Presence of lysosomal enzymes in the normal glomerular basement membrane matrix

Summary The question posed in the present study was: are there hydrolytic enzymes, including proteases, present in the extracellular matrix of the glomerular basement membrane? If these enzymes are present they may play a role in the catabolism of the glomerular basement membrane (GBM) and removal of macromolecular debris resulting from ultrafiltration. Enzymes, acid phosphatase - the marker for lysosomal enzymes - β-galactosidase, β-glucuronidase and acid protease (using albumin as substrate) were biochemically assayed in purified basement membrane preparations. It was found that all enzymes were present in significant amounts in the basement membrane. Compared to other enzymes, acid protease activity was present in much higher amounts. The pH optima of these enzymes were variable but all had significant activity at neutral pH. A method was developed to localize the marker enzyme, acid phosphatase, ultrastructurally in the basement membrane in order to substantiate the biochemical findings. Activity was shown by the presence of dense deposits of lead phosphate. Staining for acid phosphatase could also be shown on isolated, purified basement membrane. The demonstration of acid hydrolases in the GBM matrix argues for their role in (i) the extracellular turnover of basement membrane macromolecules, and (ii) clearance of debris of ultrafiltration which tend to clog the membrane pores.     

Evidence of homology in a high-sulphur protein fraction (SCMK-B2) of wool and hair α-keratins

Fractions corresponding to the S-carboxymethylated high-sulphur protein component SCMK-B2 isolated by Gillespie (1963) from Merino wool were prepared from five different wool samples and also from bovine hair. The six fractions showed great similarities in amino acid composition, and also gave very similar peptide ;maps' after tryptic and chymotryptic digestion. Some of the peptides were isolated from the different samples, and evidence is given that suggests that a sequence of at least 21 amino acids is common to all the fraction SCMK-B2 preparations. Further, all the fractions derived from the wool samples have the same acetylated heptapeptide for the N-terminal sequence, but one extra residue may be present in this N-terminal sequence in the protein from bovine hair. The general significance of these findings is discussed.     

The effect of acclimation temperature on the activation energies of state III respiration and on the unsaturation of membrane lipids of goldfish mitochondria

The influence of the acclimation temperature on the thermotropic behaviour of mitochondrial respiration and on the degree of unsaturation of mitochondrial membrane lipids has been studied. The mitochondria were isolated from red muscle, white muscle and liver of goldfish acclimated to 5, 20 and 30°C. ADP-activated succinate oxidation was measured at different temperatures and resulted in non-linear Arrhenius-plots with breaks between 10 and 23°C. As for the break-temperatures, there was found a shift downwards in preparations of decreased acclimation temperatures. This could be caused by a changed composition of membrane lipids and a simultaneous shift of the membrane phase transition temperature. Therefore, the fatty acid composition of all membrane preparations was analyzed. However, no consistent change of the degree of unsaturation due to a changed acclimation temperature could be found.     

Loss of a homologous group of proteins in a dominantly inherited ectodermal malformation.

Hair from mice bearing the dominantly inherited Naked trait (NN) and from normal (NN) mice of the same inbred strain was separated into its major protein components by standard techniques. The relative amounts of proteins in these components were then determined by a regression method from the amino acid composition of the hair samples and of the fractions into which they had been separated. The results indicated that the amount of soluble fibril in Naked-mouse hair is decreased. Polyacrylamide-gel electrophoresis of this fraction prepared from the hair of both normal and Naked mice revealed that all protein bands present in the normal are also present in the Naked mice. However, a densitometric scan of the gels at 280 nm showed that the soluble fibril fraction from Naked-mouse hair is deficient in several proteins which, on amino acid analysis, were found to contain 31% glycine and 10% tyrosine. Gel filtration of S-carboxymethylkerateine prepared from normal and mutant hair showed that the mutant hair is deficient in a heterogeneous, low-molecular-weight fraction also rich in glycine and tyrosine. Our present data do not reveal the mechanism whereby a single gene locus modulates the production of several different proteins.     

Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations

The binding of radioactive 5-methyltetrahydrofolate and folic acid was found to be greater in brush border than in basolateral membrane preparations of rat renal cortex. This appeared to be due to an increased amount of a specific folate binding protein in the brush border membrane preparations as compared to those of the basolateral membrane. The binding was saturable and inhibited by nonradioactive folic acid and, therefore, a specific, rather than nonspecific process. The Km's for folic acid binding in brush border and basolateral membrane preparations were similar and involved a single high-affinity binding site. In contrast, methotrexate was found to bind equally well to both brush border and basolateral membrane preparations. Moreover, folic acid binding was not inhibited by an equimolar amount of methotrexate. A folate binding protein could be extracted from either membrane preparation with 1% Triton X-100 and, to a lesser extent, with 0.6 M NaCl. These different extraction procedures resulted in different apparent molecular weights for folate binding protein (greater than 160,000 for Triton X-100-extracted samples and 40,000 for NaCl-extracted samples). The membrane preparation pellets remaining after NaCl extraction were able to rebind tritiated folic acid and also the 40,000-Da folate binding protein. On the other hand, membrane preparations extracted with Triton X-100 lost the ability to bind folic acid or the 40,000-Da folate binding protein. These differences in molecular weight and rebinding capacity may be explained by the existence of a receptor for folate binding protein which was extracted by Triton X-100, but not by NaCl. The greater concentration of folate binding protein in the renal tubule cell brush border membrane preparations as compared to those from basolateral membranes ascribes, for the first time, a functional role for folate binding protein in the renal reabsorption of folates which is required to prevent loss of folate in the urine and perhaps in the membrane transport of folates in general.     

The effect of acclimation temperature on the activation energies of state III respiration and on the unsaturation of membrane lipids of goldfish mitochondria.

The influence of the acclimation temperature on the thermotropic behaviour of mitochondrial respiration and on the degree of unsaturation of mitochondrial membrane lipids has been studied. The mitochondria were isolated from red muscle, white muscle and liver of goldfish acclimated to 5, 20 and 30 degrees C. ADP-activated succinate oxidation was measured at different temperatures and resulted in non-linear Arrhenius-plots with breaks between 10 and 23 degrees C. As for the break-temperatures, there was found a shift downwards in preparations of decreased acclimation temperatures. This could be caused by a changed composition of membrane lipids and a simultaneous shift of the membrane phase transition temperature. Therefore, the fatty acid composition of all membrane preparations was analyzed. However, no consistent change of the degree of unsaturation due to a changed acclimation temperature could be found.     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

An electron microscope study of kidney basement membrane changes in the mouse by lipoteichoic acid from Streptococcus pyogenes

Mice injected repeatedly, intraperitoneally or intravenously, for approximately 1 month with a total of 1.04 mg lipoteichoic acid from a nephritogenic strain of Streptococcus pyogenes lost weight. Analysis by electron microscopy revealed that they also exhibited extensive kidney changes in basement membrane morphology which resembled, in part, those observed in human poststreptococcal glomerulonephritis. For example, the glomerular basement membrane became electron dense and exhibited at least a twofold increase in width sporadically within the same preparation after exposure to lipoteichoic acid. Also, whereas appreciable loss or reduction in epithelial foot processes as a result of fusion was clearly evident, epithelial slits and slit membranes or diaphragms between normal foot processes were not selectively affected. In addition, another mostly thickened, highly coiled or serpentinelike basement membrane with amorphous nodules appeared in these preparations. This type membrane was not observed surrounding the capillary lumina and was the most pronounced abnormality apparent in almost all preparations from mice exposed to lipoteichoic acid. Likewise, the proximal tubular basement membrane became variable in width and increased in electron density in mice given lipoteichoic acid as compared with controls. In addition, this membrane was often punctuated with various morphological protrusions originating from only its thickened areas and which extended away from, and not into, the capillary space. This change was only associated with the basement membrane of the proximal tubular capillaries. All membrane changes persisted but gradually subsided, with normal kidney membrane morphology reappearing on the 4th day following the last injection of lipoteichoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative lipid analysis and structure of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

The lipid composition and structure of detergent-resistant membrane rafts from human, goat, and sheep erythrocytes is investigated. While the sphingomyelin:cholesterol ratio varied from about 1:5 in human to 1:1 in sheep erythrocytes a ratio of 1:1 was found in all raft preparations insoluble in Triton X-100 at 4 degrees C. Excess cholesterol is excluded from rafts and saturated molecular species of sphingomyelin assayed by gas chromatography-mass spectrometry determines the solubility of cholesterol in the detergent. Freeze-fracture electron microscopy shows that vesicles and multilamellar structures formed by membrane rafts have undergone considerable rearrangement from the original membrane. No membrane-associated particles are observed. Synchrotron X-ray diffraction studies showed that d spacings of vesicle preparations of rafts cannot be distinguished from ghost membranes from which they are derived. Dispersions of total polar lipid extracts of sheep rafts show phase separation of inverted hexagonal structure upon heating and this phase coexists with multilamellar structures at 37 degrees C.     

Commercial Hair Sprays as Fixatives for Hematological Cytochemistry

Commercial hair sprays have been found to be excellent cytological fixatives for a variety of enzymatic and nonenzymatic hematological staining procedures. Of the varieties evaluated, not all were found suitable for each staining procedure tested. With some preparations, excellent leukocyte morphology and preservation of reaction product was obtained after staining for carbohydrates (PAS), lipid (Sudan black), nucleic acids (methyl green-pyronin), peroxidase, M-nadi oxidase and alkaline phosphatase. These spray preparations are remarkably inexpensive, readily stored, and stable and simple to use. The fixative ability is probably related to their polyvinylpyrrolidine and alcohol content.     

Formation of diacyl- and alkylacylphosphatidylcholine by the membranes of human platelets

We have investigated the distribution and fatty acid preference of two acyl-CoA transferase activities in a human platelet mixed membrane fraction and in well-characterised surface and intracellular membrane subfractions prepared from it by high-voltage free-flow electrophoresis. One transferase inserts long-chain unsaturated fatty acids into 1-acyllysophosphatidylcholine (1-acyl-LPC) and the other into lyso-platelet-activating factor (LPAF). Both transferase activities were approx. 4-fold enriched in the intracellular membranes with respect to their specific activities in the mixed membranes. The surface membrane activities were correspondingly depleted. Using 1-acyl-LPC as the acceptor, all the intracellular membrane preparations showed transferase preference for the CoA ester of 8,11,14-eicosatrienoic acid. In contrast when LPAF was the acceptor the CoA esters of linoleic and arachidonic acid were the preferred donors.     

Subcellular Localization of Rat Brain Insulin Binding Sites

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, gamma-aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mitochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r = 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.     

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.     

Studies on the cellular and free lipopolysaccharides from Branhamella catarrhalis.

Cellular and free lipopolysaccharides obtained from Neisseria catarrhalis and Branhamella catarrhalis were found to be essentially identical. Both cellular and free lipopolysaccharides contained core-oligosaccharides of the following composition: D-glucose (4 mol), D-galactose (1 mol), 2-amino-2-deoxy-D-glucose (1 mol), and 3-deoxy-D-manno-octulosonic acid. Aldoheptose and phosphate components were below levels of detection. Several physical methods indicated that all core-oligosaccharide preparations were identical. Lipid A preparations from cellular and free lipopolysaccharides of both organisms were qualitatively and quantitatively similar; they were composed of decanoic acid, dodecanoic acid, 3-hydroxy dodecanoic acid, 2-amino-2-deoxy-D-glucose, phosphate, and ethanolamine. The results tend to justify the transfer of Neisseria catarrhalis to the genus Branhamella.     

The viscosity and lipid composition of the plasma membrane of multiple drug resistant and sensitive yeast strains.

Four different plasma membrane preparations were isolated from multiple drug resistant and sensitive isolates of two isogenic groups of Saccharomyces cerevisiae strains: zymolyase ghosts, concanavalin A ghosts, pH 4 nonaggregated vesicles, and sucrose-gradient purified vesicles. The viscosities of these preparations were determined by the use of a fluorescence polarization technique with 1,6-diphenyl-1,3,5-hexatriene. The viscosities of all four membrane preparations within an isogenic set were the same for resistant and sensitive strains. A comparison of the viscosity of zymolyase ghost liposomes showed that zymolyase ghost (glyco) proteins of resistant and sensitive strains had the same effect on viscosity. There was no difference between resistant and sensitive isolates in the mole concentration of the following lipid classes extracted from zymolyase ghosts: phospholipid, sterol, sterol ester, triglyceride, diglyceride, and free fatty acid. The fatty acid distribution of esterified and free fatty acids and the distribution of nine phospholipids was the same in zymolyase ghosts from sensitive and resistant strains. It was concluded that multiple drug resistance does not result from an alteration in plasma membrane viscosity or lipid composition.     

Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.     

A cell-free assay for the insertion of a viral glycoprotein into the plasma membrane

A cell-free assay has been developed for the delivery of influenza virus neuraminidase to the plasma membrane. Two types of postnuclear supernatant, which acted as donor and acceptor of the enzyme, were prepared from baby hamster kidney cells. Donor preparations were obtained from cells infected with influenza virus and containing neuraminidase en route to the plasma membrane. Acceptor preparations were obtained from cells containing, bound to their plasma membranes, Semliki Forest virus with envelope glycoproteins bearing [3H]N-acetylneuraminic acid. Fusion between vesicles from these two preparations permits access of the enzyme to its substrate, which results in the release of free [3H]N-acetylneuraminic acid. This release was detected through the transfer of radioactivity from a trichloroacetic acid-insoluble to a trichloroacetic acid-soluble fraction. An ATP-dependent component of release was found, which appears to be a consequence of vesicle fusion. This component was enhanced when the donor was prepared from cells in which the enzyme had been concentrated in a compartment between the Golgi complex and the plasma membrane, which indicates that a specific exocytic fusion event has been reconstituted. The extent of fusion is greatly reduced by pre-treatment of donor and acceptor preparations with trypsin, which points to the involvement of proteins in the fusion reaction.     

Improved Isolation Procedures for the Purple Membrane of Halobacterium Halobium

Techniques for purifying the purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromoprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.0.³ The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.     

The action of digitonin on rat liver mitochondria. Phospholipid content

1. The amount and types of phospholipid and the fatty acid composition of the various phospholipids were examined in intact rat liver mitochondria, in mitochondria devoid of their outer membrane (preparation A) and in very small pieces derived from the disruption of the inner-membrane complexes (preparation B). The latter two preparations were obtained by digitonin treatment and carry out oxidative phosphorylation. 2. The ratio mug.atoms of phospholipid P/mg. of protein was 0.163 for intact mitochondria, decreased to 0.118 on removal of the outer membrane and increased markedly to 0.292 on disruption of the inner-membrane complex. 3. Examination of the various types of phospholipid present showed that the molar proportions cardiolipin:phosphatidylcholine:phosphatidylethanolamine were approx. 1:6:6 for intact mitochondria and 1:3:3 for preparations A and B. 4. There was a correlation between the recovery of cardiolipin and adenosine triphosphatase activity in the conversion of intact mitochondria into preparations A and B. 5. The fatty acid contents of the various types of phospholipid purified by thin-layer chromatography were identical in all three preparations. Our results show a considerably higher content of arachidonic acid and lower content of oleic acid for phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol than have previously been reported for mitochondrial phospholipids.     

Stereocilia membrane deformation: implications for the gating spring and mechanotransduction channel

In hair cells, although mechanotransduction channels have been localized to tips of shorter stereocilia of the mechanically sensitive hair bundle, little is known about how force is transmitted to the channel. Here, we use a biophysical model of the membrane-channel complex to analyze the nature of the gating spring compliance and channel arrangement. We use a triangulated surface model and Monte Carlo simulation to compute the deformation of the membrane under the action of tip link force. We show that depending on the gating spring stiffness, the compliant component of the gating spring arises from either the membrane alone or a combination of the membrane and a tether that connects the channel to the actin cytoskeleton. If a bundle is characterized by relatively soft gating springs, such as those of the bullfrog sacculus, the need for membrane reinforcement by channel tethering then depends on membrane parameters. With stiffer gating springs, such as those from rat outer hair cells, the channel must be tethered for all biophysically realistic parameters of the membrane. We compute the membrane forces (resultants), which depend on membrane tension, bending modulus, and curvature, and show that they can determine the fate of the channel.