The localization of lysosomal enzymes in chromaffin tissue

Smith, A.D.; Winkler, Hans

doi:10.1113/jphysiol.1966.sp007859

Cited by 82 publications

(22 citation statements)

References 27 publications

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“…Though Hook et al used an adrenal chromaffin granule preparation for their enzymatic studies, this preparation contains a substantial amount of lysosomes (6,12,16,17).…”

Section: Discussionmentioning

confidence: 99%

Enkephalin convertase: purification and characterization of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal chromaffin granules.

Fricker

Snyder

1982

Proc. Natl. Acad. Sci. U.S.A.

254

141

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A specific carboxypeptidase that converts enkephalin precursors into enkephalin in adrenal chromaffin granules has been purified and characterized. In the adrenal this enzyme, designated enkephalin convertase, is uniquely localized to the chromaffin granules, which contain enkephalin and precursor peptides. Enkephalin convertase is markedly stimulated by CoC12 and inhibited by EDTA or 1,10-phenanthroline, unlike the lysosomal carboxypeptidase. The purified enzyme has a high affinity for the hexapeptides Brain enkephalin convertase shows 10-fold regional variations, unlike other carboxypeptidases, which are uniformly distributed. Enkephalin convertase appears to be associated selectively and physiologically with biosynthesis of the enkephalins.Most biologically active peptides are synthesized from large peptide precursors by sequential actions of a trypsin-like enzyme followed by a carboxypeptidase B-like activity that removes basic amino acids from the carboxyl terminus ofthe trypsin fragments (1). Large enkephalin precursor peptides have been identified in which the enkephalin sequence is surrounded by dibasic amino acid pairs (2, 3). Moreover, hexapeptides comprising the enkephalin sequence with an arginine or lysine at the carboxyl terminus have been isolated from brain and adrenal (4). It is unclear whether the biosynthesis of biologically active peptides involves carboxypeptidases uniquely concerned with the formation of a single peptide or whether general carboxypeptidases serve this function.In the adrenal, enkephalins are localized to chromaffin granules, from which they are released together with catecholamines after cholinergic stimulation (5). If enkephalin is synthesized by a unique carboxypeptidase, such an enzyme might be selectively localized to chromaffin granules and might exhibit selective affinity for enkephalin-containing hexapeptides.Conventional assays for carboxypeptidase are often nonspecific, insensitive, or, if they utilize the biologically active peptide precursor, time consuming. We have designed a novel carboxypeptidase assay using a fluorescent enkephalin analog. The assay is simple, sensitive, and specific. We now describe a unique cobalt-stimulated carboxypeptidase that, in the adrenal, is concentrated in chromaffin granules and that has selective affinity for enkephalin-containing peptides. This enzyme, designated enkephalin convertase, appears to be selectively associated with physiological synthesis of the enkephalins.MATERIALS AND METHODS Synthesis of Dansylphenylalanylleucine. L-Phenylalanyl-Lleucine (100 mg) was dissolved in 20 ml of pyridine. Dansyl chloride (200 mg) was added and the mixture was stirred for 5 hr, acidified with dilute HC1 (pH 1 to 2), and extracted with 1 liter ofCHCl3. The chloroform was dried (MgSO4) and removed at reduced pressure, yielding a bright yellow oily solid. The product was purified by TLC on 1-mm silica GF (Analtech) using chloroform/methanol (4:1). Dansyl-Phe-Leu (Rr 0.5) was eluted with hot methanol, yielding 95 mg of product.Synth...

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“…Though Hook et al used an adrenal chromaffin granule preparation for their enzymatic studies, this preparation contains a substantial amount of lysosomes (6,12,16,17).…”

Section: Discussionmentioning

confidence: 99%

Enkephalin convertase: purification and characterization of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal chromaffin granules.

Fricker

Snyder

1982

Proc. Natl. Acad. Sci. U.S.A.

254

141

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“…The contrary evidence requires us, I believe, to conclude that after releasing their contents to the cell exterior, either by frank rupture of the adherent plasmalemma and granule membranes, or by some greatly increased permeability at the site of adhesion, emptied granules may remain, for a time at least, within the cell (Fig. 1) ultimately broken down, perhaps by the lysosomal enzymes in which the chromaffin cell is rich (Smith & Winkler, 1966). Evidence of destruction of secretory granules by lysosomal activity has been obtained from electronmicrographs of other secretory cells (Smith & Farquhar, 1966).…”

Section: The Adrenal Chromaffin Cellmentioning

confidence: 99%

“…In both instances granule membranes are retained after evacuation of their soluble contents. ultimately broken down, perhaps by the lysosomal enzymes in which the chromaffin cell is rich (Smith & Winkler, 1966). Evidence of destruction of secretory granules by lysosomal activity has been obtained from electronmicrographs of other secretory cells (Smith & Farquhar, 1966).…”

Section: Depolarizationmentioning

confidence: 99%

Stimulus‐secretion coupling: the concept and clues from chromaffin and other cells

Douglas

1968

British J Pharmacology

1,360

414

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“…Protein content was determined according to the procedure of Lowry et al (1951). Density gradient centrifugation was performed a s described by Smith and Winkler (1966…”

Section: Other Analytical Proceduresmentioning

confidence: 99%

Biogenesis of Chromaffin Granules: Incorporation of Sulfate into Chromogranin B and into a Proteoglycan

Falkensammer

Fischer‐Colbrie

Winkler

1985

Journal of Neurochemistry

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The incorporation of [35S]sulfate into the soluble proteins of chromaffin granules was studied. Isolated bovine chromaffin cells were pulse-labeled with [35S]sulfate. The radioactively labeled products were characterized by one- and two-dimensional electrophoresis. Three proteins of chromaffin granules were preferentially labeled. One was identified by immunoprecipitation as chromogranin B (Mr 100,000). This result explains why during cellular synthesis the chromogranin B precursor is converted into a significantly more acidic protein. During chase periods, the newly synthesized chromogranin B was progressively degraded by endogenous proteases. A second labeled protein, much less labeled than chromogranin B, was identified as chromogranin A. The largest portion of the radioactive label was found in a heterogeneous component (Mr 86,000-100,000; pI 4.3-5.0). Digestion experiments with chondroitinase ABC demonstrated that this labeled component and a comigrating Coomassie Blue-stained spot were selectively degraded by this enzyme. This establishes that this component is a proteoglycan.

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The localization of lysosomal enzymes in chromaffin tissue

Cited by 82 publications

References 27 publications

Enkephalin convertase: purification and characterization of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal chromaffin granules.

Enkephalin convertase: purification and characterization of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal chromaffin granules.

Stimulus‐secretion coupling: the concept and clues from chromaffin and other cells

Biogenesis of Chromaffin Granules: Incorporation of Sulfate into Chromogranin B and into a Proteoglycan

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