Decrease In Staining Intensity Research Articles

Immunohistochemical localization of copper-zing and manganese superoxide dismutases in diisopropanolnitrosamine-induced rat thyroid lesions.

The immunohistochemical localization of both copper-zinc (Cu, Zn) and manganese (Mn) superoxide dismutases (SODs) in diisopropanolnitrosamine (DIPN) induced rat thyroid lesions was examined.In normal thyroid tissue, Cu, Zn-SOD was homogeneously demonstrated in both the nucleus and cytoplasm of the follicular epithelium, whereas Mn-SOD was present in the cytoplasm in a granular pattern.The lesions of diffuse hyperplasia, which are regarded as early changes induced by DIPN administration, showed almost the same pattern, but in places a partial decrease in staining intensity was observed compared to thyroid tissue of the control animals and DIPN-treated animals with no remarkable lesions. In both benign (types 1 and 2) and malignant (type 3) lesions, the overall staining intensity of Cu, Zn-SOD and Mn-SOD was decreased. However, in some malignant lesions (type 3), a focal increase in staining of SODs was observed. In foci of degeneration and necrosis, relatively strong expression of Mn-SOD was seen in the thyroid follicular epithelium as well as in mesenchymal cells. In addition, an inverse correlation was noted between Cu, Zn-SOD and Mn-SOD localization in the lesions of diffuse hyperplasia and benign and malignant nodules.These findings suggest a possible correlation between alterations of SOD activities and thyroid tumorigenesis, and the existence of regulatory mechanisms for SOD expression.

Selenium complexes in the anterior pituitary of rats exposed to L-selenomethionine

Selenium precipitates were demonstrated histochemically by silver amplification at light and electron microscopic levels in the anterior pituitary of rats exposed to L-selenomethionine (SeMeth). By electron microscopy (EM), the silver amplified selenium complexes were identified in somatotrophs, corticotrophs and gonadotrophs. Precipitates were observed mainly in the secretory granules and to a lesser extent in the lysosomes. The staining intensity increased with increasing amounts of SeMeth. Following a single injection of 3.7 mg Se/kg a substantial increase in staining was observed during the first 48 h after injection and precipitates could still be observed in the anterior pituitary after 2 weeks. During a long-term study where the rats were exposed to selenium contained in the drinking water (3.0 mg Se/l drinking water for 1, 2 or 4 weeks) an increasing amount of precipitates were observed during the first 2 weeks followed by a small decrease in staining intensity. Organic selenium, or rather a metabolite, is suggested to form bands with endogenous metal, primarily zinc, as has been suggested in the brain and anterior pituitary after exposure to sodium selenite.

Tritiated uridine labeling of the retina: Changes after retinal detachment

As part of a study designed to examine the response of photoreceptor cells to outer segment injury (retinal detachment), the pattern of RNA labeling ([ 3H]uridine incorporation) has been determined in detached cat retinas. Retinas were experimentally detached from the adjacent cellular layer (the retinal pigment epithelium: RPE) by injecting fluid into the extracellular space between the retina and RPE. Twenty-four hours before the animals were killed they received intravitreal injections of [ 3H]uridine. Autoradiograms were prepared from plastic sections 1·0 μm thick taken from detached retinal regions and, because the detachments do not encompass all of the retina, from nearby attached retinal regions. Twenty-four hours after retinal detachment there is a decrease in labeling intensity of the photoreceptors and Müller's glia in the region of detachment (compared to cells in nearby attached regions). Seventy-two hours after retinal separation, the same result is obtained in the photoreceptors, but labeling intensity is greatly increased in both the nuclei and cytoplasm of Müller's glia. The decrease in [ 3H]uridine labeling of the photoreceptors correlates with a decreased staining intensity of the cytoplasm and ultrastructural signs of necrosis. The striking change in the pattern and intensity of labeling of the Müller cells precedes extensive hypertrophy of these cells and the appearance within their cytoplasm of numerous 10-nm diameter filaments. Two weeks, and also 1 month, after detachment the pattern and labeling levels are similar to those observed 1 day after retinal separation. These data suggest a highly localized change in metabolism because the change in RNA labeling is restricted to the region of detached retina.

Immunohistochemical demonstration of carcinogen—DNA adducts in target and non-target tissues of rats given a prostate carcinogen, 3,2'-dimethyl-4-aminobiphenyl

An immunohistochemical procedure was applied which allows accurate localization of DNA lesions within organs and tissues of rats given 3,2'-dimethyl-4-aminobiphenyl (DMAB) using polyclonal antibodies against DMAB-DNA adducts. Dose-related nuclear staining was observed in organs regardless of DMAB-carcinogenic organotropism. In the male accessory sex organs, the lateral lobe of the prostate, a non-target site, demonstrated a similar staining intensity to that found for the ventral prostate and seminal vesicle, target sites. Orchiectomy and pretreatment with ethinyl estradiol resulted in a moderate to slight decrease in binding in the accessory sex organs. No observable decrease in staining intensity was evident in most organs 168 h after the administration of DMAB. These findings suggest that DNA adduct formation itself is not necessarily sufficient for tumor induction.

Glucocorticoid Receptor Immunoreactivity in the Rat Intermediate Lobe

Abstract Cells containing Type II glucocorticoid receptor (GR) immunoreactivity were identified in the rat pituitary gland by immunocytochemistry using a specific monoclonal antibody. At light microscopic level, GR immunoreactive cells were located in the intermediate lobe in addition to the well known GR-containing cell population in the anterior lobe. In both groups of cells GR appeared predominantly in the cell nuclei. Adrenalectomy resulted in a decrease in staining intensity of the anterior lobe and changed the pattern of fluorescence in a minority of cells where cytoplasmic staining became predominant. These changes appeared less marked in the intermediate lobe. Dexamethasone administration reversed the adrenalectomy-induced alterations of GR staining in both lobes. At the electron microscopic level, GR immunoreactive sites were revealed by the protein A-gold technique. In contrast to the distribution of fluorescence, GR was localized in cell nuclei as well as in the cytoplasm in both lobes. Quantitative estimates indicate that about 40% more immunoreactive sites are present in the anterior lobe than in the intermediate lobe. The presence of GR in the intermediate lobe suggests that this pituitary region, like the anterior lobe, is influenced by glucocorticoid hormones.

Differences in dopamine β-hydroxylase immunoreactivity between the brains of genetically epilepsy-prone and Sprague-Dawley rats

Biochemical studies have indicated that norepinephrine is present in lower levels in certain brain regions of genetically epilepsyprone rats (GEPR-9s) as compared to non-epileptic Sprague-Dawley (SD) rats. In this study, the immunocytochemical localization of dopamine β-hydroxylase (DBH), the synthesizing enzyme for norepinephrine, was compared between GEPR-9s and SD rats. Brain regions caudal to the inferior colliculus, such as the cerebellum and locus coeruleus, showed no differences in the distribution of DBH-like immunoreactive (DBH-I) neurons and fibers. In contrast, differences in the distribution of DBH-I fibers were observed in more rostral brain regions including the central nucleus of the inferior colliculus, thalamus, piriform, orbital and somatosensory cortices and hippocampus. In these areas, the number, and often the staining intensity, of DBH-I processes was lower in GEPR-9s as compared to SD rats. It was interesting to note that other cortical regions displayed no differences in DBH immunoreactivity between GEPR-9s and SD rats. These results provide anatomical data that support previously described biochemical results. Furthermore, the reduced number pf fibers and their decreased staining intensity in specific brain regions provide greater details to resolve the localization of deficiencies in the noradrenergic fiber plexus of GEPR-9s.

Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system.

This study describes the alterations induced by Interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta) on fibroblast-synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen-coated Terasaki wells for 3 or 6-9 weeks to create 3-dimensional cell-containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL-1 alpha or IL-1 beta (10(-9) M) at 37 degrees C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL-1 alpha and IL-1 beta induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross-sectional area and an increased density of the matrices. Three-week matrices were reduced 26% and 45% by using IL-1 alpha and IL-1 beta, respectively. Comparable values obtained by using 6-week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL-beta than with IL-1 alpha. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3-week matrix following IL-1 beta incubation. There was also a decrease in collagen type 1 staining of 9-week matrices treated with IL-1 alpha or IL-1 beta. These studies show that IL-1 has an effect on fibroblast-synthesized extracellular matrix and indicate that the effects of IL-1 alpha and IL-1 beta may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.

Analysis of experimental immune synovitis cartilage using monoclonal antibodies reactive to rabbit proteoglycan.

This study details the macromolecular changes in cartilage involving proteoglycan molecules in an animal model of rheumatoid arthritis. In experimental chronic immune synovitis, fluorescein-conjugated mouse IgG and three monoclonal antibodies (MAbs 2G2, 2E9, and 6C9) portraying differing fine antigenic specificity for rabbit cartilage proteoglycan monomer were utilized to detail alterations in cartilage proteoglycan. In normal and IgG-immune animals, fluorescein isothiocynate (FITC)-conjugated MAbs 2G2 and 2E9 stained cellular/pericellular (C/PC) region intensely. FITC-MAb 2G2 stained cartilage interterritorial matrix as well. FITC-MAb 6C9 stained only C/PC area lightly but did not stain matrix. A marked decrease in staining intensity with FITC-MAb 2G2 was noted in cartilage sections derived from animals with immune synovitis. A corresponding increase in staining of cartilage was noted with FITC-MAb 6C9. The augmented staining of articular cartilage with FITC-MAb 6C9 was most prominent in femoral condyle tissue sections, which corresponded to the cartilaginous area, with the greatest severity in gross pathology. There was a slight augmentation of staining with FITC-MAb 2E9, especially in the C/PC area of medial/femoral cartilage. In addition, the animals with immune synovitis showed abortive cartilage repair exemplified by the presence of chondrocyte cloning (up to 20 cells) which correlated with increased FITC-MAb 2G2 staining. The differential MAb staining patterns of cartilaginous tissues obtained utilizing FITC-conjugated monoclonal antibodies with known fine antigenic specificity indicates a modulation of proteoglycans involving predominantly core protein epitopes in the articular cartilage of animals with chronic immune synovitis.

Progesterone and antiprogesterone (RU 38486) modulation of estrogen-inducible glycoprotein (USP-1) synthesis and secretion in rat uterine epithelial cells.

Previous study in this laboratory revealed that estrogen stimulates synthesis and secretion of 97K glycoprotein [uterine secretory protein-1 (USP-1)] in rat uterine epithelial cells. In the present communication, the effects of progesterone and antiprogesterone (RU 38486) on estrogen-stimulated USP-1 biosynthesis were investigated. Ovariectomized rats were treated with estrogen for 4 days to induce USP-1 synthesis and secretion. Then, progesterone and RU 38486 were injected sc. After 6 and 12 h of treatment, uterine luminal fluid proteins were labeled for 6 h by direct administration of [35S]methionine into uterine lumen. By radioimmune precipitation assay using specific antibody against USP-1, it was shown that progesterone can reduce the USP-1 synthesis and secretion, which were prestimulated by estrogen. This antagonistic effect of progesterone on estrogen-induced USP-1 biosynthesis was also evident by the decreased staining intensity of USP-1 in uterine epithelial cells, as revealed by the immunohistochemical method. Although RU 38486 itself did not manifest any effect on estrogen-stimulated USP-1 biosynthesis, it completely inhibited the progesterone-induced decrease in synthesis and secretion. This system is believed to provide a useful model to analyze the progesterone and antiprogesterone actions on rat uterine epithelial cells.

The effects of conditioning on meat collagen: Part 2—Direct biochemical evidence for proteolytic damage in insoluble perimysial collagen after conditioning

The effect of proteolytic attack on unwashed and 6 M urea washed bovine perimysial collagen was examined using model systems. Pepsin and cathepsin solubilized collagen continuously over 24h ( r = 0·95 and r = 0·88 for time course of pepsin and cathepsin solubilized collagen). Insoluble perimysium treated with pepsin over 24 h resulted in little damage to the insoluble collagenous residue remaining, as evidenced by one-dimensional SDS-polyacrylamide gel electrophoresis. Insoluble perimysium treated with cathepsin resulted in changes to the major peptide bands which were evident after 24 h treatment, visible as broadening and ‘fuzziness’ of bands, decreased staining intensity, loss of high molecular weight material and a significant reduction in quantity of all peptides when compared with untreated perimysium. The effects of proteolytic action on bovine perimysial collagen in vitro and in conditioned meat were investigated by means of two-dimensional polyacrylamide gel electrophoresis, which provided a more sensitive technique for elucidating changes than the one-dimensional method. The peptide maps obtained from conditioned insoluble perimysium and from insoluble perimysium treated with cathepsin for 24 h were altered relative to the unconditioned insoluble perimysium, with the loss of some peptides and generation of others. The in vitro case was extreme, but was comparable with samples of perimysium from conditioned muscles. The results provide direct biochemical evidence for the presence of proteolytic damage in the insoluble perimysial collagen matrix of meat after conditioning.

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase.

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA.

Urinary excretion of neutral proteinases in nephrotic rats with a glomerular disease

A proliferative glomerulonephritis was induced in rats pre-immunized with rabbit IgG by injecting intravenously a sub-nephrotoxic dose of rabbit anti-glomerular basement membrane (GBM) IgG (A rats). Most rats (80%) developed a severe proteinuria (greater than 100 mg/24 hr) within two to five days after the injection of anti-GBM IgG. At the same time, microscopic examination of the kidneys revealed a glomerular infiltration by mononuclear phagocytes and a prominent decrease in the intensity of the colloidal iron reaction in glomeruli. A non-proliferative glomerular disease was induced in another group of rats (B rats) by intraperitoneal administration of aminonucleoside of puromycin. A marked proteinuria (greater than 100 mg/24 hr) occurred after six days in 90% of animals. Histochemical studies then revealed a decrease in staining intensity of glomeruli for polyanion. No glomerular hypercellularity was noted. In normal rats and in non-proteinuric A or B rats, the 24 hour urinary excretion of neutral proteinases ranged from 1.4 to 7.8 units (mean value +/- SEM, 4.69 +/- 0.60, N = 11), that of laminin ranged from 100 to 3,900 ng (mean value +/- SEM, 1,154 +/- 325, N = 10), and that of type IV collagen ranged from 160 to 420 ng (mean value +/- SEM, 306 +/- 26.5 ng, N = 8). In proteinuric rats from groups A (N = 11) and B (N = 9), the 24 hour urinary excretion of neutral proteinases significantly increased (mean values +/- SEM, 38.55 +/- 8.66 U for A rats and 42.17 +/- 7.92 U for B rats) and ran parallely with that of proteins, laminin and type IV collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

COMPARATIVE STUDIES ON THE CELL WALLS OF SEXUAL AND ASEXUAL BANGIA ATROPURPUREA (RHODOPHYTA). I. HISTOCHEMISTRY OF POLYSACCHARIDES1

ABSTRACTA comparative histochemical study of the nature and distribution of acidic and neutral cell wall polysaccharides was conducted on marine sexual and asexual filaments of the red alga Bangia atropurpurea (Roth) C. Ag. Outer and inner walls of this species were clearly partitioned according to staining and transmission electron microscopic characteristics. Neutral polysaccharides were detected in the outer coating (cuticle) but were absent from outer and inner walls of all filaments. Acidic polysaccharides were noted in the outer wall material but not in the inner wall layers of any filaments at any developmental stages. The major staining component of vegetative regions of sexual material and all regions of asexual filaments was a highly sulfated polymer. During sexual reproduction only there was a generalized change in the nature of the acidic component, characterized by a decrease in intensity of staining for sulfates in both male and female filaments and the appearance, in female filaments only, of polysaccharides which presumably were carboxylated. Spermatia attached to both male and female filaments in regions where sexual differentiation was initiated and where changes in the outer wall components commenced.

Radiation inactivation studies on the rabbit kidney sodium-dependent glucose transporter.

Rabbit kidney cortical brush-border membrane vesicles were irradiated in the frozen state with increasing doses of high energy electrons from a Van de Graaff generator. Sodium-dependent D-glucose and L-alanine transport showed a simple exponential loss of activity with increasing radiation dosage. Target size calculation based on these data gives estimates of 1.0 X 10(6) daltons for the glucose transporter and 1.2 X 10(6) daltons for the alanine transporter. A highly purified glucose transport protein extracted from rabbit kidney cortex was similarly irradiated both before and after reconstitution into liposomes. The target size of this purified glucose transporter was 343,000 daltons, based on inactivation of transport. The intensity of the major 165,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation was decreased by radiation. The decrease in staining intensity was dose-dependent, yielding a target size of 298,000 daltons, in situ. We propose that the purified glucose transporter reconstituted into liposomes is a tetramer comprised of 85,000-dalton subunits.

Inhibition by chelating agents of the formation of active extracellular proteinase byPseudomonas fluorescens32A

The effect of chelating agents on extracellular proteinase production by Pseudomonas fluorescens 32A was examined. Increasing concentrations of orthophosphate slightly stimulated growth while inhibiting proteinase synthesis. Fifty per cent inhibition was found at 35 and 28 mM-orthophosphate at 5 and 20 degrees C respectively. Extracellular protein concentration was reduced by 30% when cells were grown with 100 mM-orthophosphate. Polyacrylamide gel electrophoresis of the cell-free supernatants suggested that reduced enzyme synthesis had taken place as evidenced by the decrease in staining intensity of the protein band corresponding to the proteinase. Other phosphate compounds could replace orthophosphate as an inhibitor. Extent of inhibition was related to chain length; polyphosphates with 4-6 or 13-18 phosphorus atoms were the most effective inhibitors. EDTA (0.5 mM) completely inhibited proteinase synthesis. This inhibition could be partly reversed by Ca2+ and, to a lesser extent, Mn2+. Proteinase production at 5 degrees C in skim milk was completely inhibited by phosphate glass (P13-P18). Control experiments showed that loss of activity with chelators was not due to inhibition of preformed enzyme. The results suggest a possible role for polyphosphates in controlling proteinase production in stored milk.

The effect of oxygen-derived free radicals on gingival proteoglycans and hyaluronic acid.

The effect of a chemically induced oxygen‐derived free radical flux has been studied on extracted porcine gingival hyaluronic acid and proteoglycans as well as on cryostat sections of porcine gingivae. The result of the free radical producing system on hyaluronic acid and the proteoglycans was one of reduction of specific viscosity and molecular size. When frozen sections were subjected to the same oxygen‐derived free radical flux and subsequently stained with Alcian blue, a noticeable decrease in staining intensity was observed when compared to control sections. These results are considered to reflect the in vitro capacity of oxygen‐derived free radicals to depolymerize the two major non‐fibrous extracellular macromolecules of gingivae. It is postulated that such a mechanism may be responsible, at least in part, for the destruction of gingival proteoglycans and hyaluronic acid observed in inflammatory periodontal disease.

Nucleoprotein changes during male pronuclear development as determined by the ammoniacal silver reaction

AbstractSperm nucleoprotein changes during male pronuclear development in fertilized sea urchin (Arbacia punctulata) eggs have been examined utilizing the ammoniacal silver reaction (ASR) at the light and electronic microscopic levels of observation. Previous studies and control preparations indicated that the ASR has an affinity for basic proteins, staining intensely those rich in arginine residues. Differences in the affinity of the paternally derived chromatin to the ASR prior to, during, and following pronuclear development were observed. Relative to the female pronucleus the unincorporated sperm nucleus was densely stained. Upon its entry into the egg the sperm nucleus showed a two‐fold increase in staining, indicating an augmentation in the availability of reactive sites already present in the paternally derived chromatin or an accumulation of “new” reactive sites from the egg cytoplasm. With the dispersion of the sperm nucleus there was a progressive decrease in staining intensity of the paternally derived chromatin. Subsequent to pronuclear fusion the paternally derived chromatin, recognized by its relatively dense staining, was seen at one pole of the zygote nucleus. With time there was a gradual regression in the size and staining intensity of the paternally derived chromatin within the zygote nucleus. Changes in reactivity of sperm‐derived chromatin are discussed in reference to previous studies of chromatin transitions at fertilization.

Experimental high-velocity missile head injury

A standardized experimental high-velocity penetrating head-injury model has been produced in which pathological lesions were observed, not only in the wound track but at sites more remote from the track in the hypothalamus, brain stem and cerebellum. Diffuse subarachnoid haemorrhage was common and intraventricular haemorrhage was a constant feature. Other constant histological abnormalities were: 1. 1. Perivascular ‘ring’ haemorrhages. 2. 2. Perivascular haemorrhage with a surrounding zone of decreased staining intensity. 3. 3. Perivascular increased staining intensity. 4. 4. Areas of decreased staining intensity apparently dissociated from areas of haemorrhage. The pathogenesis of the perivascular lesions is discussed and preliminary studies suggest that these may be the site of early oedema. The implications of this experiment for military surgery and for ballistic protection of the head are discussed.

Localization of hexokinase in neural tissue: electron microscopic studies of rat cerebellar cortex.

The distribution of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in the rat cerebellar cortex has been studied at the electron microscopic level using the peroxidase-antiperoxidase procedure. Extensive staining of cytoplasmic regions, with some increased staining at mitochondrial profiles, was seen in the cell bodies of both neurons (basket, stellate, Lugaro, Golgi, and granule cells) and astrocytes. Oligodendrocytes showed little or no detectable staining. Purkinje cell perikarya were much less intensely stained than were the perikarya of other neurons. The initial portion of the Purkinje dendrite was, like the perikaryon from which it emerged, lightly stained. More intense staining was seen in the secondary and tertiary branches of the Purkinje dendrite, but the terminal branches were devoid of stain. Granule cell dendrites were well stained in their initial portions but devoid of stain in their terminal dendritic digits which form part of the cerebellar glomeruli. In contrast to the unstained granule cell dendritic digits, the central mossy fiber nerve terminal of the glomerulus exhibited intense staining of the mitochondrial profiles and of synaptic vesicles adjacent to the mitochondria. Axons of basket cells showed intense staining in the segments adjacent to the Purkinje cell soma, while terminal twigs of the basket axons in the pinceau surrounding the (unstained) initial segment of the Purkinje axon showed markedly decreased staining intensity. These results indicate that there may be substantial variation in hexokinase levels between the various regions of neuronal processes. Hexokinase was seen at both cytoplasmic and mitochondrial locations in a variety of cells. It does not appear likely that location of hexokinase can be directly correlated with cell type, i.e., with neurons versus glia.

Photodynamic action of bilirubin on the inner mitochondrial membrane. Implications for the organization of the mitochondrial ATPase

Bilirubin in the presence of O 2 and light catalyzes the photodynamic modification of the proteins of the inner mitochondrial membrane as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Numerous polypeptide bands become streaked towards higher apparent molecular weight and decrease in staining intensity while other bands remain largely unchanged. The loss in staining intensity which occurs is at least partially due to apparent cross-linking of the polypeptides to produce aggregates which cannot penetrate into the gel. The α and β bands of the mitochondrial ATPase differ markedly in their susceptibility to modification. The β subunit is rapidly modified while the α subunit is largely inert. This differential susceptibility is a consequence of the binding of the soluble F 1 ATPase to the membrane. When submitochondrial particles with their normal complement of bound F 1 are mixed with free F 1 and are photolyzed together in the presence of bilirubin and O 2, it is found that the β subunit of the membrane-bound F 1, but not the α subunit, has been modified while neither subunit of the free F 1 has been modified. This increased susceptibility of the β subunit in the membrane state may represent cross-linking to membrane components and is consistent with the β subunit making more extensive contacts with membrane components than does the α subunit.