Changes in the Respiratory, Enzymatic, and Swelling and Contraction Properties of Mitochondria from Colytedons of Phaseolus vulgaris L. during Germination

Mitochondria isolated from cotyledons of germinating wax beans (Phaseolus vulgaris L.) showed fairly good respiratory control on days 1 and 2 after planting. The respiratory control was completely lost from days 3 to 5. During this period mitochondria were shown to be very leaky, losing about 88% of their total nicotinamide adenine dinucleotide to the suspending medium in a short time. The respiratory control was partially recovered by day 7, after which it completely disappeared again. By the use of differential centrifugation, the mitochondria were divided into subfractions by sequential centrifugation: 10,000g for 5 minutes, 25,000g for 5 minutes, and 40,000g for 5 minutes. The 10,000g subfraction was responsible for the recovery of mitochondrial activity (respiratory control value, adenosine diphosphate to oxygen ratio, and rate of oxygen utilization), on day 7. Activities of succinate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, and isocitrate dehydrogenase from different mitochondrial subfractions of aging cotyledons were determined. In general, the enzyme activities, adenosine diphosphate to oxygen ratios, and the ability of mitochondria to swell and contract followed the same pattern as for respiratory control.     

Quantitation of mitochondrial DNA,RNA, and protein in starved and starved-refed rat liver

The purpose of this study was to investigate the problem of mitochondrial biogenesis in rat liver. The approach consisted of isolating mitochondria from control, 6 day starved and 6 day starved-5 day refed rats and comparing their DNA, RNA and protein content. This was performed by isolating the mitochondria by reorienting rate zonal centrifugation in sucrose gradients. It was found that six days of starvation resulted in a loss of 30% of the body weight, 55% of the liver weight, 40% of the mitochondrial protein, 60% of the mitochondrial RNA, but only 20% of the mitrochondrial DNA. It was also shown that refeeding of the rats for five days resulted in a restoration to normal or near normal levels in all the parameters measured. Further experiments employing the incorporation of ³H-TTP into isolated mitochondria indicated that the maintenance of mitochondrial DNA was not the result of continuous DNA synthesis.     

Lack of change in the composition of fetal lamb lung surfactant during gestation

Fetal surfactant from lamb lung fluids collected daily from day 114 to day 146 of gestation, was isolated by centrifugation (pellet material) and further purified by sucrose density gradient centrifugation. The concentration of the pellet material from lung fluid (crude surfactant) increased from day 125 till day 135 and fluctuated strongly from that period onwards, whereas lung fluid secretion increased linearly until a few days before parturition. The pellet phospholipid composition changed with gestational age, suggesting biochemical maturation of the surfactant-producing system. The purified surfactant fraction, of which approximately 85% was phosphatidylcholine, did not change however from day 122 onwards except for a small increase in the percentage of phosphatidylglycerol. Alveolar wash surfactant or the lamellar body material, isolated from fetal lungs at different gestational ages had the same composition as surfactant from lung fluids. Only the composition of lamellar bodies of '125 day' lungs differed slightly from that of the lung fluid surfactant. The similar characteristics of all purified surfactant fractions throughout gestation indicate that, in the fetal lamb, lung maturation is associated with an increase in surfactant production no significant changes in phospholipid composition.     

The influence of age,sex, pregnancy,starvation, and other factors on the cytoplasmic ribonucleoproteins of rat liver

Rat liver was homogenized in 0.88 M sucrose. The DNA and total RNA were determined, and the homogenate was fractionated by differential centrifugation. The pellets obtained between 30 minutes at 20,000 g and 180 minutes at 105,000 g were analyzed for RNA and nitrogen. The ribonucleoproteins were determined in the analytical ultracentrifuge. The non-pellet RNA was calculated by difference. The results are reported as amounts per 6.7 x 10(-9) mg. of DNA. In young, growing male rats the amounts of microsomal protein and ribonucleoprotein B (83S) increased with age. Non-pregnant adult females showed less non-pellet RNA and much more ribonucleoprotein C (63S) than did adult males. During pregnancy both of these cell constituents reverted to levels characteristic for male animals. Starvation for 5 days resulted in a reduction in the mass of liver tissue, the non-pellet RNA, the microsomal protein, and ribonucleoproteins B and C. During recovery from starvation the return of the liver to normal paralleled the rate at which body weight was restored. Treatment with cortisone, 25 mg. per rat per day for 5 days, caused an increase in microsomal protein and a decrease in ribonucleoprotein B. Treatment with 6-mercapto-purine, 50 mg. per kilo per day for 5 days, caused little change in liver composition in either males or females.     

A microassay for the determination of soluble and membrane-bound glutamate decarboxylase activity--influences of cations, lipid composition, and pyridoxal 5'-phosphate on the glutamate decarboxylase binding to liposomes

A radiochemical microassay for soluble and membrane-bound glutamate decarboxylase (GAD) is described. Up to 180 samples can be determined per day with a variation coefficient of 2%. The method detects newly synthesized gamma-amino-n-butyric acid in the picomole range and can easily be applied to other enzymes whose substrate and product differ by charge. In an aqueous homogenate of brain (1 + 10; w/v) about 15% of the total GAD activity are spun down by centrifugation (1 h, 100,000g) increasing to 35% of the total GAD activity in solutions with 8 mM calcium chloride or 100 mM potassium acetate. There is similar dependence on the cation concentration when GAD binds to phospholipid vesicles (liposomes) as well as dependence on lipid concentration and lipid composition. The coenzyme pyridoxal 5'-phosphate has no influence on GAD binding to liposomes.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

Virus-like particles from killer, neutral, and sensitive strains of Saccharomyces cerevisiae.

Procedures were developed for purification of virus-like particles (VLPs) from killer, neutral, and sensitive strains of Saccharomyces cerevisiae. Morphologically similar spherical VLPs measuring 40 nm in diameter were extracted from all three phenotypes. The particles were partially purified by high-speed centrifugation through a layer of CsCl (1.26 g/cm3) and sucrose density gradient centrifugation. Gradient-purified preparations contained three centrifugal species that sedimented at approximately 43, 102, and 162S. The 43S component is considered to be an artifact. Preparations from killer strains contained three double-stranded RNA (ds-RNA) components with molecular weights of 1.19 x 10(6), 1.29 x 10(6) and 2.54 x 10(6). VLPs from neutral and sensitive strains contained only the largest ds-RNA species. VLP preparations were subsequently separated into two major density components by CsCl equilibrium gradient centrifugation. The light component banding at 1.28 to 1.30 g/cm3 was void of nucleic acid, and the heavy component banding at 1.40 g/cm3 contained only the largest ds-RNA species.     

Purification of human blood burst-forming units-erythroid and demonstration of the evolution of erythropoietin receptors

To facilitate the direct study of the molecular events that control the development of human burst-forming units-erythroid (BFU-E), we have developed a method to purify BFU-E from peripheral blood. Using density centrifugation, rosetting with a mixture of neuraminidase-treated and IgG-coated sheep erythrocytes, positive panning with anti-My10 monoclonal antibody, overnight adherence to plastic dishes, negative panning with monoclonal antibodies, and density centrifugation, human blood BFU-E were purified from 0.04% to 56.6%, a 1,400-fold purification with a 13% yield. More than 90% of purified BFU-E were recombinant interleukin-3 (rIL-3) dependent, which survived for 48 h with rIL-3 in the absence of recombinant erythropoietin (rEP), and 80% gave rise to erythroid bursts of more than 500 hemoglobinized cells. rEP dependency was not evident until after 72 h of incubation in vitro. The purified cells (day 1) were incubated with rIL-3 and rEP in liquid culture for 24 (day 2), 48 (day 3), and 72 (day 4) h and then were transferred into semisolid cultures and incubated until day 15. The size of the erythroid colonies observed in semisolid cultures decreased continuously in association with the incubation time of day 1 purified cells in liquid cultures. The first appearance of colony-forming units-erythroid (CFU-E) that gave rise to colonies of 8 to 49 cells was observed after 72 h of incubation of day 1 cells in the liquid culture. 125I-rEP was incubated for 5 h at 37 degrees C with purified cells (day 1) or with the cells that had been incubated in liquid culture for an additional 24-72 h, and the presence of erythropoietin (EP) receptors was investigated using autoradiography. Specific binding of 125I-rEP was detected in 19 +/- 7% of the initial day 1 BFU-E. The percentage of 125I-rEP-binding to erythroid progenitor cells and the amount of binding continuously increased as day 1 BFU-E matured. 125I-rEP specific binding was observed with all of the erythroid progenitor cells that had been incubated in liquid culture for 72 h. These data demonstrate that primitive BFU-E have a much lower number of EP receptors than CFU-E and develop an increased concentration of EP receptors in association with their maturation and loss of proliferative capacity.     

Morphology, homogeneity and functionality of human monocytes-derived macrophages.

Primary cultures of human monocyte-derived macrophages (n = 50) were characterized in order to use this cellular model to establish a proteomic map of macrophages. Peripheral blood mononuclear cells were isolated from healthy donors' blood using density gradient centrifugation. The cell culture quality was checked in respect of several morphological and molecular aspects.The homogeneity and purity of cells was assessed after 12 days' primary culture with phase microscopy, immunocytochemistry and flow cytometry. Monocytes were completely differentiated into macrophages within 12 days as shown by phase microscopy. On day 12, all cells expressed CD68 antigen and were negative for CD3. Flow cytometry experiments showed a purity of the primary culture on day 12, in a range between 76% and 98% of CD14+ cells. The functionality of cells was characterized for the presence of ECE-1 as an intracellular marker and for the presence of MMP-9 as a marker secreted into the culture medium. This study allowed to determine criteria of quality and functionality for the primary culture of monocyte-derived macrophages. Cultures meeting these criteria will be used for the proteomic analysis and the establishment of the reference map.     

High-yield culture and purification of Chlamydiaceae bacteria

Research on intracellular bacteria of the family Chlamydiaceae, and the diseases they cause, requires large amounts of infectious elementary bodies (EB). We describe an approach that maximizes the generation of Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia abortus, or Chlamydia pecorum EBs in several replication cycles over approximately 10 days or more in a saturated equilibrium monolayer cell culture system. Buffalo Green Monkey Kidney (BGMK) cells, Human Epidermoid Carcinoma-2 (HEp-2) cells, or mouse McCoy cells were tested. BGMK cells best supported C. pneumoniae replication when cultivated in Iscove's Modified Dulbecco's Medium. From day 1 to day 9 after inoculation, C. pneumoniae genomes per ml culture medium increased from 10(5.1) to 10(8.6) in BGMK, from 10(5.6) to 10(8.1) in HEp-2, and remained at 10(5.2) in McCoy cell cultures. Three-month pre-inoculation maintenance of BGMK cells in different culture media did not influence C. pneumoniae yields. Inoculation at multiplicities of infection (MOI) of 10 or higher and supplementation of the cell culture medium on day 7 after inoculation with 0.1% glucose enhanced C. pneumoniae EB yields in harvested cell culture medium. For purification, EBs in medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Extensive sonication increased yield and infectivity of chlamydial EB. The combined method typically produced from 1000 ml infected BGMK culture medium 10 ml homogeneous, single-cell, highly infectious EB stock containing approximately 5x10(11) C. pneumoniae genomes equivalent to 4-5x10(11) inclusion forming units.     

Regulation of Glyoxysomal Enzymes during Germination of Cucumber: I. Developmental Changes in Cotyledonary Protein, RNA, and Enzyme Activities during Germination

Developmental patterns of glyoxylate cycle and photosynthetic activities have been correlated with electrophoretic profiles of cotyledonary RNA and protein in both light- and dark-grown cucumber seedlings (Cucumis sativus L.) Cytoplasmic rRNA increases 10-fold between days 0 and 5, and the steepest increase coincides with the most rapid rise in activities of the glyoxysomal enzymes, isocitrate lyase and malate synthase. Chloroplast rRNA and ribulose bisphosphate (RuBP) carboxylase begin rising at day 3, followed about a day later by increases in glyoxylate reductase activity and chlorophyll content. Of these phototrophic indicators, only chlorophyll requires light for its initial appearance. Sodium dodecyl sulfate gel electrophoresis of total and soluble cotyledonary protein showed several developmental patterns, including: (a) progressive disappearance of storage protein present initially in particulate form; (b appearance and subsequent disappearance of a family of polypeptides identified by molecular weight, developmental profile, and density gradient centrifugation as subunits of glyoxysomal enzymes; and (c) appearance and progressive increase (in both light- and dark-grown cotyledons) of the large and small subunits of RuBP carboxylase, as well as other polypeptides presumably of chloroplast and peroxisomal origin.     

Survival and growth of developing rats during centrifugation at 2G

We studied the effects of 2G hypergravity on the survival, body mass and growth of postnatal rats (Rattus norvegicus). Nursing litters comprised of either neonatal (Postnatal day [P]7) or pre-weanling (P14) rats and their mothers were exposed to 16 days of continuous centrifugation. All of the offspring survived and gained body mass, indicating that mothers nursed their young. Following the onset of centrifugation, neonatal and pre-weanling rats showed a reduction in growth relative to age-matched environmental controls (EC). At the completion of testing, body mass of the hypergravity (HG) groups was significantly less than that of controls (p<0.05). Over the course of the test, the HG-exposed P7 group showed an overall 55% gain in body mass as compared to a 71% increase in controls, while the HG-exposed P14 group showed a 62% increase relative to 75% in controls. Neonatal offspring (P7) gained body mass during centrifugation, but at significantly slower rates as compared to EC controls (p<0.05). In contrast, growth rates of pre-weanling (P14) rats were not reduced relative to controls, possibly related to the initiation of weaning, around P18 in the rat. These findings raise key issues relevant to studies of nursing mammals reared in altered gravity.     

Fertility of stallion semen processed, frozen and thawed by a new procedure

The fertility of frozen-thawed and fresh semen from three stallions was compared in a trial using a randomized block design and 90 mares for 108 cycles. Semen was collected every third day, diluted to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium, and centrifuged. The cells were resuspended at 700 x 10(6) progressively motile sperm/1.0 ml of added lactose-EDTA-egg yolk extender containing 4% glycerol, packaged by placing 0.55 ml into polypropylene straws, and frozen. Semen was thawed by immersion in 75 degrees C water for 10 sec. All of the 43 ejaculates collected were frozen, but 21 were discarded because progressive sperm motility was <35% immediately after thawing or <40% after 30 min of incubation at 37 degrees C. semen from the same stallions was collected daily for inseminations with fresh semen. Semen containing 200 x 10(6) progressively motile sperm was added to 10 ml of heated skimmilk extender. Mares were inseminated daily starting on the third day of estrus or when a >/=4-cm follicle was detected, whichever came later, and continuing through the end of estrus or for nine days. Based on palpation per rectum on day 50 postovulation, the pregnancy rates from inseminations during one estrus were 50, 56 and 61% with frozen semen and 67, 67 and 61% with fresh semen (P>0.05) from the three stallions, respectively. Thus, mean pregnancy rate with frozen semen was 86% of the rate attained with fresh semen.     

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.     

Changes in porcine,ovine, bovine and equine blood progesterone concentrations between collection and centrifugation

The aim of this study was to determine if different methods of handling porcine, ovine, bovine and equine blood between collection and centrifugation influence measurable progesterone levels. A 2 × 2 × 5 factorial experiment was conducted for each species with heparin (with or without), temperature of incubation (4 and 22°C) and time of incubation (0, 6, 12, 24 and 48 h) as the main effects. Following centrifugation, plasma and serum samples were stored at ?20°C until progesterone concentrations were determined by radioimmunoassay. Method of handling porcine and equine blood between collection and centrifugation did not affect the levels of progesterone. However, heparinized blood held at 4°C resulted in the most consistent levels of progesterone over time. Progesterone levels were fairly consistent across time in the ovine blood by all methods of handling except heparinized blood incubated at 22°C. By 24 h after collection, plasma progesterone concentrations decreased by 50% for the ovine blood incubated at 22°C with heparin. Decreases were detected by all the methods of handling the bovine blood between collection and centrifugation. The rate of decline, however, was considerably faster for blood held at 22°C than blood held at 4°C. At 12–48 h after collection, the concentrations of progesterone averaged only 5% of the time 0 sample for blood incubated at 22°C. In contrast, at least 30% of the progesterone values in the time 0 sample were detected between 12 and 48 h of incubation for the blood held at 4°C.     

TRICORE, a novel bead-based specimen concentration method for the culturing of Mycobacterium tuberculosis

Centrifugation is a necessary concentrating step for the detection of Mycobacterium tuberculosis in a liquid culture. However, centrifugation is biologically hazardous and presents an obstacle in the development of an automated culture system. A bead-based bacterial concentration method, TRICORE, was recently developed by Genetein Co., Ltd. We compared the efficacy of TRICORE and conventional centrifugation for concentrating M. tuberculosis in clinical sputum specimens by using liquid and solid culture systems. Among 90 pretreated clinical sputum specimens, 51 (57.3%) and 55 (61.8%) M. tuberculosis isolates were recovered by the MGIT culture system by using the centrifugation and TRICORE methods, respectively (chi-square test, p=0.5413). The detection time for the centrifugation method was 359.3±117.0 h, while that for the bead-based concentration method was 377.6±162.3 h (p=0.5637). However, the number of colonies recovered on solid media were significantly higher with the TRICORE method (p=0.003). In particular, among the smear-negative specimens, culture positivity of the TRICORE method was 39.6%, while that of the centrifugation method was 15.1%. The TRICORE bead-based concentration method was considered equivalent to centrifugation and enabled efficient collection of paucibacillary specimens in solution. Thus, the new noncentrifugation concentration method could yield more positive culture results.     

Development under the control of insulin of lipogenic enzymes,lipoprotein lipase,isoproterenol and glucagon sensitivity in differentiating rat preadipocytes in primary culture

In preadipose cellular fractions (I, II and III) isolated by density gradient centrifugation from the inguinal tissue of young rats, we followed the activity of fatty acid synthetase, ATP citrate lyase and lipoprotein lipase during differentiation in culture. 1.5 nM insulin when added at confluence markedly induced the activity of ATP citrate lyase and fatty acid synthetase in the cells derived from the lighter fractions (I and II). The magnitude of this response was 25–50-fold the initial value 15 days after plating. In the cells of the heaviest fraction (III) both enzymes exhibited low activity which was slightly stimulated by the presence of insulin, VLDL and heparin. In contrast, the activity of lipoprotein lipase appeared before confluence in cells from all three fractions and peaked at day 6 after plating. This early emergence was independent of the addition of insulin to the medium. However, insulin slightly enhanced the peak activity in post-confluent cells. The development of cAMP production in response to isoproterenol (100 μM) and to glucagon (0.3 μM) was determined in the cells of fraction II in the same culture conditions. The responsiveness to isoproterenol was present very early in these cells and rose rapidly during the exponential growth phase, reaching a peak value at day 8 after plating. In contrast, the development of glucagon sensitivity occurred only during late differentiation. The stimulatory effect of glucagon was enhanced when VLDL and heparin were added with insulin to the medium.     

High concentration active enzyme centrifugation studies with pig kidney phosphofructokinase. Detection of 9.8 S, 25 S, and 53 S active polymeric forms

This laboratory has carried out the first detailed studies of the active polymeric forms of phosphofructokinases over the concentration region of 1 to 1200 micrograms/ml. This includes the concentration range in which the enzymes exist in vivo and the concentration range in which their association-dissociation equilibria shift to yield various polymeric forms. Previously, active enzyme centrifugation experiments were limited to the concentration range below a few micrograms per ml. The present experiments were made possible by the recent development in this laboratory of a new technique called high concentration active enzyme centrifugation (Wei, G. J., and Deal, W. C., Jr. (1979) Biochemistry 18, 1129). We report here three new active polymeric forms of pig kidney phosphofructokinase which have been observed in high concentration active enzyme centrifugation experiments. These include: 1) a 9.8 S form (Mr = 2.6 X 10(5)); 2) a 25 S form (Mr = 1.01 X 10(6)); and 3) a 53 S form (too asymmetric to estimate Mr). In addition, a 5.4 S form is predicted from the Mr (8.8 X 10(4)) of the polypeptide chain obtained from sodium dodecyl sulfate gel electrophoresis; it is not known whether or not it is active. The 9.8 S value is the limiting sedimentation coefficient value observed in active enzyme centrifugation experiments. The 25 S form is indicated by a plateau in the 50 to 200 micrograms/ml region of the s versus c curve. The 53 S form is observed as a plateau in the 600 to 1000 micrograms/ml region of the s versus c curve.     

Isolation, homogeneity, and properties of core particle from pyocin R1.

By a mild alkaline treatment of pyocin R1, the core particle was released from the contracted sheath. After sucrose density-gradient centrifugation, core-rich fractions were treated with anti-sheath serum and by a second density-gradient centrifugation, purified core particles were isolated. Homogeneity of the preparation was confirmed by observation under the electron microscope, immuno-precipitation reaction, and sucrose density-gradient centrifugation. The core particle exhibited a sedimentation coefficient of 37S. The quaternary structure of the core consists of a single kind of subunit protein with a molecular weight of 18,000. No contamination by other proteins was detected by SDS-disc electrophoreses. Amino acid analysis revealed that the core is rich in glycine, alanine, valine, leucine, aspartic acid (or asparagine), glutamic acid (or glutamine), and serine. This amino acid composition bears some resemblance to that of T-even bacteriophage tail-core.     

Effects of starvation on vacuolar apparatus of cardiac muscle tissue determined by electron microscopy, marker-enzyme assays and electrolyte studies

The effects of a six day starvation regimen on rats' hearts were studied by electron microscopy in combination with marker-enzyme assays of density-sedimentation (rho-S) zonal centrifugation fractions, and with Na+, K+ and Ca++ determinations of sera and heart homogenates. The evidence suggested that massive intracellular cardiac destruction occurred by two pathways. One pathway was seen by electron micrography in which proliferation of lysosomal populations was demonstrated. The finding was confirmed biochemically by increased activities of lysosomal acid hydrolases, particularly cathepsin D. The second pathway was deduced from biochemical and electrolytic data. It was believed to have been initiated by cellular K+ retention, which provided the acid milieu required for intracellular Ca++ retention. It is postulated that the resulting increase in Ca++ activated the loosely-bound membrane neutral (pH 7.4), and alkaline (pH 8.5) proteases, causing subcellular autolysis, particularly involving mitochondria, myofibrils and sarcoplasmic reticulum.