Cytochromes of Microsomal Particles: Cytochrome b5 of Microsomes from Animal Tissues

121

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ∼ 12 per cent of the protein N and ∼ 35 per cent of the phospholipide of the whole mitochondria and accounted for ∼ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.

Section: Resultsmentioning

confidence: 99%

Section: Table VII Effect Of Antimycin a On Succinoxidase Activity Ofmentioning

confidence: 99%

Cytochemical Studies of Mitochondria

Siekevitz

Watson

1956

121

“…On the basis of the absorption peaks at 556 to 557 m/~, the concentration of the hemoprotein in pancreatic microsomes was estimated to be only 10 per cent of its concentration in hepatic microsomes. The hemochromgen concerned was found to occur together with the enzyme DPNH-cytochrome c reductase in microsomes separated from liver (32, 1), intestinal mucosa, and mammary gland (33). Their presence in the same cell fraction was interpreted as indicative of a functional association of the cytochrome and the enzyme in electron transfer in microsomes (32,33).…”

Section: Pancreatic Microsomesmentioning

confidence: 97%

“…The hemochromgen concerned was found to occur together with the enzyme DPNH-cytochrome c reductase in microsomes separated from liver (32, 1), intestinal mucosa, and mammary gland (33). Their presence in the same cell fraction was interpreted as indicative of a functional association of the cytochrome and the enzyme in electron transfer in microsomes (32,33). Since pancreatic microsomes do not show DPNH-cytochrome c reductase activity 4, although they contain a small though measurable amount of hemoprotein, the findings here reported do not support the hypothesis of functional association mentioned above.…”

Section: Pancreatic Microsomesmentioning

confidence: 99%

Pancreatic Microsomes

Palade

Siekevitz

1956

348

108

The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its orderly disposition in the basal region of the cell. In addition to the small, (∼15 mµ), dense particles attached to the limiting membrane of the endoplasmic reticulum, numerous particles of similar appearance are found freely scattered in the cytoplasmic matrix. The various cell structures of pancreatic exocrine cells can be satisfactorily identified in pancreatic homogenates. The microsome fraction consists primarily of spherical vesicles (80 to 300 mµ), limited by a thin membrane (7 mµ) which bears small (∼15 mµ) dense particles attached on its outer surface. The content of the microsomal vesicles is usually of high density. Pancreatic microsomes derive by extensive fragmentation mainly from the rough surfaced parts of the endoplasmic reticula of exocrine cells. A few damaged mitochondria and certain dense granules (∼150 mµ) originating probably from islet cells, contaminate the microsome fraction. Pancreatic microsomes contain RNA, protein, and a relatively small amount of phospholipide and hemochromogen. They do not have DPNH-cytochrome c reductase activity. In six experiments the RNA/protein N ratios were found grouped around two different means, namely 0.6 and 1.3. Pancreatic microsomes are more labile than liver microsomes but react in a similar way to RN-ase-(loss of the particulate component and RNA), and deoxycholate treatment (loss of the membranous component and of phospholipide, hemochromogen, and most of the protein). Postmicrosomal fractions consisting primarly of small (∼15 mµ), dense particles of ribonucleoprotein (RNA/protein N ratio = 1 to 2) were obtained by further centrifugation of the microsomal supernatant. The small nucleoprotein particles of these fractions are frequently found associated in chains or clusters.

“…This hemochromogen had spectral properties similar to those of the cytochrome b5 reported from microsomal membranes of rat liver and bovine mammary gland. While THE JOURNAL OF CELL BIOLOGY 9 VOLUME 73, 1977 9 pages 223-241 Bailie and Morton assumed that the particles associated with the milk fat globules (MFG) were derived directly from microsomal, i.e., endoplasmic reticulum (ER), membranes of mammary epithelial cells (3,4), further morphological and biochemical studies have shown that the milk fat globule membrane (MFGM) is budded from the apical plasma membrane during milk lipid globule secretion (e.g. 5; for additional references, see 25).…”

mentioning

confidence: 99%

Redox constituents in milk fat globule membranes and rough endoplasmic reticulum from lactating mammary gland.

Jarasch

Bruder

Keenan

et al. 1977

Milk fat globule membranes (MFGM) and rough endoplasmic reticulum (RER) membranes were isolated from milk and lactating mammary gland from the cow and were characterized by biochemical and electron microscope methods in terms of gross composition (proteins, phospholipids, neutral lipids, cholesterol, RNA, and DNA) and purity. Both fractions contained significant amounts of a b-type cytochrome with several properties similar to those of cytochrome b5 from liver, as well as a rotenone-insensitive NADH-and NADPH-cytochrome c reductase. The b-type cytochrome content in the apical plasma membrane-derived MFGM was of the same order of magnitude as it was in RER membranes. It was characterized by a high resistance to extraction by low-and high-salt concentrations and nonionic detergents. MFGM contained much more flavin and much higher activities of xanthine oxidase than the RER membranes. The same redox components were found in MFGM and mammary RER from women, rats, mice, and goats, but in absolute contents great differences between the species were noted.The cytochromes described here differed from liver cytochrome b5 in some spectral pr6perties. The a-band of the reduced hepatic cytochrome b5 is asymmetric with a maximum at 555 nm that is split into two distinct peaks at low temperatures. The a-band of the b-type cytochromes from MFGM and mammary RER appears as one symmetrical peak at about 560 nm that is not split at low temperatures. When treated with cyanide, MFGM and mammary microsomes showed difference spectra of a reduced b-type cytochrome. Under the same conditions, liver microsomes gave a completely different spectrum.These findings demonstrate the presence of a b-type cytochrome and associated redox enzymes in MFGM, i.e., a derivative of the apical cell surface membrane that is regularly used for envelopment of the milk fat globule during secretion.In 1955 Bailie and Morton (3) reported the discovery of a hemochromogen in a particulate fraction from bovine milk. This hemochromogen had spectral properties similar to those of the cytochrome b5 reported from microsomal membranes of rat liver and bovine mammary gland. While