Errata

Insulin treatment of adipocytes increased the amount or activity of a low molecular weight, acid stable material which, when isolated and partially purified from intact adipocytes, yielded a single active fraction which stimulated pyruvate dehydrogenase. IM-9 lymphocytes contained a similar active material but in contrast to the adipocyte, insulin treatment of IM-9 lymphocytes caused a reduction in the amount or activity of the material either by decreasing production of the material or by increasing production of an inhibitory substance. These findings are consistent with the reported biological effects of insulin on these two cell types and suggest that the active material from these two cell types is the putative insulin chemical mediator.     

Purification of antineoplastic factor from eggs of a sea hare

An antineoplastic factor inducing tumor lysis was purified to apparent homogeneity from the supernatant of a homogenate of eggs of the sea hare Aplysia kurodai. On gel filtration, the purified substance gave a single band of material of 250 kDa containing 3 different subunits and this band coincided with cytolytic activity. The purified factor was half-maximally active at 10-60 ng protein/ml and lysed all tumor cells tested, but not normal cells.     

Purification and biologic properties of fibroblast somatomedin

Cultured human fibroblasts produce a peptide growth factor that cross-reacts with antisera to human somatomedin-C (Sm-C). To determine the identity of this species and compare its molecular properties to pure Sm-C, 2 liters of conditioned medium derived from human fibroblast monolayers were concentrated (X10) by ultrafiltration. The concentrated conditioned medium was purified further by CM-Sephadex ion-exchange chromatography. Following elution in 1.0 M NaCl, pH 8.0, the active material was purified by gel filtration on Sephadex G-150. The active fractions which eluted at Kd 0.45 (Mr estimated at 32,000) were further purified by isoelectric focusing. Two peaks of activity electrofocused at pI 5.4 and 7.2, respectively. The pI 5.4 peak contained only binding protein activity. The active fractions from the neutral pool were further purified by reverse-phase high pressure liquid chromatography on a C-18 Bondapak with a linear gradient of acetonitrile (10-60%). The active single peak which eluted at 55% acetonitrile gave a single band when analyzed by polyacrylamide gel electrophoresis. This material stimulated [3H]thymidine incorporation into human fibroblast DNA with approximately 3.2 times the potency of pure Sm-C but was equipotent in stimulating BALB/c 3T3 fibroblasts. It was degraded by fibroblast cultures at a slower rate compared to Sm-C, although it had a similar affinity for Sm-C-binding protein. We conclude that human fibroblasts produce two peptides that react with anti-Sm-C antibody but are chemically distinct from Sm-C. The greater response to fibroblast somatomedin may be due to its affinity for somatomedin-binding protein and slower degradation. These findings may have implications for understanding the regulation of human fibroblast replication.     

Specificity and structural analysis of a guinea pig transfer factor-like activity.

A transfer factor-like activity was prepared by Sephadex G-25 chromatography of immune guinea pig leukocyte lysates. This isolated material leads to antigen-dependent migration inhibition and thymidine uptake by nonimmune lymphoid cells. Tests of the "transfer factor" from guinea pigs immunized to either ovalbumin or bovine gamma-globulin demonstrated the donor specificity of the in vitro activity. The activity is susceptible to heat (56 degrees C), alkali (0.5 M sodium hydroxide), pronase, and phosphodiesterase. The pronase susceptibility is blocked by traysylol, a protease inhibitor; the phosphodiesterase susceptibility is not bocked by traysylol. The guinea pig factor was purified further by alkaline phosphatase treatment. Sephadex G-25 chromatography, and DEAE-cellulose chromatography. The final product, active in vitro, represents about 0.03% of the cellular material absorbing 260 nm light, and contains polymerized amines and phosphate. Gel electrophoresis of the fluram-reactive components suggests a limited heterogeneity of the DEAE-cellulose-purified material. These data are consistent with the active "transfer factor" molecule including both peptide and phosphate-containing components.     

Intracellular proclotting enzyme in limulus (Tachypleus tridentatus) hemocytes: its purification and properties

A proclotting enzyme associated with the hemolymph coagulation system of limulus (Tachypleus tridentatus) was highly purified from the hemocyte lysate. The first step of purification was performed by chromatography of the lysate on a pyrogen-free dextran sulfate-Sepharose CL-6B column, which was essential for separation of the proclotting enzyme from its activator, named factor B. The following steps consisted of column chromatographies on DEAE-Sepharose CL-6B, Sephadex G-150, benzamidine-CH-Sepharose and Sephacryl S-300. Through these procedures, 1.4 mg of the purified material was obtained from 630 ml of the lysate and approximately 300-fold purification was achieved. The preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence and absence of 2-mercaptoethanol. The single-chain proclotting enzyme was a glycoprotein with an apparent molecular weight of 54,000, and no gamma-carboxyglutamic acid was detected. The proclotting enzyme was converted to its active form by purified factor B or by trypsin. The resulting clotting enzyme had a molecular weight of 54,000, consisting of a heavy chain of Mr = 31,000 and a light chain of Mr = 25,000. The serine active site of the clotting enzyme was found in the heavy chain. The chemical analyses of the isolated heavy and light chains indicated that the activation of the proclotting enzyme to its active form by factor B or trypsin is induced by a limited proteolysis, yielding two chains bridged by a disulfide linkage(s).     

Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture

Medium harvested from cultures of mouse L-cells contains “conditioning factor activity” (CFA) that may be detected by its ability to stimulate colony formation by mouse marrow cells in culture. The active material has been characterized and appears to be a glycoprotein with properties similar to those reported for the colony-stimulating activity in human urine. Characterization of trypsin-digested material indicated that only part of the molecule is essential for biological activity. The CFA has been partially purified from serum-free L-cell conditioned medium, and evidence has been obtained that radioactivity derived from labelled valine or glucosamine may be incorporated into the factor. L-cell conditioned medium appears to be a convenient source of partially-purified, highly active material, suitable for use in studies on its mechanism of action.     

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity

An extract of human seminal plasma was found to have inhibin-like activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2–4 residues at the C-terminus, which could not be determined.     

Purification of a Ciliary Neurotrophic Factor from Bovine Heart

A neurotrophic factor that promotes the survival of cholinergic parasympathetic ciliary neurons has been purified approximately 20,000-fold from bovine cardiac tissue under nondenaturing conditions using heparin-affinity chromatography. Up to 22 micrograms of purified factor having a specific activity of 4 X 10(5) trophic units/mg can be obtained from 250 g of heart muscle. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified material show a broad band that is sometimes resolvable into a closely spaced pair of bands of 22 and 23 kilodaltons. Partially purified factor can be resolved into two peaks of activity (pI 5.6 and 5.0) by high-resolution anion-exchange chromatography and chromatofocusing, although these procedures have not proved useful as purification methods because of the large losses of activity incurred. It is likely that these two peaks represent the two bands seen on SDS-polyacrylamide gels. The bovine cardiac factor(s) differs from similar factors purified from chick optic tissues and pig brain in that it is irreversibly denatured by SDS.     

Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate.

A factor of low M(r) with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M(r) 10,000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M(r) of 192. The embryotrophic factor in the low M(r) extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.     

Purification of a lymphokine: Osteoclast activating factor from human tonsil lymphocytes

Osteoclast activating factor is a lymphokine produced by mitogen-stimulated human lymphocytes. The current studies describe purification to essential homogeneity of the major form of osteoclast activating factor present in supernatants of phytohemagglutinin stimulated lymphocyte cultures. Preliminary chemical and biological characterization of the purified material was carried out. The active factor is a peptide which migrates in polyacrylamide gel electrophoresis as an α-2 fraction in native gels and as a 9,000-dalton species in sodium dodecyl sulfate-urea gels. The purified fraction stimulates bone resorption $\text{in}\text{vitro}$ at doses between 0.1 and 500 ng/ml, with half-maximal stimulation at approximately 1 ng/ml.     

Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes

An intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr = 64,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr = 64,000, 40,000, and 25,000) were detected on SDS-PAGE after reduction. This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr of 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64,000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.     

Isolation of a stimulatory factor for nuclear DNA replication

Aqueous extracts of isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. The stimulatory factor is present throughout the mitotic cycle, and its activity is unaffected by prior exposure of plasmodia to cycloheximide. The stimulatory substance has been partially purified by heat treatment, precipitation with ethanol, chromatography on DEAE cellulose, and gel filtration. The purified material contains both carbohydrate and protein, and exhibits a molecular weight of about 30 000. The active substance increases the rate and overall extent of DNA replication in S-phase nuclei, but does not trigger the initiation of DNA synthesis in nuclei isolated from G2-phase plasmodia. The stimulatory material contains little or no deoxyribonuclease or DNA polymerase activity, and it does not affect DNA polymerase activity assayed using a purified DNA template.     

Biochemical analysis of the ligand for the neu oncogenic receptor.

The neu protooncogene (also called HER2 and c-erbB2) encodes a cell-surface tyrosine kinase structurally related to the receptor for the epidermal growth factor (EGF). We have previously reported that a candidate ligand for the neu receptor is secreted by ras-transformed fibroblasts. Biochemical analyses of the neu stimulatory activity indicate that the ligand is a 35-kDa glycoprotein that is heat stable but sensitive to reduction. The factor is precipitable by either high salt concentrations or acidic alcohol. Partial purification of the molecule by selective precipitation, heparin-agarose chromatography, and gel filtration in dilute acid resulted in an active ligand, which is capable of stimulating the protooncogenic receptor but is ineffective on the oncogenic neu protein, which is constitutively active. The purified fraction, however, retained the ability to stimulate also the related receptor for EGF, suggesting that these two receptors are functionally coupled through a bidirectional mechanism. Alternatively, the presumed ligand interacts simultaneously with both receptors. The presented biochemical characteristics of the factor are expected to enable a completely purified factor with which to explore these possibilities.     

Partial Purification from Chick Embryos of a Factor which Promotes Myoblast Proliferation and Delays Fusion

Chick embryo extract (EE) contained an activity which promoted myoblast proliferation and delayed fusion. Various tissue extracts prepared from 12-day embryos and adult chicken also showed the activity. We partially purified this active substance from 12-day embryos, following procedures which included extraction at pH 3.5, CM-Sephadex C–50 ion exchange and Sephadex G-75 gel filtration. Judging from the dose-response analyses, the factor was purified by some hundred-fold when EE was used as the starting material. The activity was associated with a macro-molecular substance (MW > 300K daltons) at first, but the apparent molecular weight of the active substance was estimated to be between 16 and 20K daltons at the final step of the preparation. It promoted myoblast proliferation and delayed myotube formation, and was active for both avian and rat myoblasts.
Since bovine pituitary gland fibroblast growth factor (FGF) showed the same activity, the factor may be FGF-related.     

Purification of a skeletal growth factor from human bone

A skeletal growth factor was isolated and purified from demineralized human bone matrix. A dose of 6 micrograms/mL of the purified factor significantly increased the proliferation rate of embryonic chick bone cells in serum-free culture (292% of controls, p less than 0.0001) but had no effect on embryonic chick skin cells plated at the same initial density. The factor is sensitive to inactivation by trypsin and urea, but not by collagenase, 20% butanol, or 1% mercaptoethanol. It is also resistant to inactivation by heat (stable for 15 min at 75 degrees C) and extremes of pH (stable for 30 min at 4 degrees C from pH 2.5 to 10.0). Purification of the active factor by selective heat and acid precipitations, molecular sieve column chromatography, and preparative polyacrylamide gel electrophoresis provided a material that was homogeneous by the criteria of high-pressure liquid chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. The apparent molecular weight is 83 000. The purified factor increases bone cell proliferation at doses comparable to other mitogens: 0.3 microgram/mL (3.6 nM) significantly increases DNA synthesis to 231% of controls (p less than 0.001). The purified factor was also active on cultured embryonic chick bones, enhancing the growth rate of tibiae and femurs, as measured by increased dry weight (185% of controls, p less than 0.025) and [3H]proline incorporation (164% of control, p less than 0.001), respectively.     

Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies

Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor alpha

A gene encoding human tumour necrosis factor alpha (TNF-alpha) has been chemically synthesized, cloned and expressed to yield a biologically active protein in Escherichia coli. The 480-bp gene was assembled by enzymic ligation of 32 oligonucleotides, cloned directly into M13mp18 for sequence verification and expressed in the broad host range high-level expression vector pMMB66EHST. Expressed recombinant TNF-alpha was shown to have the correct molecular weight, processed N-terminal sequence, antibody cross-reactivity and tumour cell killing activity. The expression product of the synthetic gene has been purified to homogeneity by a two-step ion-exchange procedure and the purified material shown to be active.     

Characterization of Novel Forms of Coagulation Factor XIa: independence of factor XIa subunits in factor IX activation

Factor XI is the zymogen of a dimeric plasma protease, factor XIa, with two active sites. In solution, and during contact activation in plasma, conversion of factor XI to factor XIa proceeds through an intermediate with one active site (1/2-FXIa). Factor XIa and 1/2-FXIa activate the substrate factor IX, with similar kinetic parameters in purified and plasma systems. During hemostasis, factor IX is activated by factors XIa or VIIa, by cleavage of the peptide bonds after Arg145 and Arg180. Factor VIIa cleaves these bonds sequentially, with accumulation of factor IX alpha, an intermediate cleaved after Arg145. Factor XIa also cleaves factor IX preferentially after Arg145, but little intermediate is detected. It has been postulated that the two factor XIa active sites cleave both factor IX peptide bonds prior to releasing factor IX abeta. To test this, we examined cleavage of factor IX by four single active site factor XIa proteases. Little intermediate formation was detected with 1/2-FXIa, factor XIa with one inhibited active site, or a recombinant factor XIa monomer. However, factor IX alpha accumulated during activation by the factor XIa catalytic domain, demonstrating the importance of the factor XIa heavy chain. Fluorescence titration of active site-labeled factor XIa revealed a binding stoichiometry of 1.9 +/- 0.4 mol of factor IX/mol of factor XIa (Kd = 70 +/- 40 nm). The results indicate that two forms of activated factor XI are generated during coagulation, and that each half of a factor XIa dimer behaves as an independent enzyme with respect to factor IX.     

A diffusible factor responsible for the determination of cholinergic functions in cultured sympathetic neurons. Partial purification and characterization

A macromolecule has been partially purified which influences the choice of the neurotransmitter synthesized by sympathetic neurons. Previous studies showed that culture medium conditioned by incubation on certain types of non-neuronal cells increased [3H]acetylcholine synthesis and accumulation from [3H]choline in primary cultures of neurons dissociated from neonatal rat superior cervical ganglion and grown in the virtual absence of non-neuronal cells. A concomitant decrease of [3H]catecholamines production was observed (Patterson, P. H., and Chun, L. L. Y. (1977) Dev. Biol. 56, 263-280). The active cholinergic factor in conditioned medium from C6 glioma or primary rat heart cultures has been purified about 1500-fold by a sequence of ammonium sulfate precipitation, DEAE-, CM-cellulose, and Sephadex G-100 chromatography. The partially purified factor is active at 1 microgram of protein/ml of culture medium and is eluted from Sephadex columns as a single peak of apparent Mr = 40,000-45,000. This material is insensitive to 0.2 M 2-mercaptoethanol, but is inactivated by 1 mM Na periodate. Its activity is partially decreased by treatment with a protease from Streptomyces griseus but is unaffected by neuraminidase. Material purified through the ammonium sulfate, DEAE- and CM-cellulose steps contains large amounts of trypsin- and chymotrypsin-inhibiting activities.