (undifferentiated), outer cortical (differentiating), inner cortical (mature) and nuclear (aged) regions of individual lenses. Thermal stability of the enzyme activity obtained from the outer cortical and nuclear regions of the same lenses was also determined. Specific activity and thermal stability were found to decrease as a function of lens cell development. Changes with cell development represent the effects of both differentiation and increasing cell age. To determine the effects of cell age alone, activity was determined in the same population of aged, fully differentiated cells in lenses of different ages. Specific activity decreased as a function of cell age alone. Changes with animal age were determined by comparing cells of the same developmental stage from animals of different ages (e.g., differentiating cells of the cortex in animals 3 months to 15 years old). Specific activity for the cortical region increased with animal age while specific activity in the nuclear region appeared to remain constant or decrease slightly with increasing animal age. Thermal stability of the enzyme activity from the cortex was different in young and adult lenses. The change in stability occurred early in the lifespan and was therefore more closely related to animal development than to aging.

A synthetic endopeptidase substrate hydrolyzed by the bovine lens neutral proteinase preparation

Lens neutral proteinase is thought to exhibit primarily endopeptidase activity. We have identified a synthetic endopeptidase substrate which is hydrolyzed by the bovine lens neutral proteinase preparation. Among 11 fluoro- and chromogenic endopeptidase substrates, only carbobenzoxy-glycylglycyl-L-leucyl-p-nitroanilide is effectively hydrolyzed. The activity hydrolyzing this substrate co-elutes with neutral proteinase activity upon gel filtration and specifically attacks the leucyl-p-nitroaniline bond. Optimal hydrolysis of the synthetic substrate is at neutral pH and high temperature (53 degrees C), analogous to the alpha-crystallin protein substrate obtained from lens. The rate of hydrolysis of the synthetic substrate increased proportionally with temperature between 20 and 60 degrees C, in contrast to alpha-crystallin. The rate of hydrolysis was linear for at least 1 h at 37 degrees C and there was no evidence of enzyme activation at high temperature.

Post-translational arginylation in the bovine lens

This study demonstrates post-translational arginylation of bovine serum albumin and endogenous lens proteins by bovine lens arginyl-tRNA:protein transferase. This reaction has been proposed to be the first step in marking specific proteins for degradation by the non-lysosomal, ATP-dependent, ubiquitin-mediated proteolytic pathway. The transferase was obtained by the method used for isolation of the same enzyme from reticulocytes (Ferber and Ciechanover, 1987, Nature 326, 808-11). Incorporation of [3H]Arg was linear for at least 2 hr at 37 degrees C. The amount of incorporation was directly proportional to the amount of lens enzyme or substrate added. Arginylation was ATP-dependent. A requirement for tRNA was demonstrated by inhibition upon pretreatment of the enzyme preparation with nuclease to hydrolyse endogenous tRNA, and restoration of activity upon replacement of tRNA. [3H]Leu, [3H]Lys and [3H]His were not incorporated, demonstrating specificity of the reaction for arginine. This is the first demonstration of post-translational modification of proteins by arginylation in the lens.

Bovine Lens Multicatalytic Proteinase Complex

The ocular lens grows by laying down new cells on top of old in a differentiation process that results in loss of protein-synthesizing capacity, but preservation of the cells themselves for the lifetime of the organism. The transparency and refractive index of the lens depend on protein integrity and longevity, yet proteolysis is needed for normal growth and development. Therefore, control of proteolysis must be stringent. Here we review the structural features and major proteolytic enzymes of the lens and the properties of the bovine lens multicatalytic proteinase complex, including native and SDS-PAGE patterns, and activation and inhibition by cations, amphiphilic molecules and temperature.

Covalent labelling of bovine lens multicatalytic proteinase complex with (3H)Di-isopropyl fluorophosphate

Enzymatically active lens multicatalytic proteinase complex bound [3H]iPr2P-F after incubation for 3 hours at ambient temperature. Label was associated with the lowest molecular weight band (Mr 22,000) on sodium dodecyl sulfate polyacrylamide gels. This binding was inhibited by preincubation of the enzyme with the cysteine-directed reagent, p-chloromercuribenzoate, which inhibits all three hydrolytic activities of the enzyme. Leupeptin, which inhibits the arginyl-hydrolyzing component, but not the iPr2P-F-inhibitable leucyl-hydrolyzing component of the enzyme, does not inhibit [3H]iPr2P-F binding. These data suggest that the leucy-hydrolyzing component of the lens multicatalytic proteinase complex is localized to the 22,000 Mr subunit and is a member of the thiol-dependent subclass of serine proteinases.

Conserved N-terminal sequences in homologous subunits of the multicatalytic proteinase complex (proteasome)

The bovine lens multicatalytic proteinase complex (MPC) (MW 700 kDa) comprises at least twelve subunits in the molecular mass range 22-35 kDa. Three of the subunits, L1 (27 kDa), L2 (24 kDa) and L3 (29 kDa), were purified by reverse phase HPLC. Their amino acid composition and N-terminal sequences indicate that they are not identical. L1 and L2 subunits show very high (greater than 90%) sequence homology with specific subunits of rat liver and human reticulocyte MPC and these are considered to be homologous components of the MPC which are highly conserved in evolution.