Marjorie A. Jones

Immunoadsorbent affinity purification of the fifth component (C5) of human complement and development of a highly sensitive hemolytic assay

The fifth component of complement (C5) has been isolated from human serum in fully hemolytically active form by immunoadsorbent and anion exchange column chromatography. The immunoadsorbent column was prepared by the covalent coupling of the purified IgG fraction obtained from monospecific goat anti-human C5 antiserum to CNBr activated Sepharose 4B. Establishment of appropriate conditions for the dissociation and elution of functionally active C5 from the immunoadsorbent column was of central importance in the development of this purification procedure. The C5 preparations exhibited final yields of 20--50% with 570--710-fold purification factors based on recovery of specific hemolytic activity. These preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide slab gel electrophoretic and immunochemical criteria. A C5-depleted reagent (C5D) was generated from the non-adsorbed protein containing fractions obtained subsequent to the passage of freshly drawn NHS plus 10 mM EDTA through the monospecific anti-C5 Sepharose 4B column. Upon reconstitution of C5D with Ca2+, Mg2+, and C1q, this reagent was utilized for the detection and quantitation of C5 hemolytic activity. The purified C5 preparations contained 1.5--2.5 x 10(12) effective molecules/mg protein and NHS expressed 0.5--2.0 x 10(11) effective molecules/ml.

A rapid and simple microprocedure for myelin isolation.

In order to conduct enzymatic studies on myelin from discrete regions of brain or spinal cord, we required a method capable of yielding purified myelin from tissue samples weighing 50 mg or less. Microprocedures that have been reported such as that of Detering and Wells (1976) or Fagg et al. (1978) were not suitable for rapid and routine myelin preparations. In this communication, the results of experiments in which we were able to isolate myelin from as little as 5 mg of whole brain are reported. Data are presented to show that the myelin isolated by this procedure is comparable to the myelin isolated by the macro‐scale procedure of Norton and Poduslo (1973a).

Effect of indomethacin in vivo on prostaglandin content of several rabbit tissues.

The prostaglandin (PG) content of several tissues and fluids from 6 day pregnant rabbits was evaluated following treatment with indomethacin or vehicle in vivo. PGE and PGF were measured by radioimmunoassay. More complete depletion of PGE and PGF was accomplished by 3 injections of indomethacin (s.c.) given during the 18 h before sacrifice at a dose of 10 mg indomethacin per kg body weight than was accomplished by 1 injection of the same amount of indomethacin (i.v.) 1.5 h before sacrifice. Levels of PGF were more easily depressed by indomethacin than were those of PGE. PG levels in the kidney and blastocysts were depressed to a greater extent by indomethacin than were those in the uterus, uterine fluid or peritoneal fluid. Evaluation of the effect of indomethacin on a particular physiological function should be interpreted with caution unless the extent of PG depletion in that tissue is also measured.

A biochemical investigation of spinal cord after contusive injury.

Abstract A closed spinal cord contusive injury system which is easily and reproducibly inflicted is described. At various times after injury, homogenates from individual segments along the cord were evaluated for total protein, lipid soluble galactose, (galactolipid), ceramide, 2′,3′-cyclic nucleotide 3′-phosphohydrolase, and myelin. Homogenates and myelin were also evaluated by SDS-polyacrylamide gel electrophoresis. In addition, the tissues were evaluated by light and electron microscopy. The results indicated that there was little change, in comparison with normal spinal cord tissue, in any biochemical components studied as long as 48 h after contusive injury. This was especially evident when the data were normalized on the basis of protein and/or galactolipid per tissue slice. This was in marked contrast to the progressive tissue necrosis and unraveling of the myelin sheath evident by light and electron microscopic examination of the injury site. The data suggest that at short times after injury, the architecture of the myelin sheath is disorganized yet the biochemical building blocks are still present.

A micromethod for the determination of ceramide

An enzymatic micromethod for the determination of ceramide is presented. The enzyme, E. coli diglyceride kinase was used to phosphorylate ceramide, as well as diglyceride with high specific activity gamma-[32P]ATP, and the two products are differentiated by their alkali stability. This method was applied to the detection of endogenous phospholipases and sphingolipases in several membrane systems and could have wide application.

The Presence of the Fifth Component of Complement (C5) in Rabbit Uterine Flushings in Relation to Reproductive State

The fifth component of complement (C5) has been detected in rabbit uterine flushings. The C5 activity was evaluated using a hemolytic assay which requires the use of a C5-depleted reagent (C5D) prepared by affinity chromatography of normal human serum. In the absence of C5D, there was no hemolysis of antibody-sensitized erythrocytes by rabbit uterine flushings, whereas the presence of the C5D reagent resulted in substantial hemolysis. The amount of hemolysis was correlated with the reproductive state of the rabbits, with higher amounts of hemolysis (expressed per mg uterine flushing protein) evident in estrous rabbits. In addition, the amounts of immunoglobulin G (IgG), albumin, and total protein were also determined in the uterine flushings. The amounts of total protein and IgG were increased in day-6 pregnant animals compared to estrus while the amount of albumin per ml uterine flushing was not significantly changed.

Residual complement activity after incubation with myelin isolated from both the central and peripheral nervous tissues

The relative ability of isolated central and peripheral nervous system myelin to interact with the complement system of plasma proteins was studied. The myelin used was a highly pure form, devoid of contamination by any subcellular organelles or membranes. Residual complement activity was a linear function of increasing quantities of myelin from 10 to 40 micrograms of myelin protein. Central and peripheral nervous system myelin showed identical residual complement activity at various temperatures above 7 degrees C and also after various time periods of incubation. The results show that central and peripheral nervous system myelin show equal ability to interact with complement, in spite of their different origin and differences in morphology and protein composition.