MICROSOMAL AND LYSOSOMAL ACID PHOSPHATASE ISOENZYMES OF MOUSE KIDNEY CHARACTERIZATION AND SEPARATIONLIN, CHI-WEI; FISHMAN, WILLIAM H.
doi: 10.1177/20.7.487pmid: 4338762
Biochemical methods have been employed to characterize and separate acid phosphatases from lysosomal and microsomal fractions in order to decide whether different isoenzymes reside in these subcellular locations. Microsomal and lysosomal fractions of mouse kidney homogenate were isolated by differential centrifugation. Acid phosphatase of lysosomal fraction goes into solution after lysosomes have been repeatedly frozen and thawed, whereas acid phosphatase of microsomal fraction is firmly bound to the membrane and is freed of contamination by lysosomal enzyme after ultrasonication and centrifugation. The membrane-bound microsomal acid phosphatase is labile at 37°C, pH 4.9, more active toward phenolic substrates (phenyl phosphate and p-nitrophenyl phosphate) than β-glycerophosphate, α-naphthol phosphate or naphthol AS-BI phosphate. It also has a higher pH optimum (6.3), is resistant to l-tartrate and oxalate inhibition and has a slower electrophoretic migration rate in Triton X-100-impregnated polyacrylamide gels. The free lysosomal acid phosphatase is relatively heat-stable, is less active against phenolic substrates, is sensitive to l-tartrate and oxalate inhibition, has a lower pH optimum (5.6) and has a faster migration rate in electrophoresis. These two acid phosphatases can also be separated by diethylaminoethyl-cellulose chromatography. This study thus demonstrated the existence of an acid phosphatase isoenzyme in the microsomal membrane with different biochemical properties from the lysosomal isoenzyme of acid phosphatase.
MODIFICATION OF THE GELATIN SUBSTRATE PROCEDURE FOR DEMONSTRATION OF ACROSOMAL PROTEOLYTIC ACTIVITYPENN, ARTHUR; GLEDHILL, BARTON L.; DARŻYNKIEWICZ, ZBIGNIEW
doi: 10.1177/20.7.499pmid: 4556624
In situ proteolytic activity in the heads of sperm of seven mammalian species has been demonstrated using autoradiographic film as a gelatin substrate. The film is first exposed and processed and then coated with the sperm sample. Proteolytic activity is monitored by following the appearance of "halos," which are areas of gelatin digestion and are found to surround the heads of reacting sperm. The proteolytic factor appears to be released from the acrosome and may be the protease with trypsin-like activity that has been found associated with the acrosome in biochemical studies. l-Chloro-3-tosylamido-7-amino-2-heptanone and diisopropyl fluorophosphate, both of which inhibit trypsin activity, apparently interact with a substance released from the acrosome but do not interfere with the formation of halos. Sperm treated with trichloroacetic acid, urea or formalin do not form halos.
THE NATURE OF NONSPECIFIC ESTERASES A SUBUNIT CONCEPTCHOUDHURY, S. ROY
doi: 10.1177/20.7.507pmid: N/A
To investigate the nature of esterase polymorphism, nonspecific esterases were investigated in a large number of organs obtained from rat. In starch gel electropherograms the capacity of ester hydrolysis was seen to decline predictably with successively longer chain carbon substrates. An increasing susceptibility to organophosphate inhibition was observed with progressive lengthening of the acyl chain in the substrate molecules. Attempts to hybridize an organophosphate-sensitive esterase with a resistant type yielded a few additional esterase species. Studies on an esterase fraction isolated by column chromatography revealed the highly relative nature of organophosphate inhibition. These suggested that nonspecific esterases all belong to one enzyme system that does not justify the present compartmentation into acetyl-, aryl- or carboxylesterases on grounds of substrate hydrolysis or organophosphate sensitivity. It is likely that esterase species are each built on a subunit structure and exhibit overlapping specificities through sharing of common subunits. The concept that a whole range of esterase subunits exist, each varying in its capacity to hydrolyze esters of up to an optimal chain length and arranged according to the dictates of specific tissue requirements, is in accord with the present findings.
ELEMENTAL ANALYSIS OF PRECIPITATES FORMED IN NUCLEI BY ANTIMONATE-OSMIUM TETROXIDE FIXATIONSPICER, S. S.; SWANSON, A. A.
doi: 10.1177/20.7.518pmid: 4338763
Elements retained in cervical lymph nodes, isolated hepatic nuclei and salt-impregnated gels by fixation with antimonate- or pyrophosphate-containing and other osmium tetroxide solutions were assayed by nuclear activation analysis or by atomic absorption spectrophotometry. Salts preserved by the antimonate-osmium tetroxide fixative in lymph nodes, isolated nuclei and a KCl-enriched gel consisted almost entirely of potassium antimonate. The K+ in the precipitates in these specimens appeared to derive partially from that in the fixative solution and partially from that in the specimen. Salts preserved by the antimonate-osmium tetroxide fixative in an NaCl-supplemented gel consisted partly of potassium antimonate derived from the fixative as in unsupplemented gels and partly of sodium antimonate. The Na+ precipitated in this gel amounted to less than one-half that originally present. In comparison the pyrophosphate-osmium tetroxide solution retained higher levels of K+ in lymph nodes, nuclei and the KCl gel, but the potassium pyrophosphate was not evident as electron-opaque precipitates. The latter fixative was less effective in preserving Na+ in the NaCl gel. The pyrophosphate-containing fixative, which was about twice as efficient as the antimonate-containing solution in retaining the divalent cations, preserved 70% of the Mg++ and 100% of the Ca++ added to gels.
IMMUNOPEROXIDASE LOCALIZATION OF ANTIHAPTEN ANTIBODIES IN RATS RESPONDING UNEQUALLY TO ANTIGENIC STIMULATIONFRANÇOIS, DOMINIQUE; ORIOL, RAPHAËL; BINAGHI, RUBEN A.
doi: 10.1177/20.7.527pmid: 5037425
The present work reports on an electron microscopic study of the primary response of two strains of rats to the subcutaneous injection of the hapten 2,4-dinitrophenyl (DNP). When this substance is injected into the two strains used, there is either a weak or a strong production of circulating anti-DNP antibodies. Satellite lymph nodes were fixed in glutaraldehyde, incubated in a solution of horseradish peroxidase labeled with DNP and treated with diaminobenzidine + H2O2. A specific positive reaction was observed: (a) in the perinuclear cisternae of numerous lymphocytes and lymphoblasts; (b) in the perinuclear cisternae as well as in the peripheral cisternae of some plasmablasts; and (c) in a recently described rare type of cell known as a "lymphoplasmacyte." In certain lymphocytes, there was an accumulation of the reaction product in the perinuclear cisternae, and sometimes in the peripheral cisternae, which, in some places, provoked a dilation of the cisternae. There was no significant qualitative difference between the two strains of rats.
THE HISTOCHEMICAL DEMONSTRATION OF POLYADENYLIC ACID HYDROLASES IN RAT LIVER DURING AZO DYE CARCINOGENESISDAOUST, R.
doi: 10.1177/20.7.536pmid: 4338764
Films of polyadenylic acid (poly-A) were exposed to liver sections from 4-dimethylaminoazobenzene (DAB)-fed rats in order to determine whether the nucleases acting on these films, like the ribonucleases (RNases), are depressed during carcinogenesis. Normal liver parenchyma gave a positive reaction which was particularly intense in periportal areas. Livers from animals fed the basal control diet showed a similar distribution of enzyme activity but were generally more active than normal livers. In DAB-fed rats, the nodules of hepatic tissue gave intense reactions while the trabeculae of bile ducts and connective tissue, as well as the necrotic areas, were negative. The formation of hyperbasophilic foci at later stages of DAB feeding was accompanied by a loss of enzyme activity. The hepatomas, which apparently derived from such foci, showed weak or negligible activity. Thus the changes in RNases and poly-A hydrolases occur at different stages of the carcinogenic process. The loss of RNase activity precedes the neoplastic transformation while the decrease in the activity of poly-A hydrolases is closely associated with tumor formation, but the induced tumors are deficient in both types of nuclease activity.
QUARTZ FIBER ULTRAMICROBALANCEMOSS, M. L.
doi: 10.1177/20.7.545pmid: 4338765
Design and operation of a quartz fiber cytoanalytical balance are described. The 2-µ fiber, 3-4 mm long, is mounted in a V-shaped notch in an aluminum slide for use on an ordinary microscope. The deflection is measured with the focusing adjustment by viewing through thin glass windows, which are flush with the surfaces of the slide. Since the opening to the balance faces away from the operator, weighings can be made without closing the balance and removing time manipulator needles employed for handling the specimens. Transverse sections of single muscle cells, 0.1 µg after freeze-drying, can be weighed to within 3% under ordinary atmospheric conditions.