Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Gratzl, Manfred

doi:10.1016/0003-2697(84)90529-3

Cited by 15 publications

(18 citation statements)

References 29 publications

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“…Differential centrifugation and subsequent application of isotonic (Percoll) and hypertonic (sucrose) gradients yield highly purified chromaffin vesicles [10]. While mitochondria and microsomes can easily be removed using Percoll gradients [9], further purification of chromaffin vesicles and separation from the remaining lysosomal contamination require an additional sucrose density gradient.…”

Section: Resultsmentioning

confidence: 99%

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

Schilling

Gratzl

1988

FEBS Letters

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Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.p38/synaptophysin; Secretory vesicle; Immunoblotting; Quantification; (Adrenal medulla)

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Section: Resultsmentioning

confidence: 99%

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

Schilling

Gratzl

1988

FEBS Letters

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“…BoNTIA Chromaffin vesicles were isolated as described [26]. Briefly, bovine adrenal glands were perfused with 5 mM HEPES (pH 7.0), 150 mM NaC1, 5 mM EDTA.…”

Section: Purification Of Chromaffin Vesicle Membranes and Incubation mentioning

confidence: 99%

“…Centrifugation was done for 1 h at 40000 rpm in a 50.2 Ti rotor (Beckman). Absorbance at 280 nm after precipitation of the protein with 10% (w/v) TCA was measured to conveniently locate chromaffin vesicles in the gradient while arylsulfatase served to identify fractions containing lysosomes and other subcellular membranes [26]. The fractions enriched in chromaffin vesicles (fractions [11][12][13][14] were recovered from the gradient, diluted 1:4 with 20 mM MOPS (pH 7.0), 5 mM EDTA and centrifuged for 1 h at 100 000 x g. The purified chromaffin vesicles were osmotically lysed by addition of a 10-fold excess of 20 mM MOPS (pH 7.0) with 5 mM EDTA.…”

Section: Purification Of Chromaffin Vesicle Membranes and Incubation mentioning

confidence: 99%

Adrenal chromaffin cells contain functionally different SNAP‐25 monomers and SNAP‐25/syntaxin heterodimers

Höhne-Zell¹,

Gratzl²

1996

FEBS Letters

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“…2 5 , N O . 1 5 , 1 9 0 6 4403 described for a similar gradient centrifuged in a swing out rotor (Gratzl, 1984). After centrifugation, the secretory vesicles were concentrated around the 1.8/2.0 M sucrose interface.…”

Section: A " B I N D I N G T O C H R O M a F F I N V E S I C L E M mentioning

confidence: 99%

Calcium binding to chromaffin vesicle matrix proteins: effect of pH, magnesium, and ionic strength

Reiffen¹,

Gratzl²

1986

Biochemistry

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Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-77651. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 p M (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 p M (n = 0.8 pmol/pmoi) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 pM). At p H 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 p M and after addition of 35 m M K+ a dissociation constant of 170 pM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 p M and after addition of 15 mM Na' by a Kd of 340 pM. The similar affinity of Ca2+ for protein and ATP, especially at pH 6, in media of increased ionic strength and after addition of Mg2+, points to the possibility that the intravesicular medium determines whether Ca2+ is preferentially bound to ATP or the chromaffin vesicle matrix proteins. Purified chromogranin A, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stains with a carbocyanine dye ("Stains-all") and, following blotting onto nitrocellulose, binds to 45Ca2+. A spectrophotometric analysis of dye binding to chromaffin vesicle matrix proteins revealed a strong absorption band at 615 nm for the dye-protein complex. Since the observed spectral changes were unaffected by the presence of Ca2+ (100 p M free), the sites interacting with the dye and Ca2+ must be regarded as different.

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Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Cited by 15 publications

References 29 publications

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

Adrenal chromaffin cells contain functionally different SNAP‐25 monomers and SNAP‐25/syntaxin heterodimers

Calcium binding to chromaffin vesicle matrix proteins: effect of pH, magnesium, and ionic strength

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