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Glycoproteins can be cleared from circulation if they carry oligosaccharide structures that are recognized by specific receptors. High-mannose type and asialo complex oligosaccharides are cleared by the mannose and asialoglycoprotein receptors, respectively. This paper presents the protein and terminal saccharide characterization for nine batches of a glycoprotein developed for pharmaceutical use. Each of these batches was evaluated in human pharmacokinetic (PK) studies, and had similar terminal elimination half-lives, but the initial clearance of this glycoprotein varied between batches. The protein is lenercept, an immunoadhesin comprising the Fc domain of human IgG1 and two tumor necrosis factor (TNF) binding domains derived from the extracellular portion of the TNFR1(p55). Lenercept is manufactured in Chinese hamster ovary (CHO) cells and is extensively N-glycosylated but is devoid of high-mannose glycans. The pharmacokinetic variability between these lots only correlated with terminal N-acetylglucosamine and not with terminal galactose, sialic acid or any polypeptide related parameter. The data emphasize the need for appropriate analytical methods for the characterization of glycoproteins, especially those designed for long half-lives, and show that assessment of the content of all three terminal saccharides is sufficient to ensure consistency of their PK performance properties.

A method has been validated, according to the Guidelines of the International Conference on Harmonization (ICH), for precise quantitation of the biological activity of recombinant human nerve growth factor (rhNGF) for lot release testing. The assay is based on the survival of a subclone of rat pheochromocytoma PC12 cells (PC12-CF) in response to rhNGF. Cell survival is measured by monitoring the reduction, by living cells, of the alamarBlue dye into a red form which is highly fluorescent. The assay is simple, has high throughput (performed in 96-well microtiter plates) and shows reproducible dose-response curves in the concentration range of 0.2-50 ng/ml. The method was validated for its linearity, accuracy, precision, robustness, and to meet current regulatory requirements. The assay demonstrated good linearity, yielding a coefficient of determination of 0.9902. Sample recovery studies demonstrated an accuracy ranging from 96 to 98%. The repeatability of the assay and intermediate precision had coefficients of variation (CV) of <9%. The assay was stability-indicating since it was able to detect changes in rhNGF samples degraded by protease treatment and in a number of isolated rhNGF variants. Robustness was demonstrated by the relative insensitivity of the assay to small deliberate changes in key method parameters. The validation data, provided in this manuscript, indicate that the newly described bioassay for rhNGF is robust, accurate, precise, and suitable for lot release potency testing of rhNGF.

Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.

Overexpression of the HER2/neu protooncogene has been shown to correlate with poor clinical prognosis. A murine monoclonal antibody (4D5) directed against the extracellular domain (ECD) of p185HER2 has been shown to inhibit in vitro and in vivo growth of carcinomas overexpressing HER2 and has been humanized (rhuMAb HER2). The objective of the study was the identification of an agent which might be useful for in vitro studies, tumor imaging and/or radioimmunotherapy by linking beta-emitting radionuclides to these HER2-targeted antibodies. Murine 4D5 and humanized rhuMAb HER2 were radiolabeled with 125I, 131I or 186Re. Physical characteristics (TCA precipitability, SDS-PAGE, size exclusion chromatography), binding affinities to the HER2 ECD (in an ELISA and on SK-BR-3 cells) and antiproliferative activities of the radiolabeled antibodies were determined. Although 131I-4D5 and 131I-rhuMAb HER2 usually retained > 85% ECD binding, they exhibited increased aggregation and fragment content, drastically reduced antiproliferative activities and poor stability upon storage at 4 degrees C. For these antibody preparations, conservation of binding did not necessarily correlate with preservation of bioactivity indicating the importance of bioactivity determinations in radiolabeled antibody studies. Conversely, 4D5 and rhuMAb HER2 labeled with 125I or 186Re maintained physical properties, ECD binding, antiproliferative activities and were stable upon storage at 4 degrees C for at least 8 days. The superior retention of physical and biological characteristics of 186Re-labeled 4D5 and rhuMAb HER2 compared with their 131I-labeled counterparts suggests the potential for their use as radioimaging and radioimmunotherapeutic agents in the treatment of HER2 overexpressing tumors.

Alanine scanning mutagenesis of the charged amino acids of melanoma growth stimulating activity (MGSA) was used to identify specific residues that are involved in binding to the human erythrocyte Duffy antigen/chemokine receptor (DARC) and to the type B interleukin-8 receptor (IL-8RB) on neutrophils. Receptor binding and biological studies with the alanine scan mutants of MGSA demonstrate that MGSA binds to DARC and the IL-8RB through distinct binding regions. One of the MGSA mutants, E6A, binds to human erythrocytes and is able to inhibit malaria invasion as efficiently as wild type MGSA but has a severely reduced ability to bind to or signal through the IL-8RB. Mutant chemokines like E6A could prove to be useful therapeutically for the design of receptor blocking drugs that inhibit erythrocyte invasion by Plasmodium vivax malaria.

The product of the HER2 protooncogene, p185HER2, represents an attractive target for cancer immunotherapies. We have prepared anti-p185HER2 immunoliposomes in which Fab' fragments of a humanized anti-p185HER2 monoclonal antibody with antiproliferative properties (rhuMAb-HER2) were conjugated to either conventional or sterically stabilized liposomes. These immunoliposomes bind specifically to p185HER2-overexpressing breast cancer cells (SK-BR-3 and BT-474). High-affinity binding of anti-p185HER2 immunoliposomes is comparable to that of free rhuMAbHER2-Fab' or the intact antibody. Empty immunoliposomes inhibit the culture growth of p185HER2-overexpressing breast cancer cells, and this antiproliferative effect is superior to that of free rhuMAbHER2-Fab', indicating that liposomal anchoring of these anti-p185HER2 Fab' fragments enhances their biological activity. Efficient internalization of anti-p185HER2 immunoliposomes, demonstrated by light and electron microscopy, occurs by receptor-mediated endocytosis via the coated pit pathway and also possibly by membrane fusion. Doxorubicin-loaded anti-p185HER2 immunoliposomes are markedly and specifically cytotoxic against p185HER2-overexpressing tumor cells in vitro. Anti-p185HER2 immunoliposomes administered in vivo in Scid mice bearing human breast tumor (BT-474) xenografts can deliver doxorubicin to tumors. These results indicate that anti-p185HER2 immunoliposomes are a promising therapeutic vehicle for the treatment of p185HER2-overexpressing human cancers.

Recently, we reported the development of fully humanized bispecific F(ab')2 antibodies with dual binding specificities to human T-lymphocytes and to tumor cells overexpressing HER2. These antibodies were shown to effectively mediate targeted HER2-overexpressing tumor cell killing by freshly isolated human T-cells. In this report we extend our studies to describe the interaction of the bispecific antibody with activated T-lymphocytes (ATL) maintained in culture for an extended period of time. A microtiter plate radioreceptor assay was used to elucidate the affinity of bispecific antibody binding to ATL. The data show that ATL maintained in vitro for up to 5 weeks continued to express high-affinity CD3 surface markers that bound to bispecific antibody with a Kd of 2.49 nM and exerted cytolytic activities against targets overexpressing HER2. In addition, we demonstrated the specific localization of HER2 x CD3 bispecific antibody to HER2-overexpressing tumor xenografts in nude mice. Furthermore, HER2 x CD3 bispecific antibody has the ability to inhibit the proliferative activities of breast tumor (SKBR-3) cells in vitro. The clinical implications of these data are discussed.

The humanized anti-p185HER2 antibody, humAb4D5-8, has completed Phase II clinical trials for p185HER2-overexpressing breast cancer. Here, this antibody is used as a building block to engineer a disulfide-linked Fv (dsFv) beta-lactamase fusion protein for use in antibody-dependent enzyme-mediated prodrug therapy using cephalosporin-based prodrugs. Three Fv variants were designed with an interchain disulfide bond buried at the VL/VH interface and secreted from Escherichia coli. One variant, dsFv3 (VL L46C VH D101C0, has similar affinity for antigen (Kd = 0.7 nM) as the wild-type Fv and was used to construct a fusion protein in which beta-lactamase, RTEM-1, is joined to the carboxy terminus of VH. The dsFv3-beta-lactamase fusion protein secreted from E. coli efficiently activates a cephalothin doxorubicin prodrug (PRODOX, kcat/km = 1.5 x 10(5) s-1 M-1). PRODOX is approximately 20-fold less toxic than free doxorubicin against breast tumor cell lines SK-BR-3 and MCF7, which express p185HER2 at elevated and normal levels, respectively. Prebinding the dsFv3-beta-lactamase fusion protein specifically enhances the toxicity level of PRODOX to that of doxorubicin against SK-BR-3 but not MCF7 cells. The fusion protein retains both antigen-binding plus kinetic activity in murine serum and is cleared rapidly as judged by pharmacokinetic analysis in nude mice (initial and terminal half-lives of 0.23 and 1.27 h, respectively). Development and characterization of the dsFv3-beta-lactamase fusion protein is an important step toward targeted prodrug therapy of p185HER2-overexpressing tumors.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.

Bovine insulin-like growth factor 2 (bIGF2) was produced in inclusion bodies in the cytoplasm of Escherichia coli and accumulated at high levels: 20-25% of total Coomassie-stained bacterial protein. The level of accumulation of bIGF2 was affected by the choice of codons in the 5' end of the coding sequence and by a rpoH mutation in the host cells. Purified recombinant bIGF2 had the native N terminus and the same mitogenic activity as that of bIGF2 purified from bovine serum.

The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti-p185HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185HER2. The monoclonal antibody is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function. In vitro assays have shown that muMAb 4D5 can specifically inhibit the growth of tumor cells only when they overexpress the HER2 protooncogene. MuMAb 4D5 has also been shown to enhance the TNF-alpha sensitivity of breast tumor cells that overexpress this protooncogene. Relevant to its clinical application, muMAb 4D5 may enhance the sensitivity of p185HER2-overexpressing tumor cells to cisplatin, a chemotherapeutic drug often used in the treatment of ovarian cancer. In vivo assays with a nude mouse model have shown that the monoclonal antibody can localize at the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185HER2. Modulation of p185HER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. Together with the demonstrated activity of muMAb 4D5 in nude mouse models, these results support the clinical application of muMAb 4D5 for therapy of human cancers characterized by the overexpression of p185HER2.

The proto-oncogene HER2/neu encodes a protein tyrosine kinase (p185HER2) that is homologous to the human epidermal growth factor receptor. Amplification and/or overexpression of HER2/neu occurs in multiple human malignancies and appears to be integrally involved in progression of some breast and ovarian cancers. Because of this fact, HER2/neu is an intriguing target for specific cancer therapeutic strategies. One such strategy is active specific immunotherapy, in which the immune system is targeted at specific antigens expressed by tumor cells. We have employed a transfected cell line that secretes the extracellular domain of p185HER2 as a source of HER2-derived immunogen in a guinea pig model. The immunized animals developed a cellular immune response, as monitored by delayed-type hypersensitivity, and antisera derived from immunized animals specifically inhibited the in vitro growth of human breast tumor cells overexpressing p185HER2. These data provide support for an immunotherapeutic approach to cancers characterized by overexpression of the HER2/neu proto-oncogene.

The HER2/neu proto-oncogene encodes a receptor that belong to the tyrosine-specific protein kinase family. Amplification of the HER2 gene in patients with breast and ovarian cancer has been shown to predict poorer survival rates. In order to understand the role of HER2 in malignant and normal cells, it is necessary to devise assays that can quantitate expression levels of the HER2 gene product (p185HER2) in production samples, biopsy specimens and biological fluids. We have developed a simple, quantitative ELISA that uses two monoclonal antibodies directed against the extracellular domain of the HER2 gene product, p185HER2 (HER2 ECD). The assay has a detection range of 0.25-120 ng/ml, is precise and sensitive. The ability of this assay to detect biologically active rHER2 ECD is demonstrated by its correlation to a growth inhibitory bioassay (r = 0.92). The sandwich ELISA can also accurately quantitate rHER2 ECD in mouse and monkey serum. This assay should be useful for quantitating low levels of circulating rHER2 ECD in animals in which rHER2 ECD is being used as antigen for immunotherapy and in patients which 'shed' receptor.

Sometribove (SB) is a synthetic form of bovine somatotropin (BST) whose amino acid sequence is the same for 190 of the 191 amino acids in BST. Administration of 500 mg of SB to dairy cows every 14 d increases the efficiency of milk production. Regulatory agencies have authorized a zero (0) milk and meat withdrawal time for investigational use of SB. The scientific basis for this authorization is as follows: 1) BST and other non-primate somatotropins are not active in humans, due to differences in the amino acid sequence from human somatotropin, which limits the ability of BST to bind to receptors on human tissues. 2) SB is not orally active, as it is degraded like other proteins when eaten. Administration of 50,000 microgram/kg/d SB to rats for 90 d produced no growth response. 3) Residual levels of SB in meat/milk are very low (ppb) and comparable to endogenous BST levels. 4) Residual levels (ppb) of insulin-like growth factor I (IGF-I) in meat and milk are only marginally increased by SB treatment (somatotropin stimulates local production of IGF-I in tissues to mediate some of its biological effects. 5) IGF-I was not orally active when fed to rats at doses ranging from 200 to 2,000 microgram/kg for 14 d.

We have developed a statistically standardized bioassay for quantifying the effect of swine serum on the proliferation rate of cultured L6 myogenic cells. The intra-assay coefficient of variation for this assay is 2.5%. Over 29 experiments, the relationship between the assays response to 2.5% control swine serum and its response to 2.5% serum plus 10(-7) M insulin is linear (r2 = .9). This relationship can be used to adjust experimental data obtained in different experiment to a common control value so that valid inter-assay comparisons can be made. The inter-assay coefficient of variation is reduced four- to five fold by this adjustment procedure. We believe this standardized assay provides a useful system for identifying and isolating unknown growth factors that affect muscle growth rate in an economically important species.

alpha-Lactalbumin undergoes a calcium-dependent electrophoretic shift at pH 8.3. When Ca2+ is removed by a chelator, the mobility of the protein increases, reflecting the exposure of negative electrical charges. The shift, however, is not observed by electrophoresis in the presence of SDS, which demonstrates that alpha-lactalbumin does not undergo a measurable conformational change upon debinding of Ca2+. Relative electrophoretic mobilities vary from 1.0 (no shift) to 1.4 among alpha-lactalbumins of different orders of mammals. The differences suggest a variable number of gram atoms of Ca2+ bound to alpha-lactalbumin or substitution of amino acid Ca2+ ligands in the calcium-binding loop.

Partial misincorporation of Lys for Arg has been observed for the Arg residues of IGF-1 when the molecule is expressed in Escherichia coli using a synthetic gene with the low frequency AGA codon encoding all six Arg residues and yeast preferred codons encoding the remaining residues. The Lys for Arg substitution at these residues could not be detected when a gene containing E. coli preferred codons, with the codon CGT coding for all Arg residues, was used for the expression of the protein. Similarly, no misincorporation of Lys for Arg could be detected when a gene containing Escherichia coli preferred codons at all positions, except for an AGA codon at Arg (36), was utilized.

When microsomes were prepared in 2-mercaptoethanol Vmax for 17beta-hydroxysteroid oxidoreductase (17beta-HSD) was greater, the Km for NAD+ was greater and the Km for testosterone lower than in its absence. During storage at 4 degrees Vmax increased in the presence of 2-mercaptoethanol and decreased in its absence; Km values for testosterone and NAD+ increased during storage in both cases. The presence or absence of 2-mercaptoethanol did not affect the extent or time-course of inactivation of 17beta-HSD by trypsin or phospholipase A. Furthermore, no differences were detected in sedimentation properties on sucrose density gradients suggesting that the differences and changes in the kinetic behavior of 17beta-HSD reflect a conformational flexibility at the active site and are not due to extensive changes in the structure of the microsomes. 17beta-HSD exposed to 2-mercaptoethanol was subject to substrate inhibition by testosterone, a type of inhibition not previously reported for this enzyme.

Exposure of guinea pig liver microsomes to phospholipase A2 resulted in the nearly complete loss of 17 beta-hydroxysteroid oxidoreductase (17 beta-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17 beta-HSD indicating that they may contribute to the inactivation by phospholipase A2. If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17 beta-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis per se results in a destabilization of 17 beta-HSD resulting in the subsequent activity loss. The inactivation of 17 beta-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism in vivo.

Bovine calf liver was homogenized in 0.1M NH4Ac, pH 7.4, and centrifuged. The supernatant was lyophilized and resuspended in 44 mM NaHCO3. After size exclusion chromatography on sephacryl S-300, a fraction of approximately 100 to 160 kDa was shown to inhibit the proliferation of rat L6 myoblasts in culture. The inhibitory activity was abolished when the resuspended preparation was heated at 70 C for 30 min before testing in L6 cell proliferation assay. Addition of 10(-9)M IGF-I did not influence the inhibitory response. Two IGF-I-binding proteins, 30 and 36 kDa, were identifiable in this fraction. These two proteins were more evident in other fractions in which no inhibitory activity was found. Inhibitory activity was not associated with IGF-I binding proteins.

Pregnant rabbits were injected with either 0.8 mg of betamethasone or with a comparable volume of saline on days 24 and 25 of gestation and delivered by cesarean section on day 26. There was a significant reduction in the weight of fetuses and in the weights of fetal brain, lungs, liver, and placenta in betamethasone-treated animals. With respect to the fetal brain, the concentration of DNA, the average of cell number, cell size, cell weight, and cell phospholipids were unaffected whereas the concentrations of phospholipids and protein were elevated in the treatment group.

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.

The active sites of bovine insulin-like growth factor (IGF) I and II fragments were studied. Overlapping fragments of IGF I (residues 1-25, 11-35, 21-45, 31-55, and 41-70) and of IGF II (residues 1-24, 10-34, 20-44, 30-54, and 40-67) were chemically synthesized. The activity of the fragments was measured by stimulating the proliferation of rat L6 myoblast cells. Two fragments of IGF I (residues 21-45 and 31-55) and two fragments of IGF II (residues 20-44 and 30-54) were active while the other fragments were inactive in stimulating cell proliferation. Although the activity of these fragments was observed only at a high concentration of 0.1 mM, the results imply that the active site is located around residues 31-45 for IGF I fragments and residues 30-44 for IGF II fragments. Consequently, an IGF I fragment (residues 26-50) having a five-residue extension to both the N- and C-terminal sites of residues 31-45 also stimulated the proliferation of L6 myoblast cells. Furthermore, the substitution of Ile-35 in two IGF II fragments (residues 21-45 and 31-55) by Ser inactivated these fragments. This suggests that Ile-35 is an essential residue for IGF II fragment activity. Ser-35, which was reported in the original sequencing of bovine IGF II, is incorrect in the sequence and furthermore has been consistently found to be an Ile-35 in our hands.(ABSTRACT TRUNCATED AT 250 WORDS)

alpha-Lactalbumin, the modifier protein of galactosyl transferase in the synthesis of lactose by the mammary gland, has been shown to undergo a Ca2+-dependent electrophoretic shift. Such shifts, characteristic of most calcium modulated proteins, are related to gross conformational changes upon binding calcium when detected in the presence of detergent (SDS-PAGE). However, we detected the calcium shift for alpha-lactalbumin using non-denaturing PAGE (ND-PAGE) where electrical charge changes are observed upon binding calcium. In order for a shift to be observed between the apo and calcium bound protein, calcium ion binding to proteins must have minimal dissociation constants (Kdiss) of 10(-7) M; alpha-lactalbumin is reported to bind calcium at Kdiss = 10(-10) to 10(-12) M. The electrophoretic shift identifies alpha-lactalbumin in complex milk whey patterns of many species of mammals.

The synthesis, processing and secretion of insulin-like growth factor-1 (IGF-1 or somatomedin-C) fused to LamB and OmpF secretion leader sequences in Escherichia coli have been investigated. Expression and secretion of IGF-1 was achieved. The major portion of this secreted IGF-1 accumulated in the periplasmic space as insoluble aggregates. A small amount of IGF-1 was found folded in its native conformation in the medium. The lamB and ompF signal sequences were fused to the 5' coding sequence of IGF-1. Fusion of the lamB signal sequence directly to IGF-1 (lamB-IGF-1) resulted in accumulation of 16-20 micrograms/A550/ml of correctly processed IGF-1 in the periplasmic space. The processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in an E. coli strain bearing a prlA4 mutation. Amino acid sequence analysis of IGF-1 secreted into the periplasm and exported into the medium confirmed the precise removal of the LamB or OmpF signal sequence. IGF-1 synthesized in E. coli was demonstrated to be active in a cell proliferation bioassay.

When guinea-pig liver microsomes were exposed to phospholipase C the rate of phospholipid hydrolysis exceeded the rate of decrease in 17 beta-hydroxysteriod dehydrogenase (17 beta-HSD) activity. The time-course of the decrease in 17 beta-HSD activity was biphasic. An initial more rapid decrease (30-50% of total) was associated with the major extent (85%) of phospholipid hydrolysis. Subsequently, a second, slower phase of 17 beta-HSD inactivation was observed. The addition of purified phospholipids did not reactivate 17 beta-HSD but did protect against the inactivation seen in the second phase. The diacyglycerides produced by phospholipase C action remained associated with the microsomes. It is proposed that the differences in the rates of 17 beta-HSD inactivation reflect variations in the distribution of a single form of 17 beta-HSD among differing membrane fractions rather than the existence of multiple enzyme forms. The stabilizing effects of phospholipids may be due to their ability to prevent changes in lipid-lipid, lipid-protein and protein-protein interactions resulting from diacylglyceride formation. Resuspended microsomal lipids (chloroform-methanol extracts) inactivated 17 beta-HSD suggestive of the presence of endogenous lipid modulators of enzymatic activity.

The proliferation-promoting activity of sera obtained from pigs before and after porcine growth hormone injections was tested in a muscle cell culture bioassay. For 3 d, purified porcine growth hormone (pGH) was administered by intramuscular injection to crossbred barrows. Two levels of pGH were administered: 18 micrograms pGH X kg-1 body weight X d-1 (low dose) or 143 micrograms pGH X kg-1 body weight X d-1 (high dose). Multiple blood samples were withdrawn from jugular catheters for 3 d prior to the injection, during the injection period and for 6 d after the last injection. Although serum pGH levels in low-dose pigs were raised from two to three times pre-injection levels, there was no significant change in serum proliferation-promoting activity or somatomedin-C (SmC), insulin or cortisol levels during or after administration of pGH. In contrast, the proliferation-promoting activity of sera obtained during and after the high-dose pGH injections was higher (P less than .005) than the pre-injection levels. Serum pGH levels were increased approximately 30-fold by 4 h after each injection, and increases in SmC levels were observed 10 to 16 h after the pGH injection. During the injection period SmC levels increased from 1.7 to 4 times pre-injection levels. Insulin and cortisol levels did not change significantly during the 3-d treatment period. We believe that this muscle cell culture bioassay system will be a useful addition to traditional radioimmunoassays and whole animal studies in elucidating the mode of action of pGH in pituitary-intact swine.

When human placental microsomes were heated in boiling water or exposed to trypsin, 30 to 40% of the 5-ene,3-ketosteroid isomerase activity was stable. Aqueous suspensions of chloroform:methanol extracts of microsomes also catalyzed isomerization of 5-pregnene-3,20-dione, activity being associated with the polar lipid fraction. The trypsin- and heat-stable activities, as well as that of resuspended microsomal lipids, showed a dependence on buffer composition and concentration. Little activity was detected in water at pH 7.0. Relative activities in various buffers were Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) greater than Pipes (1,4-piperazinediethanesulfonic acid) greater than potassium phosphate greater than Mes(4-morpholineethanesulfonic acid). The data suggest that the occurrence of membrane lipid-dependent nonenzymatic catalysis could contribute to the isotope exchange with solvent observed in previous studies of the mechanism of isomerization catalyzed by placental microsomes. The ability of the membrane lipid phase to catalyze steroid isomerization under certain conditions and the fact that this activity is subject to modifications by exogenous agents may have more general implications for an understanding of possible effects of xenobiotics on steroid hormone formation and action in vivo.