Alan G. Ferguson

Primary structure similarities in canine cardiac cathepsin D polypeptide chains

Abstract Canine cardiac cathepsin D was purified approximately 1000-fold to homogeneity by sequential acid precipitation, affinity chromatography and gel filtration. The purified enzyme was homogeneous on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and consisted of two polypeptide chains of mol. wt 29 000 ± 2500; n = 6 (designated the A chain) and 13 000 ± 1000, n = 6 (designated the B chain). Separation of the A and B polypeptide chains using gel filtration in the presence of guanidine hydrochloride (6 mol/l) followed by amino acid analysis revealed the presence of a similar amino acid composition in each polypeptide. N-terminal analysis showed that glycine was the sole N-terminal amino acid residue in each chain, while tryptic digestion and subsequent peptide mapping gave 17 major peptides and 6 major peptides for the A and B chains respectively. Three peptide fragments were found to be common to both polypeptide chains. Extracts of canine cardiac tissue did not contain free A or B chains of cathepsin D, however a high molecular weight form of the enzyme (mol. wt 130 000) was observed with a specific activity (U/μg immunoreactive cathepsin D) similar to that of the active undissociated (mol. wt 42 000) enzyme. Immunodiffusion studies using antibodies raised against the whole enzyme showed a reaction of complete identity between the A and B polypeptide chains and undissociated enzyme. Use of the A and B chains in a radioimmunoassay developed for measurement of the undissociated cathepsin D revealed that the A chain bound antibody raised against the undissociated enzyme equally as well as the undissociated enzyme, while the B chain was approximately 100 times less effective at binding the antibody. The results suggest that there may be some degree of similarity in primary structure between the A and B polypeptide chains of canine cardiac cathepsin D. This similarity in primary structure may represent the suggested areas of sequence homology surrounding the active site aspartate residues known to be characteristic of this particular class of acid proteases.

Regulation of Cathepsin D Metabolism in Rabbit Heart: EVIDENCE FOR A ROLE FOR PRECURSOR PROCESSING IN THE CONTROL OF ENZYME ACTIVITY

Production of active lysosomal enzymes may involve limited proteolysis of inactive high molecular weight precursors. Precursor processing potentially regulates lysosomal enzyme activity. To test whether rabbit cardiac cathepsin D is first synthesized as a precursor and whether prolonged fasting (a condition affecting both cathepsin D and total cardiac protein turnover) influences precursor processing, rates of cathepsin D synthesis and processing were compared in left ventricular slices of control and 3-d-fasted rabbits incubated in vitro with [(35)S]methionine. (35)S-labeled cathepsin D was isolated by butanol-Triton X-100 extraction, immunoprecipitation, and dodecyl sulfate-polyacrylamide gel electrophoresis. Total cardiac protein synthesis was measured by tracer incorporation and normalized for differences in precursor pool size by direct measurement of [(35)S]aminoacyl-tRNA-specific radioactivity. Relative cathepsin D synthetic rates were obtained by comparing (35)S incorporation into cathepsin D with (35)S incorporation into all cardiac proteins. Enzyme processing was assessed in pulse-chase experiments and assayed by autoradiography. The results indicate that (a) rabbit cardiac cathepsin D is synthesized as a precursor (53,000 mol wt) that is processed to a 48,000-mol wt form, (b) rates of both cathepsin D and total cardiac protein synthesis are similar in control and fasted rabbits, suggesting that decreased enzyme degradation rather than increased synthesis is responsible for the elevated levels of cardiac cathepsin D in starvation, and (c) cathepsin D processing in hearts of fasted animals is incomplete, with accumulation of the precursor during pulse-chase experiments of 6 h duration. Based upon these results, a three-stage model for the regulation of cathepsin D activity in rabbit heart is proposed.

A method for the determination of amino acid incorporation into protein and the specific activity of tissue amino acid in small cardiac muscle samples

Abstract A method for the determination of amino acid incorporation into protein and the specific activity of the tissue amino acid pool in cardiac muscle samples weighing as little as 1 mg is described. This technique is sufficiently sensitive to measure the specific activity of an amino acid in the picomole range and is based on the reaction of [3H]dansyl chloride with 14C-tracer amino acid. With this procedure, apparent alterations in protein synthesis noted in muscles incubated in media containing tracer amino acid of differing specific activities were normalized when the relative specific activity of tissue amino acid was considered.

Quantitative analysis of myofibrillar protein subunits: Demonstration of large molar excesses of myosin light chains in rabbit ventricular myocardium

We describe a method for the complete solubilization and quantitative analysis of individual myofibrillar proteins in whole tissue homogenates of ventricular myocardium using gradient dodecyl sulfate polyacrylamide gel electrophoresis and staining with 125I-labeled Coomassie brilliant blue. The procedure allows for the simultaneous quantification of myosin heavy chain, myosin light chain, phosphorylatable myosin light chain and actin from as little as 50 mg of tissue. Within-assay and between-assay variations range from 8.0% to 12.6% for each protein subunit. The method was applied to the determination of the subunit stoichiometry of purified myosin, and to the measurement of myosin and actin concentrations in the neonatal and adult rabbit heart. Furthermore, we provide quantitative biochemical evidence for the existence of large molar excesses of myosin light chains in tissue homogenates of both neonatal and adult rabbit ventricular myocardium.

Determination of 35S-aminoacyl-transfer ribonucleic acid specific radioactivity in small tissue samples

Rate determination of protein synthesis utilizing tracer amino acid incorporation requires accurate assessment of the specific radioactivity of the labeled precursor aminoacyl-tRNA pool. Previously published methods presumably useful for the measurement of any aminoacyl-tRNA were unsuccessful when applied to (/sup 35/S)methionine, due to the unique chemical properties of this amino acid. Herein we describe modifications of these methods necessary for the measurement of /sup 35/S-aminoacyl-tRNA specific radioactivity from small tissue samples incubated in the presence of (/sup 35/S)methionine. The use of (/sup 35/S)methionine of high specific radioactivity enables analysis of the methionyl-tRNA from less than 100 mg of tissue. Conditions for optimal recovery of /sup 35/S-labeled dansyl-amino acid derivatives are presented and possible applications of this method are discussed.

Cathepsin D catalyzed degradation of cardiac mitochondria. A study of enzyme inactivation and ultrastructure

Abstract The ability of cathepsin D to catalyze degradation of intact mitochondria in vitro was used as a probe to investigate the possible role of this protease in the initiation of ischemia-induced mitochondrial injury. Cathepsin D mediated protein degradation was monitored using: 1. (1) Degradation of gross mitochondrial protein as assessed by changes in trichloroacetic acid soluble peptide formation. 2. (2) Inactivation of specific mitochondrial enzymes vis monoamine oxidase (MAO), adenylate kinase (AK), creatine kinase (CK), Ca2+-Mg2+ adenosine triphosphatase (ATPase) and malate dehydrogenase (MDH) in intact and solubilized mitochondrial preparations. 3. (3) Electron microscopy. The pH-activity profile for cathepsin D mediated mitochondrial protein degradation revealed an optimum at pH 3.2 to 3.4 with 12 and 5% optimal activity remaining at pH values of 5.5 and 6.0 respectively. Incubation of intact mitochondria with cathepsin D (pH 5.7 at 37°C) revealed that MAO was inactivated to a small extent (4.5% loss in activity following a 60-min incubation) while other enzymes were unaffected. In similar experiments with solubilized mitochondria however, MAO, AK and ATPase were moderately inactivated (10, 20 and 15% at 60 min respectively) while CK and MDH were unaffected. Pepstatin A was found to inhibit the observed inactivation indicating that cathepsin D and not a contaminating protease was responsible for the observed effects. Results from electron microscopy, paralleled those of the incubation studies indicating that there was little effect of cathepsin D on the intact organelle. The results suggest that while mitochondria contain proteins which are susceptible to cathepsin D catalyzed hydrolysis, the accessibility of cathepsin D to these proteins is limited in intact mitochondria. If cathepsin D is responsible for either the physiologic turnover of mitochondria or the mitochondrial destruction characteristic of ischemic injury in myocardial tissue, a concomitant disruption of the mitochondrial membrane by other groups of lysosomal or non-lysosomal enzymes must be postulated.